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WO2022251679A1 - Nitroxide radicals for use as antiviral treatment for coronavirus infection - Google Patents

Nitroxide radicals for use as antiviral treatment for coronavirus infection Download PDF

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WO2022251679A1
WO2022251679A1 PCT/US2022/031405 US2022031405W WO2022251679A1 WO 2022251679 A1 WO2022251679 A1 WO 2022251679A1 US 2022031405 W US2022031405 W US 2022031405W WO 2022251679 A1 WO2022251679 A1 WO 2022251679A1
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oxyl
coronavirus
tetramethylpiperidine
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composition
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Tracey A. Rouault
William Marston LINEHAN
Nunziata MAIO
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US Department of Health and Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • Coronaviruses are a group of RNA viruses known to cause disease in mammals and birds.
  • the novel coronavirus severe acute respiratory syndrome-coronavirus 2 (SARS- CoV-2), has caused a global pandemic known as coronavirus disease (COVID-19).
  • SARS- CoV-2 severe acute respiratory syndrome-coronavirus 2
  • COVID-19 coronavirus disease
  • Other disease caused by coronaviruses include, e.g., SARS, MERS, and the common cold.
  • Vaccines are available for some coronaviruses (e.g., SARS-CoV2); however, a need remains for antiviral treatments for patients that are infected with coronaviruses, at risk for infection with coronaviruses, and/or patients that have been exposed to coronaviruses.
  • SARS-CoV2 coronavirus-CoV2
  • the invention provides a method of treating a subject, the method comprising administering to the subject an effective amount of an anti -viral agent, wherein the anti -viral agent comprises a nitroxide radical, and wherein the antiviral agent treats or prevents infection by a coronavirus in the subject.
  • the inventive method further comprises the administration of an iron chelator to the subject.
  • the invention also provides a composition for use in treating a subject, the composition comprising an effective amount of an anti-viral agent and a pharmaceutically acceptable carrier, wherein the anti-viral agent comprises a nitroxide radical, and wherein the composition treats or prevents infection by a coronavirus when administered to the subject.
  • the inventive composition further comprises an iron chelator.
  • Figure 1 A-E collectively show that Fe-S cluster incorporation into nspl2 occurs through its interactions with components of the Fe-S biogenesis machinery.
  • Figure 1 A depicts representative Coomassie blue staining of pull-down assays performed with purified proteins.
  • 0.25 pg of purified nspl2-FLAG or the variants wherein either or both LYR motifs were replaced by alanines (VYR-AAA, LYR-AAA and VYR/LYR-AAA, respectively) were combined with 0.25 pg of HSC20, as indicated.
  • Immunoprecipitations (IPs) were performed with anti-FLAG antibody to immunocapture nspl2 proteins.
  • HSC20 aka HSCB
  • Figure 1C depicts mass spectrometry identification of affinity purified interacting partners of nspl2 that are components of the Fe-S cluster biogenesis pathway (see Table SI for a complete list).
  • Figure ID depicts levels of radiolabeled iron ( 55 Fe) incorporated into nspl2 WT or the variants in control cells transfected with non-targeting siRNAs (NT-siRNAs) and in cells transfected with siRNAs directed against the main scaffold protein ISCU (si-ISCU).
  • NT-siRNAs non-targeting siRNAs
  • siRNAs directed against the main scaffold protein ISCU si-ISCU
  • Figure IE depicts representative Coomassie staining showing levels of nspl2 WT or variants in control and ISCU-depleted cells that were quantified in (ID) for their iron content. Immunoblots to ISCU, showing the efficiency of its silencing (knock-down), and to a-tubulin, used as a loading control, are also presented.
  • Figure 2A-F collectively show evidence for ligation of two Fe-S metal cofactors by nspl2.
  • Figure 2A depicts UV-vis spectra of nspl2 WT or variants of the cysteine residues in the two metal ligating centers.
  • the order of traces/lines is, from top to bottom, WT, C301S-C306S-C310S, C847S-C645S-C646S, and C301/306/645/646-S.
  • Figure 2B depicts representative Coomassie blue staining of purified nspl2 WT or variants analyzed in Figure 2A.
  • FIG. 2C depicts Mossbauer spectra of nspl2 WT and variants exhibited the parameters typical of [Fe4S4] clusters. For each of the two nspl2 cys-to-ser variants, approximately 95% of iron was still associated with a quadrupole doublet that matched parameters of WT nspl2.
  • Figure 2E depicts conserved zinc-binding motifs in SARS-CoV-2 nspl2 (PDB ID: 7BTF) rendered in the ribbon representation. H295-C301-C306-C310 ligate zinc at the interface between the NiRAN and the catalytic domain, whereas the C487-H642-C645-C646 residues ligate zinc in the catalytic domain.
  • Figure 2F depicts levels of radiolabeled iron ( 55 Fe) incorporated into nspl2 WT or variants, as indicated.
  • Significance was determined by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Mean ⁇ 95% CF ***/ > ⁇ o.001.
  • the order of the bars is, from left to right: WT, LYR-AAA, C301S-C306S-C310S, C847S-C645S-C646S, C301-306-645-646-S, and EDTA.
  • Figure 3A-C collectively show that Fe-S cluster sites in nspl2 are important for activity and interactions with nspl3.
  • Figure 3B depicts schematic of the complex required for coronaviral replication in which the two Fe-S clusters and their coordination spheres are highlighted.
  • Figure 4A-F collectively show that the stable nitroxide TEMPOL potently inhibits the RdRp by causing disassembly of its Fe-S clusters and blocked viral replication in cell culture models of SARS-CoV-2 infection.
  • Figure 4A depicts UV-vis spectra of nspl2 anoxically purified from Expi293F control cells and from cells treated with TEMPOL.
  • the order of lines (traces) is, from top to bottom: nspl2, npl2 + Tempol 0.2 mM, and nspl2 + Tempol 0.5 mM.
  • Figure 4B depicts UV-vis spectra of as purified nspl2 and of purified nspl2 incubated with TEMPOL (1: 2 ratio nspl2: TEMPOL) for 10 min.
  • Figure 4C depicts RNA polymerase activity of the RdRp complexes anoxically purified from control and TEMPOL-treated Expi293F cells.
  • Figure 4D depicts representative Coomassie staining of the RdRp complexes analyzed for the activity in Fig. 4C.
  • Figure 5 provides multiple sequence alignments of the residues ligating zinc in the crystal structure of the RdRp of SARS-CoV-2. Multiple sequence alignments of the catalytic subunit of the RdRp, nspl2, from SARS-CoV-2 and other coronaviruses, as indicated, showing conservation of the residues ligating 2 zinc ions in the crystal structure.
  • the conserved zinc-binding motifs in SARS-CoV (PDB ID: 6NUR) and SARS-CoV-2 (PDB ID: 7BTF) nspl2 are rendered in the ribbon representation. The coordinate details of the zinc binding residues are shown in stick representation.
  • the SEQ ID NOs are, from top to bottom, for the upper grouping SEQ ID Nos: 27-40 and for the lower grouping, SEQ ID Nos: 41-47.
  • Figure 6A-D collectively show that Fe-S cluster incorporation into nspl2 requires its interactions at LYR motifs with the Fe-S cluster biogenesis machinery.
  • Figure 6A depicts multiple sequence alignments of nspl2 from SARS-CoV-2 and other coronaviruses showing conservation of the VYR and LYR motifs that are present in the NiRAN and in the catalytic domains of the polymerase, respectively (UniProt entries are indicated).
  • the SEQ ID Nos are, from top to bottom, SEQ ID Nos: 48-62.
  • Figure 6B depicts proposed model of the chaperone/cochaperone-mediated transfer of nascent Fe-S clusters, assembled on the main scaffold protein, ISCU, through the direct binding of the cochaperone HSC20 to LYR motifs present in recipient apoproteins.
  • ATP hydrolysis by the HSC20-cognate chaperone, HSPA9 is proposed to facilitate cluster transfer to recipient proteins, while concomitantly driving folding of the recipient protein into its final conformation.
  • Figure 6C depicts Coomassie blue staining of 3xFLAG-nspl2 proteins recombinantly expressed and anoxically purified from Expi293F mammalian cells at different time points after transfection, as indicated. Optimal expression was achieved 48h post transfection.
  • Figure 7A-D collectively show that ligation of two Fe-S metal cofactors by nspl2 in sites occupied by zinc in the structure of the protein purified aerobically.
  • Figure 7A depicts UV-vis spectra of nspl2 wild type or variants, as indicated.
  • the spectrum of nspl2 treated with the chelator EDTA was included to show complete loss of absorbance at 420nm, which was similar to the loss of absorbance of the variant in which cysteines in both metal-ligating centers were replaced by serines (nsp 12 C ’ I) I S_C ’ I)6S_C645S_C646S )
  • serines nsp 12 C ’ I) I S_C ’ I)6S_C645S_C646S
  • Figure 7B depicts Coomassie blue staining of nspl2 proteins analyzed in Fig. 7A.
  • Figure 7C depicts X-bnd EPR spectra, recorded at 20 K, of as purified nspl2 wild type and Cys-to-Ser variants of the two metal-ligating sites.
  • Figure 7D depicts X-band EPR spectra, recorded at 20 K, of nspl2 WT and variants exhibited the parameters typical of [Fe4S4] + clusters upon reduction with sodium dithionite.
  • Figure 8A-E collectively show that markedly increased RNA-binding and polymerase activities of the RdRp complex containing the Fe-S clusters compared to the zinc-RdRp.
  • the assay performed upon addition of only [a- 32 P]ATP enabled extension of the primer of a single nucleotide, thereby providing a way to assess template binding affinities of the [FeS] versus the Zn-containing RdRp complex.
  • Figure 8C depicts representative Coomassie blue staining of the anoxically purified ([FeS]) and aerobically, zinc-reconstituted (Zn) RdRp complexes.
  • Figure 8D depicts UV-vis spectra of the [FeS]-nspl2 alone and in complex with the accessory factors nsp7 and nsp8, required for the activity, showing that the presence of nsp8 and nsp7 did not perturb the [Fe4S4] cluster typical absorbance at 420nm. As expected, the Zn-containing RdRp complex lacked the absorbance at 420nm.
  • Figure 8E depicts gel mobility Vshift assay to detect RdRp binding to RNA.
  • Either the Fe-S- or the zinc-RdRp complex (9pg) was incubated with increasing amounts of template/primer RNA (0, 0.3, 0.6, 1.2 and 2 pg).
  • 9 pg of purified nspl2 were combined with 1.5 pg template-primer RNA. Strikingly, the Fe-S-nspl2 bound the template/primer complex even in the absence of nsp7/nsp8, although less efficiently than the full RdRp complex (nspl2/nsp7/nsp8).
  • Figure 9A-F collectively show that the doses of TEMPOL that disabled the RdRp in HEK293 cells did not impair mitochondrial function or diminish activity of the cytosolic Fe-S enzyme DP YD.
  • Figure 9A presents, from top to bottom, immunoblots to FLAG, cytosolic IRPl and DPYD on lysates from control (CTRL) HEK293 cells transfected with the backbone vector (empty) or with 3xFLAG-nspl2 and from cells transfected with 3xFLAG-nspl2 and treated with the indicated doses of TEMPOL.
  • CTRL control
  • In-gel activity assays of cytosolic (ACOl) and mitochondrial (AC02) aconitases showed loss of cytosolic, but not mitochondrial, aconitase activity, upon TEMPOL treatment.
  • respiratory complexes I and II activity assays were unaffected by TEMPOL treatment.
  • TOM20 was used as a loading control for the mitochondrial fractions.
  • Figure 9B depicts schematic representation of respiratory complexes I-IV showing the Fe-S clusters and heme centers required for their activities.
  • FIG. 9C depicts oxygen consumption rates (OCRs) of cells treated as in Figure 9A.
  • Figure 9D depicts schematic representation of the reaction catalyzed by the cytosolic Fe-S enzyme DPYD.
  • DPYD converts [4- 14 C]-thymine to [4- 14 C]-dihydrothymine.
  • Figure 9E depicts ribbon representation of the crystal structure of DPYD, which assembles into a dimer, containing a total of 8 [Fe4S4] clusters.
  • Figure 9F depicts DPYD-mediated conversion of [4- 14 C]-thymine to [4- 14 C]- dihydrothymine in control and TEMPOL-treated HEPG2 or Expi293F cells, as indicated, assayed by thin layer chromatography (TLC) and autoradiography.
  • the reaction mix containing the substrate of the reaction [4- 14 C]-T without cell extract was loaded as a negative control (no extract) to visualize the substrate (4- 14 C-thymine) by TLC.
  • Figure 10A-G collectively show that the doses of Tempol that disabled the RdRp in VeroE6 cells did not adversely affect mitochondrial function or impair activity of the cytosolic Fe-S enzyme DPYD.
  • Figure 10A provides, from top to bottom, immunoblots to FLAG, IRPl and TOM20 in cells transfected with 3xFLAG-nspl2 and treated with TEMPOL, as indicated.
  • gel activity assays of respiratory complexes I, II and IV showing intact mitochondrial respiration in cells treated with TEMPOL.
  • Figure 10B depicts OCR measurements confirming that TEMPOL treatment did not affect the activities of the mitochondrial respiratory chain complexes.
  • Figure IOC depicts immunoblots to the Fe-S subunit of complex I, NDUFS1, the core subunit of complex III (UQCRC1), the heme subunit of complex IV (MTCOl), and the Fe-S subunit of complex II, SDHB, showed that TEMPOL treatments did not affect protein levels of the indicated subunits of the respiratory complexes.
  • TOM20 was used as a loading control.
  • Figure 10D depicts in-gel activity assay of cytosolic (ACOl) and mitochondrial (AC02) aconitases showed loss of cytosolic, but not mitochondrial, aconitase activity, upon TEMPOL treatment.
  • Figure 10E depicts levels of cytosolic IRPl, IRP2, transferrin receptor (TFR1), ferritin heavy chain (FTH), the CIA component, CIAOl, and the Fe-S enzyme DPYD in control and TEMPOL treated cells.
  • Figure 10F depicts DPYD-mediated conversion of [4- 14 C]-thymine to [4- 14 C]- dihydrothymine in control or TEMPOL treated VeroE6 cells for the indicated time points, assayed by thin layer chromatography (TLC) and autoradiography.
  • TLC thin layer chromatography
  • Figure 11 A-D collectively show that binding of nspl2 to the components of the Fe-S cluster biogenesis machinery was not affected by TEMPOL treatment.
  • WT DFO cells expressing wild type nspl2 treated with IOOmM of the iron chelator DFO.
  • WT_T cells expressing wild type nspl2 treated with 0.5mM Tempol.
  • Figure 1 IB depicts representative Ponceau S staining of blotting membrane used in Fig. 11 A.
  • FIG. 1 ID depicts representative Ponceau S staining of blotting membrane used in (C) for western.
  • Figure 12A-B collectively show that the nitric oxide donor diethylamine nonoate (DEA/NO) at ImM inhibited the activity of the Fe-S-RdRp by 60%.
  • DEA/NO diethylamine nonoate
  • Figure 12A depicts representative polymerase activity assay of the RdRp complexes anoxically purified from control and DEA/NO-treated cells at the different doses indicated.
  • Figure 14 depicts the Synergistic effect of a combination of Tempol and RDV-TP in inhibiting the activity of the SARS-CoV-2 RdRp complex.
  • Activity of the RdRp complex anoxically purified from Expi293F cells and treated with the indicated doses of DEA/NO, RDV-TP, TEMPOL or a combination of RDV-TP and TEMPOL in vitro.
  • Figure 15 is a comparison of remdesivir efficacy on the FeS RdRp vs. the Zn- containing RdRp complex in vitro.
  • RNA polymerase activity of the RdRp complex anoxically purified from Expi293F cells and treated with the indicated doses of DEA/NO, RDV-TP and TEMPOL.
  • the activity of the Fe-S-containing enzyme was compared to the activity of the RdRp purified aerobically and reconstituted with zinc.
  • TL TEMPOL
  • DFP deferiprone
  • n 4 biological replicates
  • Figure 17 presents a plot representing the quantification of the RdRp assay intensities of the bands from the gel represented by Figure 16.
  • band intensities of the products of the primer extension assay (Elongated RNA) in control (CTRL), TEMPOL-, DFP-, and TEMPOL/DFP- treated samples were assessed by IMAGE J and the results were plotted using PRISM 9. Multiple comparisons were performed using One-way Anova Sidak's multiple comparisons test.
  • Coronaviruses are a group of RNA viruses known to cause disease in mammals and birds. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirale s, and realm Riboviria. Exemplary coronaviruses include, e.g., SARS-CoV2, SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63. Diseases caused by coronavirus infection include, e.g., SARS, MERS, COVID-19 and the common cold.
  • the coronavirus is SARS-CoV2, which causes COVID-19.
  • the coronavirus is other than a SARS-CoV2 coronavirus, for example SARS-CoV, or MERS-CoV.
  • the coronavirus may be human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63, which are known to cause the common cold.
  • Coronaviruses employ a multi-subunit machinery for replication and transcription.
  • a set of nonstructural proteins (nsps) produced as cleavage products of the ORFla and ORFlab polyproteins assembles to facilitate viral replication and transcription.
  • the core component of this complex is the catalytic subunit (nspl2) of an RNA-dependent RNA polymerase (RdRp), which catalyzes the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of SARS-CoV-2, with the assistance of nsp7 and nsp8 as accessory factors.
  • RdRp nspl2-nsp7-nsp8 complex
  • RdRp of SARS-CoV-2 was proposed to contain zinc ions ligated in the same locations as those observed in SARS-CoV in highly conserved metal binding motifs composed of H295- C301-C306-C310 and C487-H642-C645-C646 (see Fig. 5).
  • the inventors have surprisingly discovered that the catalytic subunit of the RdRp, nspl2, ligates two iron-sulfur metal cofactors in the sites that were previously thought to contain zinc ions. These metal binding sites are essential for replication and for interaction with the viral helicase. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.
  • an anti-viral agent comprising a nitroxide radical can be used to inhibit RdRp, and, thus, can be used to treat coronaviruses that rely on RdRp, including coronaviruses other than SARS-CoV2.
  • the coronavirus may be a coronavirus having a metal binding motif in the nspl2 subunit of RdRp.
  • coronaviruses having the highly conserved metal binding motif include SARS-CoV, MERS- CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63 as illustrated in sequence alignments presented in Fig. 5.
  • the present invention relates to antiviral agents.
  • Antiviral activity refers to the ability of a compound or composition to prevent, inhibit, or lessen replication of a virus.
  • the antiviral agent prevents, inhibits or lessens the replication of a coronavirus in a host, for example a human subject.
  • the anti-viral agents according to the present invention may block viral replication by oxidizing iron sulfur (Fe-S) metal cofactors ligated by the catalytic sub-unit of RNA-dependent RNA polymerase (RdRp), thereby inhibiting the RdRp and preventing, inhibiting and or lessening viral replication.
  • Fe-S iron sulfur
  • RdRp RNA-dependent RNA polymerase
  • Nitroxide radicals include compounds having the general formula R2NO, wherein the R groups may be the same or different.
  • the nitroxide radical is a cyclic nitroxide.
  • the antiviral agent may comprise, consist of, or consist essentially of a nitroxide radical.
  • Various nitroxide radicals are known in the art.
  • Nitroxide radicals include, for example, piperidine nitroxide derivatives such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol); 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l- piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo- Tempo).
  • piperidine nitroxide derivatives such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol); 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l- piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo- Tempo).
  • Nitroxide radical compositions can also include other substituted variants of Tempo (typically in the 4 position) such as, for example, 4-(2-bromoacetamido)-2, 2,6,6- tetramethylpiperidine- 1 -oxyl; 4-ethoxyfluorophosphonyloxy-2,2,6,6-tetramethylpiperidine- 1 - oxyl; 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl, 4-isothiocyanato-2, 2,6,6- tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(4- nitrobenzoyloxyl)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-phosphonooxy-2,2,6,6- tetramethylpiperidine-l-oxyl; and the like.
  • the nitroxide radical also includes other compounds known
  • the nitroxide radical is 4-hydroxy-2, 2,6,6- tetramethylpiperidine- 1 -oxyl (Tempol).
  • Suitable nitroxide radical compounds include 2-ethyl-2,5,5-trimethyl-3- oxazolidine-l-oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3- aminomethyl-Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-cyano-Proxyl); 3-Carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3 -Carbarn oyl-Proxyl); and 3- Carboxy-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy-Proxyl).
  • the coronavirus when the coronavirus is SARS-CoV2, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) or (b) when the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol), the coronavirus is other than SARS-CoV2.
  • the activity of the nitroxide radical in treating or prophylaxis of a coronavirus infection can be enhanced by an iron chelator.
  • Example 7 herein demonstrates that a synergistic effect is realized when the nitroxide radical, tempol, is employed in conjunction with the iron chelator, deferiprone.
  • any suitable iron chelator can be used, and the iron chelator can be administered with the nitroxide radical either within the same formulation (composition) or in a separate formulation, which can be administered prior to, concurrently with, or subsequent to the nitroxide radical.
  • An exemplary iron chelator contemplated for use in the present invention comprises deferiprone (DFP), which is currently approved by the United States Food and Drug Administration as a solution comprising either 80 or 100 mg/ml deferiprone or as a tablet comprising either 1 g or 500 mg deferiprone as an active agent, both formulations for oral administration.
  • DFP deferiprone
  • FERRIPROX ® FERRIPROX ®
  • Another exemplary iron chelator contemplated for use in the present invention comprises deferasirox, which is currently approved by the United States Food and Drug Administration as orally-administrable granules (approved dosages being 90 mg, 180 mg, or 360 mg) or tablets (approved dosages being 90 mg, 180 mg, or 360 mg).
  • deferasirox is currently approved by the United States Food and Drug Administration as orally-administrable granules (approved dosages being 90 mg, 180 mg, or 360 mg) or tablets (approved dosages being 90 mg, 180 mg, or 360 mg).
  • a granule formulation of deferasirox is marketed for pharmaceutical use under the tradenames JADENU ® and JADENU ® SPRINKLE.
  • Another exemplary iron chelator contemplated for use in the present invention comprises desferrioxamine, which is administered intravenously.
  • Another orally active investigational iron chelator contemplated for use in the present invention comprises CN128 (see Chen et al., “CN128: A New Orally Active Hydroxypyridinone Iron Chelator,” ./. Med. Chem ., 63, 4215-4226 (2020), which is incorporated herein in its entirety).
  • an iron chelator in which an iron chelator is employed, preferably one is selected that is orally active. Furthermore, a dosage preferably is selected to minimize the risks of side effects associated with the use of such agents.
  • iron chelators are administered to treat patients with iron overload. For use in the present invention, however, lower dosages than those approved to treat iron overload are preferred.
  • a suitable dosage of the iron chelator, DFP, contemplated for antiviral use in conjunction with Tempol in accordance with the present invention, can be lower than the approximately 75 mg/kg dosage currently employed for DFP when it is employed to treat patients with iron overload.
  • Exemplary dosages of DFP for use in the context of the present invention include less than or equal to 70 mg/kg, such as less than or equal to 60 mg/kg, such as less than or equal to 50 mg/kg, such as less than or equal to 40 mg/kg, such as less than or equal to 30 mg/kg, such as less than or equal to 20 mg/kg, and even lower dosages may be appropriate.
  • subject refers to an animal, e.g., preferably, a mammal and, more preferably, a human.
  • the invention provides a method for treating a subject, e.g., a human, that has, is suspected to have, or is at risk for contracting a viral infection.
  • a subject e.g., a human
  • the subject has been exposed to a viral infection.
  • the viral infection is due to a coronavirus, for example SARS-CoV2.
  • the terms “preventing” and “treating,” “treatment,” and the like are used herein to refer to obtaining a desired pharmacological and physiological effect.
  • the effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition associated with viral infection and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect associated with viral infection.
  • preventing and “treating,” as used herein, covers any prevention or treatment of a disease in a mammal, such as a human, and includes: (a) preventing the disease from occurring in a subject which may be at risk for contracting the disease but has not yet been diagnosed as having it, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be at risk for contracting the disease but does not yet experience or display symptoms of the disease; (b) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and (c) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions.
  • inventive methods can provide any amount of any level of treatment or prevention in a subject.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the diseases described herein being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • an “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response, e.g., treating the condition.
  • the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject.
  • An effective amount encompasses therapeutic and prophylactic treatment.
  • a “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • Aspects of the invention include the administration of the antiviral agent prophylactically. “Prophylactically” means that the antiviral agent is administered to prevent a condition or to prevent one or more symptoms associated with a condition.
  • a “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • a subject may be considered to have been exposed to coronavirus infection if the subject has recently (e.g. within a week or two weeks) been in contact with individuals known or suspected to have a coronavirus infection. Such contact will be of sufficient duration and under conditions sufficient to allow the coronavirus to spread from one subject to another. Exposure may be determined via techniques known in the epidemiology field, for example contact tracing. For example, a human subject may be considered to have been exposed to coronavirus infection if one or more members of the subject’s family or members of the subject’s household have recently been diagnosed with a coronavirus infection. The human subject may also have been exposed to coronavirus at a place of employment.
  • the human subject may be considered to have been exposed to coronavirus infection if the subject is a health care provider who recently treated or examined an individual or individuals known to have a coronavirus infection.
  • the exposed subject has not been tested for coronavirus infection.
  • the antiviral agent is administered to a subject that has been exposed to a coronavirus infection.
  • a subject may be considered at risk for coronavirus infection when the individual is likely to have come into contact with one or more individuals having a coronavirus infection. Risk may be assessed via techniques known in the epidemiology field, e.g. statistical methods and/or models of coronavirus infection.
  • the at-risk subject may live and/or work in an area in which a coronavirus infection is prevalent.
  • a human subject may be considered at risk for coronavirus infection if one or more members of the subject’s family or members of the subject’s household have recently exhibited symptoms consistent with a coronavirus infection.
  • the at-risk subject may be a health care provider that frequently examines or treats patients that may have a coronavirus infection.
  • An at-risk subject may be a health care provider who recently treated or examined an individual or individuals presenting with symptoms consistent with a coronavirus infection.
  • a human subject may be considered at risk if the subject regularly comes into contact with numerous individuals in a region where coronavirus infections are known to be present.
  • the antiviral agent is administered prophylactically to a subject that is at risk for coronavirus infection.
  • test types include, for example, reverse transcriptase polymerase chain reaction tests, antigen tests, and antibody tests. Techniques, material and procedures used to perform these tests are known in the art.
  • the subject has tested positive for a coronavirus infection. In other aspects of the invention, the subject has not tested positive for a coronavirus infection.
  • Coronavirus infection may cause various symptoms in a subject. Symptoms may range from mild to critical. For example, mild symptoms may include symptoms generally associated with respiratory infection, and include nasal congestion, runny nose, cough, muscle pain, sore throat, headache, fever, breathing difficulties, mild pneumonia, loss of smell, and loss of taste. Severe symptoms of coronavirus infection may include, for example, dyspnea and hypoxia. Critical symptoms may include, for example, respiratory failure (e.g., acute respiratory distress syndrome (ARDS)), shock and organ failure.
  • ARDS acute respiratory distress syndrome
  • the subject to be treated may have mild or severe symptoms, but not critical symptoms. In other aspects of the present invention, the subject to be treated may have mild symptoms, but no severe or critical symptoms. For example, the subject may not be suffering from ARDS at the time of treatment.
  • the subject e.g. the human subject
  • a subject may be asymptomatic.
  • a subject is considered asymptomatic if the subject does not exhibit clinical symptoms of an infection sufficient to lead one skilled in the art to conclude that an infection may be present, although infection might be confirmed by serological examination or other biological test for infection.
  • the subject may present with no symptoms associated with respiratory infection.
  • a subject may present without nasal congestion, runny nose, cough, muscle pain, sore throat, headache, fever, breathing difficulties, mild pneumonia, loss of smell, or loss of taste.
  • the asymptomatic subject may have been exposed to coronavirus or may be at risk for coronavirus infection.
  • the antiviral agent may be administered prophylactically to a patient who has not yet been infected with a coronavirus, or administered to treat an asymptomatic coronavirus infection.
  • the effective amount (i.e., dose) of an anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) to be administered to a subject can be determined depending upon, for example, age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment.
  • Exemplary doses can be from about 0.01 to about 1000 mg, by oral administration.
  • Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.50, 1, 5, 10, 25, 50, 100, 125, 150, 200, or 250 mg to a maximum dose of about 300, 400, 500, 600, 700, 800, 900, or 1000 mg, wherein an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses.
  • Specific examples of particular effective amounts contemplated via oral administration include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55,
  • the oral dose can be administered once daily, twice daily, three times daily, or more frequently.
  • the method comprises administering Tempol in a dose of about 500-1000 mg (e.g., about 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg) once daily, twice daily, or three times daily over a suitable period to have the desired effect (e.g., 2, 3, 4, or 5 weeks).
  • the method comprises administering an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like), which, in some embodiments, can be administered using currently-approved dosages as noted above (for agents that are approved). However, as noted, such approved dosages are approved for treatment of patients with iron overload.
  • an iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • currently-approved dosages as noted above (for agents that are approved).
  • approved dosages are approved for treatment of patients with iron overload.
  • a nitroxide radical such as Tempol
  • lower dosages of the iron chelator are preferred, to minimize potential side effects.
  • dosages of DFP suitable for use in the context of the present invention include less than or equal to 70 mg/kg, such as less than or equal to 60 mg/kg, such as less than or equal to 50 mg/kg, such as less than or equal to 40 mg/kg, such as less than or equal to 30 mg/kg, such as less than or equal to 20 mg/kg, and even lower dosages may be appropriate.
  • an iron chelator can be administered in accordance with the present invention once daily, twice daily, or three times daily over a suitable period to have the desired effect (e.g., 2, 3, 4, or 5 weeks).
  • One dosing regimen contemplated for application in the context of the present invention comprises administering Tempol (or another nitroxide radical) and deferiprone (or another iron chelator) in a single combined oral dosage form (e.g., a tablet or capsule) twice daily over a five to seven day course.
  • Tempol or another nitroxide radical
  • deferiprone or another iron chelator
  • an anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) for use in parenteral administration (preferably intravenous administration) is generally from about 0.01 to about 300 mg/kg body weight.
  • a nitroxide radical e.g., Tempol
  • an iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • parenteral administration preferably intravenous administration
  • Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.50, 1, 5, 10, 25, 50, or 100 mg/kg body weight to a maximum dose of about 125, 150, 175, 200, 250, 275, or 300 mg/kg body weight, wherein an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses.
  • particular effective amounts contemplated include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,
  • Continuous intravenous administration is also contemplated for from 1 to 24 hours per day to achieve a target concentration from about 0.01 mg/L blood to about 100 mg/L blood.
  • Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.25, 0.50, 1, 5, 10, or 25 mg/L blood to a maximum dose of about 40, 45, 50, 55, 60,
  • an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses.
  • Specific examples of particular effective amounts contemplated via this route include about 0.02, 0.03,
  • the dose to be used can depend upon various conditions, and there may be cases wherein doses lower than or greater than the ranges specified above are used.
  • the nitroxide radical is administered in a dose sufficient to produce 200-400 micromolar concentration of the antiviral agent in tissues of the subject.
  • the dose may be sufficient to produce a 200-400 micromolar concentration in multiple tissues throughout the body of the subject, for example the pharynx, trachea, lungs, blood, heart, vessels, intestines, brain and/or kidneys.
  • aspects of the present invention further provide a pharmaceutical composition
  • a pharmaceutical composition comprising a compound as described above (nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like)) and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount, e.g., a therapeutically effective amount, including a prophylactically effective amount, of the compound of the present invention.
  • the pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemi co-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical compositions; the compound of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
  • compositions described herein for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compound and one which has no detrimental side effects or toxicity under the conditions of use.
  • compositions of the present invention are merely exemplary and are in no way limiting.
  • the anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) may be administered in the form of, for example, solid compositions, liquid compositions, or other compositions for oral administration, injections, liniments, or suppositories for parenteral administration.
  • Solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules.
  • Capsules include hard capsules and soft capsules.
  • the nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • an excipient e.g., lactose, mannitol, glucose, microcrystalline cellulose, or starch
  • combining agents e.g., hydroxypropyl cellulose, polyvinyl pyrrolidone, or magnesium metasilicate aluminate
  • disintegrating agents e.g., cellulose calcium glycolate
  • lubricating agents e.g., magnesium stearate
  • stabilizing agents agents to assist dissolution (e.g., glutamic acid or aspartic acid), or the like.
  • the agents may, if desired, be coated with coating agents (e.g., sugar, gelatin, hydroxypropyl cellulose, or hydroxypropylmethyl cellulose phthalate), or be coated with two or more films. Further, coating may include containment within capsules of absorbable materials such as gelatin.
  • a preferred composition for administering the nitroxide radical and the iron chelator comprises a combined oral dosage form (e.g., a tablet, capsule, syrup, and the like) comprising both agents formulated together in a single dosage form.
  • Liquid compositions for oral administration include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs.
  • the nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • a commonly used diluent e.g. purified water, ethanol, or mixture thereof.
  • such liquid compositions may also comprise wetting agents, suspending agents, emulsifying agents, flavoring agents (e.g., flavor-masking agents) sweetening agents, perfuming agents, preserving agents, buffer agents, or the like.
  • Injections for parenteral administration include solutions, suspensions, emulsions, and solids which are dissolved or suspended.
  • the nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • the solvents are, for example, distilled water for injection, physiological salt solution, vegetable oil, propylene glycol, polyethylene glycol, alcohol such as ethanol, or a mixture thereof.
  • the injections also can include stabilizing agents, agents to assist dissolution (e.g., glutamic acid, aspartic acid, or POLYSORBATE 80TM), suspending agents, emulsifying agents, soothing agents, buffer agents, preserving agents, etc.
  • the compositions are sterilized in the final process or manufactured and prepared by sterile procedure.
  • the compositions also can be manufactured in the form of sterile solid compositions, such as a freeze-dried composition, and can be sterilized or dissolved immediately before use in sterile distilled water for injection or some other solvent.
  • compositions for parenteral administration include liquids and ointments for external use, endermic liniments, compositions for inhalation, sprays, suppositories for rectal administration, and pessaries for vaginal administration, which compositions include a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) and are administered by methods known in the art.
  • a nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • compositions for inhalation or sprays may comprise additional substances other than diluents, such as, e.g., stabilizing agents (e.g. sodium sulfite hydride), isotonic buffers (e.g. sodium chloride, sodium citrate or citric acid). See, for example, the methods described in U.S. Patents. 2,868,691 and 3,095,355.
  • the nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • a self-propelling composition that includes a solution or dispersion of the nitroxide radical in micronized form.
  • an effective dispersion of finely divided drug particles can be accomplished with the use of very small quantities of a suspending agent, present as a coating on micronized drug particles. Evaporation of the propellant from the aerosol particles after spraying from the aerosol container leaves finely divided drug particles coated with a fine film of the suspending agent.
  • the average particle size can be less than about 5 microns.
  • the propellant composition can employ, as the suspending agent, a fatty alcohol such as oleyl alcohol.
  • Propellants that may be employed include hydrofluoroalkane propellants and chlorofluorocarbon propellants. Dry powder inhalation also can be employed.
  • Nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • compositions suitable for inhalation may be used, for example, in a method of the invention to treat or reduce the remodeling of the pulmonary vasculature associated with prolonged alveolar hypoxia, e.g., in subjects that have, are suspected to have, or are at risk for pulmonary arterial hypertension, and cor pulmonale.
  • the anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be administered as a supplement with food or drink.
  • the nitroxide radical e.g., Tempol
  • iron chelator e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like
  • the composition is administered in combination with one or more additional active agents.
  • the additional active agent or agents may be antiviral agents.
  • the additional antiviral agent may be a nucleoside analog, for example remdesivir.
  • a method of treating or preventing a coronavirus infection in a subject comprising administering to the subject an effective amount of an anti -viral agent comprising a nitroxide radical, and the antiviral agent treats or prevents the coronavirus infection in the subject.
  • the coronavirus is SARS-CoV2
  • the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol)
  • the coronavirus is other than SARS-CoV-2.
  • nitroxide radical is 2, 2,6,6- tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l-piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo-Tempo), 4-(2- bromoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-ethoxyfluorophosphonyloxy- 2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-l- oxyl, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6- tetramethylpiperidine-l-oxyl; 4-(4-nitrobenzo
  • the coronavirus is other than SARS-CoV-2
  • SARS-CoV SARS-CoV
  • MERS-CoV human coronavirus HKU1
  • human coronavirus OC43 human coronavirus 229E
  • human coronavirus NL63 human coronavirus NL63.
  • compositions for use in treating or preventing a coronavirus infection in a subject comprising an effective amount of an anti -viral agent comprising a nitroxide radical and a pharmaceutically acceptable carrier.
  • the coronavirus is other than SARS-CoV-2 [0137] 20.
  • composition for use of aspect 31, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
  • a method of treating or preventing a coronavirus infection in a subject comprising administering to the subject a combination comprising an effective amount of 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and an effective amount of an iron chelator, wherein the administration of the combination treats or prevents the coronavirus infection in the subject.
  • the coronavirus is SARS-CoV-2.
  • compositions for use in treating or preventing a coronavirus infection in a subject comprising (a) an effective amount of 4- hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and a pharmaceutically acceptable carrier and (b) an effective amount of an iron chelator and a pharmaceutically acceptable carrier.
  • the coronavirus is SARS-CoV-2.
  • compositions for use of aspect 34 wherein the compositions comprise a single composition.
  • HEK293, HEPG2 and VeroE6 cells were purchased from ATCC. Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5g/L glucose, supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C and 5% C02 in a humidified incubator. Expi293F cells used for mammalian cell expression of the RdRp of SARS-CoV-2 were purchased from ThermoFisher Scientific (catalog number: A14635).
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • Expi293F cells used for mammalian cell expression of the RdRp of SARS-CoV-2 were purchased from ThermoFisher Scientific (catalog number: A14635).
  • IREs internal ribosome entry site
  • Takara bicistronic pIRES expression vector
  • Expression of the SARS-CoV-2 helicase, nspl3 was obtained from pCMV3-nspl3 (Sino Biologicals, Cat. No. VG40596-UT).
  • Point mutations into nspl2 were introduced using the QuikChange II site- directed mutagenesis kit (Agilent Technologies), following the manufacturer’s instructions. All clones were verified for the insertion of the desired mutation(s) by Sanger sequencing at Eurofms USA. Plasmid transfections into mammalian cells, except Expi293F cells (see below), were performed with Lipofectamine 2000 (ThermoFisher Scientific), according to standard procedures.
  • Expi293F cells at a final density of 3 c 106 viable cells/mL (2.25 x 109 cells at the time of transfection) were inoculated in 800 mL of fresh Expi293TM Expression Medium supplemented with 300 mM L-cysteine and 40 mM FeC13 or 57FeC13 (for Mossbauer and EPR analyses) and transfected with the following combinations of constructs: 840 pg P3xFLAG-SARS-CoV-2-nspl2 alone (for Mossbauer and EPR studies) or 420 pg P3xFLAG-SARS-CoV-2-nspl2 and 840 pg of pIRES-nsp7/nsp8-Strep II (for the RdRp activity assays), which were combined with 48 ml of Opti-MEMTM I Reduced Serum Medium (ThermoFisher Scientific) and incubated for 5 min at room temperature.
  • the plasmid DNA mix was then combined with a mix containing 2.6 ml ExpiFectamineTM 293 Reagent and 45 ml Opti-MEMTM I Reduced Serum Medium and incubated at room temperature for additional 20 min prior to addition to the cell suspension. Forty-eight hours after transfection, cells were spun down and washed twice with cold PBS prior to being taken inside an argon recirculated glove box operated at ⁇ 0.2ppm 02 for lysis.
  • Cells were lysed in 100 ml of lysis buffer (150 mM NaCl, 50 mM Na-HEPES pH 7.4, 10% (v/v) glycerol, 3 mM MgCE, 2 mM TCEP, 0.5% NP-40, EDTA-free protease inhibitor cocktail) and homogenized by gently pipetting up and down until no cell clumps were visible. After clearing up the insoluble fractions by centrifuging the homogenate at 40,000 x g for 30 min, 4 ml of pre equilibrated FLAG-M2 beads were added to the lysate. Recombinant protein complexes were immunoprecipitated for 3 hours at room temperature, before being packed into a column.
  • lysis buffer 150 mM NaCl, 50 mM Na-HEPES pH 7.4, 10% (v/v) glycerol, 3 mM MgCE, 2 mM TCEP, 0.5% NP-40, EDTA-free protease
  • the resin was washed with 10 column volumes of lysis buffer, followed by 20 column volumes of lysis buffer supplemented with 300 mM NaCl.
  • the FLAG-tagged nspl2 proteins or the 3xFLAG-nspl2/nsp7/nsp8-Strep tag II complexes were then eluted from the FLAG-M2 beads with 3xFLAG peptide dissolved in lysis buffer for 1 hour at room temperature.
  • the eluates at ⁇ 50 mM were concentrated to -250 mM using a 30 kDa molecular weight cut-off filter.
  • mitochondria from HEK293, Expi293F or VeroE6 cell pellets were isolated from the cytosolic fractions after cell permeabilization with a buffer containing 0.1% digitonin in 210 mM mannitol, 20 mM sucrose, and 4 mM HEPES.
  • the pellets after centrifugation at 700 x g for 5 min contained mitochondria, which were isolated by differential centrifugation and solubilized in lysis buffer I, containing 50 mM Bis-Tris, 50 mM NaCl, 10% w/v Glycerol, 0.001% Ponceau S, 1% lauryl maltoside, pH 7.2 and protease inhibitors.
  • C-terminally 3xFLAG-nspl2 wild type or variants of the LYR motifs and C- terminally HA-tagged human HSC20 were individually expressed and purified from Expi293F cells.
  • Affinity purification of 3xFLAG-nspl2 proteins was performed using M2- FLAG beads (Sigma), as indicated under “Protein Production and Purification”. Proteins were eluted, following extensive washing of the beads with lysis buffer supplemented with 100 pg/ml of FLAG peptide.
  • Affinity purification of HA-tagged HSC20 was performed using anti -HA agarose beads (Pierce). HSC20 protein was eluted with 20 pg/ml of HA peptide.
  • nspl2-FLAG wild type or the variants of either one or both LYR motifs were combined with 0.25 pg of HSC20.
  • Immunoprecipitations (IPs) were performed with M2 -FLAG beads (Sigma) to immunocapture nspl2 proteins.
  • the presence of HSC20 co-eluted with nspl2 proteins was analyzed by SDS-PAGE and Coomassie staining. Aliquots corresponding to 20% of the inputs were run on the gel for comparison.
  • Proteins ( ⁇ 10 pg per sample) were run on SDS-PAGE and stained with Coomassie Blue G-250. The gel bands were excised and washed overnight in 50% methanol with 10% acetic acid. Proteins were reduced using 10 mM Tris(2-carboxy ethyl) phosphine hydrochloride at room temperature for 1 hour, then alkylated with 10 mM N-ethylmaleimide (NEM) for 10 minutes and digested with trypsin (Promega) 1:20 (w/w) at 37 °C for 18 hours. Tryptic digests were extracted from the gel and cleaned with an Oasis HLB microplate (Waters).
  • NEM N-ethylmaleimide
  • the desalted peptides were injected into a Dionex UltiMate 3000 RSLCnano HPLC instrument (Thermo Fisher Scientific) with an ES802 nanocolumn (Thermo Fisher Scientific). The column temperature was set at 45 °C.
  • Mobile phase A and B (MPA, MPB) contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively.
  • the peptides were eluted at a flow rate of 300 nL/min using the following gradients: 3% to 22% MPB for 66 min, 22% to 33% MPB for 6 min, 33% to 80% MPB for 4 min.
  • Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) was used for data acquisition.
  • the LC- MS/MS data were acquired in data-dependent mode.
  • the MSI scans were performed in orbitrap with a resolution of 120K at 200 m/z with a mass range of 375-1500 m/z and an automatic gain control (AGC) value of 2 x le5.
  • the quadrupole isolation window was 1.6 m/z. Ions with an intensity greater than 1 x le4 were fragmentated by CID method with collision energy fixed at 30%.
  • the MS2 scans were conducted in ion trap with and AGC target of 3 x le4.
  • the Proteome Discoverer software version 2.4 was used for protein identification and quantitation. Raw data were searched against Uniprot Human Database. The mass tolerances for precursor and fragment were set to 10 ppm and 0.6 Da, respectively. Up to 2 missed cleavages were allowed for trypsin digestion. NEM on cysteines was set as fixed modification. Variable modifications include Oxidation (M), Met-loss (Protein-N-term) and Acetyl (Protein N-term). Peptides were validated based on q-values using percolator algorithm. The search results were filtered by a false discovery rate (FDR) of 1% at the protein level. The protein abundances were calculated by summing the abundance of the connected peptides.
  • FDR false discovery rate
  • the maximum allowed fold change value was set to 100.
  • the nspl2/control ratios were set to 100.
  • the nspl2/control ratios were set to 0.01.
  • ANOVA Individual Proteins
  • RNA oligonucleotides were purchased from Dharmacon (Horizon Discovery). RNA primers were radiolabeled at the 5' end with [g- 32 R] ATP (Perkin Elmer) using the T4 polynucleotide kinase from the KinaseMax 5' End-Labeling Kit (Thermo Fisher Scientific). Two distinct pairs of primer/template duplexes were used in the RNA extension assays.
  • a shorter pair of primer/template oligonucleotides consisted of the 4-mer 5'- rArCrGrC-3' (SEQ ID NO: 26) and the 14-mer 5'-rUrUrUrUrUrUrGrUrCrUrGrCrGrU-3' template (SEQ ID NO: 15) and a longer pair consisted of the 13-mer primer 5'- rArGrGrUrArArUrArArArArArUrU-3' (SEQ ID NO: 16), and the 29-mer template 5'- rUrUrUrUrArArArCrGrArArArArUrUrUrArUrArCrCrU-3' (SEQ ID NO: 17).
  • oligonucleotides were mixed at equal molar ratios in annealing buffer (50 mM NaCl and 10 mM Na-HEPES pH 7.5), denatured by heating to 75°C for 5 min and then slowly cooled to 4°C.
  • annealing buffer 50 mM NaCl and 10 mM Na-HEPES pH 7.5
  • the SARS-CoV-2 RdRp complex anoxically purified from Expi293F mammalian cells co-expressing 3xFLAG-nspl2 and nsp7/nsp8-Strep II at a final concentration of 1 mM was incubated with 3 mM dsRNA in the presence of 1.2 U/pl RNase inhibitor in reaction buffer containing in 100 mM NaCl, 20 mM Na-HEPES pH 7.5, 5% (v/v) glycerol, 10 mM MgCF and 0.5 mM TCEP, which were prepared in DEPC-treated water.
  • RNA extension was initiated by addition of NTPs (300 pM UTP, GTP and CTP, and 100 pM [a-32P]ATP (Perkin Elmer)). The total reaction volume was 10 pi. Reactions were stopped by the addition of 2 c stop buffer (7 M urea, 50 mM EDTA pH 8.0, 1 c TBE buffer). Samples were digested with proteinase K (New England Biolabs) and RNA products were separated on 20% acrylamide gels in 1 x TBE buffer supplemented with 8M urea and imaged by autoradiography on Carestream BioMax Light films.
  • NTPs 300 pM UTP, GTP and CTP, and 100 pM [a-32P]ATP (Perkin Elmer)
  • the total reaction volume was 10 pi. Reactions were stopped by the addition of 2 c stop buffer (7 M urea, 50 mM EDTA pH 8.0, 1 c TBE buffer). Samples were digested with proteinase K (New England Biolab
  • a gel mobility assay was performed to detect RNA binding by the RdRp complex.
  • the binding reaction contained 25 mM HEPES pH 7.4, 100 mM sodium chloride, 2 mM magnesium chloride and 1 mM TCEP, 9 pg RdRp complex with increasing amounts of template-primer RNA (0, 0.3, 0.6, 0.9, 1.2 and 2 pg).
  • the binding reaction was combined with 1.5 pg of template-primer RNA. Reactions were incubated for 1 hour at room temperature and resolved on 20% acrylamide gels in 1 x TBE buffer at 90 V for 1 hour on ice. The gel was stained with SYBR Safe RNA staining reagent (Thermo Scientific), according to the manufacturer’s instructions and visualized on a Bio-Rad Chemidoc imager.
  • On-TARGET Plus siRNA pools against human ISCU (Cat. No. L-012837-01- 0005) and the control nontargeting (NT si-RNAs) pool (Cat. No. D-001810-10-05) were purchased from Dharmacon.
  • Knockdown of ISCU in Expi293F cells was achieved by transfecting cells twice with siRNAs at a 48-hour interval using ExpiFectamineTM 293 Transfection Kit (ThermoFisher Scientific) according to manufacturer’s instructions.
  • ExpiFectamineTM 293 Transfection Kit ThermoFisher Scientific
  • Cytosolic extracts were subjected to immunoprecipitation with anti -FLAG to immunocapture 3XFLAG-nspl2 proteins 48h post-transfection. Samples collected after competitive elution (with 3X FLAG peptide at 100pg/ml) were analyzed by scintillation counting to assess 55Fe content. The background levels corresponding to 55Fe measurements on eluates after anti-FLAG immunoprecipitations on cytosolic extracts isolated from cells transfected with the empty vector, p3XFLAG-CMV-14 (Sigma, Cat.
  • the ferrozine based colorimetric assay (Sigma- Aldrich, Cat. No.: MAK025- 1KT) was used to determine the concentration of iron (Fe 2+ ) in preparations of purified nspl2 WT and variants each at 250 mM protein concentration, according to the manufacturer’s instructions.
  • the iron released by the addition of an acidic buffer was reduced to measure both Fe 2+ and Fe 3+ and colorimetrically determined at 593 nm after reaction with a chromogen proportional to the iron present in the samples.
  • Iron concentrations in nspl2 WT, nspl2C301S-C306S-C310S and nspl2C487S-C645S-C646S, each at 250 mM, were 1875 ⁇ 87.5 mM, 950 ⁇ 52.5 mM and 917.5 ⁇ 82.5 mM (n 5), respectively, corresponding to 7.5 ⁇ 0.35 iron/protomer for nspl2 WT, 3.8 ⁇ 0.21 iron/protomer for nspl2C301S-C306S-C310S and 3.67 ⁇ 0.33 iron/protomer for nspl2C487S-C645S-C646S.
  • the dihydropyrimidine dehydrogenase (DPYD) activity was determined by thin layer chromatography (TLC), following a previously described protocol (K. S. Kim, N. Maio, A. Singh, T. A. Rouault, Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAOl -mediated transfer to recipients. Hum Mol Genet , (2016)); O. Stehling et al., MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity. Science 337, 195-199 (2012)), with the following modifications.
  • Cell lysates containing 150 pg of proteins isolated from control or TEMPOL-treated cells were applied to 50 pi of a reaction mix containing 25 mM Tris-HCl (pH 7.5), 0.1% digitonin, 2.5 mM MgCF, 2mM DTT, 10 mM [4- 14C]-thymine (O.lmCi/ml Moravek Inc. CA, USA), 10 mMNADPH. After 4 hours of incubation at 32 °C, the reaction was stopped by addition of 10 pi of perchloric acid (10% v/v). Reaction mixtures were centrifuged at 20000 x g for 5 minutes and the supernatants analyzed by TLC.
  • the gel was incubated in 50 mM phosphate buffer pH 7.4 containing lmg/ml DAB (3,3’- diaminobenzidine) and lmg/ml cytochrome c at room temperature for 30-45 min.
  • UV-vis Ultraviolet-visible Absorption Spectroscopy and Amino Acid Analysis
  • AAA was performed by Alphalyse Inc. to precisely quantify the purified nspl2 wild type and variants.
  • second set of EPR samples were prepared by incubating wild type nspl2 and its variants at a final concentration of 220 mM with 5 mM sodium dithionite for 15 min prior to freezing.
  • Continuous-wave EPR (CW-EPR) spectra were acquired on an ESP300 Bruker X-band spectrometer equipped with an ER 410ST resonator. The temperature was held at 20 K by an ER 4112-HV Oxford Instruments (Concord, MA) variable-temperature helium flow cryostat.
  • The38pectrumeter was controlled through EWIN 2012 software on an external personal computer with a GPIB interface.
  • the modulation amplitude was 5 G
  • the microwave frequency was 9.439 GHz
  • the microwave power was 20 mW.
  • the “pepper” routine from the EasySpin package 5.2.30. was used to simulate the EPR spectra.
  • nspl2 For full reconstitution with zinc, which yielded a protein containing 2 zinc ions per protom er, aerobically purified nspl2 was incubated with 5 mM dithiothreitol and 10 equivalents of ZnCF at room temperature for 2 h and subsequently passed through a PD G-25 column (GE Healthcare).
  • Quantification of Zinc Content of Aerobically Purified and Reconstituted Nspl2 [0188] The QuantiChromTM Zinc Assay Kit (BioAssay Systems) was used for quantitative determination of zinc content in nspl2 purified aerobically and reconstituted with ZnCF to promote full occupancy with zinc of all the metal ligating sites.
  • OCRs Oxygen Consumption Rates
  • OCRs were examined using the XF96 Seahorse Metabolic Analyzer from Seahorse Biosciences. Briefly, HEK293 or VeroE6 cells were plated at a seeding density of 3 x 104 cells/well in 200 pL of complete media in an extracellular flux tissue culture plate. Cells were incubated with or without the indicated doses of TEMPOL for 24h. Prior to metabolic test the media was removed and replaced with Seahorse XF assay media (Agilent; Cat. No.
  • Antibodies in this study were as following: anti-HSC20 westerns were performed either with a custom-made antibody (Genscript) or with a commercial antibody (Sigma #HPA018447). Anti-CIAOl (sc-374498), NFS1 (sc-81107) and DPYD (sc-376681) were from Santa Cruz Biotechnology. Anti-nspl3 (NBP2-89168) was from Novus Biologicals. Anti-HSPA9 (HPA000898) and a-tubulin (T9026) were from Sigma. Anti- FAM96B (20108-1-AP), MMS19 (16015-1-AP), TOM20 (11802-1-AP) and ISCU (14812-1- AP) were from Proteintech.
  • Anti-FLAG antibody was from Origene (TA50011).
  • Mouse monoclonal antibody to TFR1 was from ThermoFisher Scientific (13-6800).
  • Anti-Strep II (ab 184224), ferritin heavy chain (ab65080), NDUFSl (abl69540), UQCRC1 (abl97055), MTCOl (abl4705) and SDHB (abl4714) were from abeam.
  • pairwise comparisons between two groups were analyzed using the two-tailed unpaired Student’s t test. Significance for multi-group comparisons were analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test. All tests were performed with GraphPad Prism 7, and data were expressed as mean ⁇ 95% confidence interval (Cl) except where indicated otherwise.
  • Vero E6 cells were inoculated at a multiplicity of infection (moi) of 0.01 in DMEM supplemented with GlutaMAX, sodium pyruvate, 2% heat-inactivated fetal bovine serum,
  • Vero E6 cells for SARS-CoV-2 infection studies were maintained in complete DMEM supplemented with GlutaMAX, sodium pyruvate, 10% heat-inactivated fetal bovine serum, 100 U/ml Penicillin, 100 pg/ml Streptomycin (D10 media). The day before infection, cells were plated at a density of 2x 105 cells per well in 24-well plates (Coming) supplemented with increasing concentrations of TEMPOL (range 0.1 pM to lOOOpM). DMSO was used as a solvent and included as the negative control. The next day, plates were washed twice with D2 media. Cells were infected at an moi of 0.1 or 0.01 with SARS-CoV-2 in 400pl.
  • the primary sequences of SARS-CoV-2 proteins were analyzed to investigate whether any of the proteins might incorporate Fe-S clusters.
  • the presence of Fe-S cofactors in candidate proteins can be predicted based on the identification of specific amino acid sequence motifs.
  • the analysis identified two highly conserved LYR (leucine-arginine- tyrosine)-like motifs (Fig. 6A) in the nspl2 subunit of RdRp, which are potential binding sites for the cochaperone HSC20 (aka HSCB) of the Fe-S biogenesis machinery, which facilitates Fe-S cluster transfer from the main scaffold protein, ISCU, to recipient proteins (Fig. 6B).
  • nspl2 coordinated a Fe-S cluster
  • 55Fe incorporation into the expressed protein was quantified in cells transfected with either a pool of nontargeting siRNAs (NT) or with si-RNAs against the initial Fe-S biogenesis scaffold, ISCU.
  • NT si-RNAs nontargeting siRNAs
  • nspl2 WT bound radiolabeled iron (8312 ⁇ 775 cpm/mg of cytosolic proteins, Figs. 1D-E)
  • nspl2 that lacked the LYR motifs did not interact with HSC20 and bound significantly less iron (250 ⁇ 92 cpm/mg of cytosolic proteins, Figs. 1D-E).
  • Nspl2 expressed in cells silenced for ISCU failed to incorporate iron (Figs. 1D-E).
  • This example describes the characterization of the function of the two Fe-S clusters in SARS-CoV2 RdRp.
  • Fe-S enzymes involved in DNA and RNA metabolism have often been mischaracterized as zinc-containing proteins, as Fe-S clusters readily undergo oxidative degradation during standard aerobic purification procedures of proteins, allowing zinc to coordinate the same cysteine residues.
  • zinc-containing enzymes have been shown to retain activity in vitro on short templates, which previously supported the conclusion that zinc was the physiological cofactor of these enzymes.
  • Fe-S clusters in nucleic acid metabolism enzymes have not been thought to participate directly in catalysis, but rather in modulating binding of the enzyme to the template and/or to other components of the replication complex, as well as in increasing processivity and enabling repair through a proposed charge transfer mechanism. Consistent with the notion that zinc is likely not the physiological cofactor in several viral replicases that have so far been crystallized with chelated zinc ions, supplementation with zinc has been reported to inhibit replication in several cell culture models of viral infection.
  • This example describes the use of a nitroxide radical anti-viral to prevent coronavirus replication.
  • TEMPOL treatment of cells did not impact the activities of several mitochondrial Fe-S enzymes, including the respiratory complexes and mitochondrial aconitase (AC02), and the cytosolic Fe-S enzyme, DP YD (Fig. 9A-F and 10A-F), nor it caused any cytotoxicity at doses up to 5 mM (Fig. 10G).
  • TEMPOL treatment also did not affect the interactions of nspl2 with the components of the Fe-S and CIA biogenesis machinery from which nspl2 acquires its Fe-S clusters (Figs. 11 A- D). Accordingly, it may be inferred that TEMPOL directly reacts with Fe-S clusters in RdRp, leading to their degradation.
  • DEA/NO a nitric oxide donor that readily reacts with Fe-S clusters to form dinitrosyl complexes with diminished absorbance, also inhibited the RdRp (Figs. 4E, 12A-B), although less effectively than TEMPOL.
  • TEMPOL was found to be both a more potent RdRp inhibitor (Fig. 13) and synergized with remdesivir (RDV) (Fig. 14), a nucleoside analog that has been used to target the replication of SARS-CoV-2. RDV was notably less effective against the Fe-S-RdRp than the zinc-RdRp (Fig. 15).
  • This example describes the antiviral activity of TEMPOL against live SARS- CoV2 viral replication.
  • This example demonstrates the activity of TEMPOL as an antiviral for SARS- CoV-2, SARS-CoV, and MERS-CoV.
  • SARS-CoV-2, SARS-CoV and MERS-CoV were measured using cytopathic effect (CPE) assays using Vero 76 cells.
  • SARS coronavirus (strain: Urbani) was tested in Vero 76 cells (ATCC CRL- 1587TM), with M128533 being used as a control drug at 0.1-100 pg/ml MERS coronavirus (strain: EMC) was tested in Vero 76 cells, with M128533 being used as a control drug at 0.1- 100 pg/ml.
  • SARS-CoV-2 (strain: USA_WAl/2020) was tested in Vero 76 cells, with EIDD- 1931 being used as a control drug at 0.1-100 pg/ml.
  • TEMPOL was dissolved in DMSO and tested in the concentration range of 25-1000 pM for SARS-CoV-2 and MERS-CoV and 0.1- 100 pM for SARS-CoV, with DMSO being used as a vehicle control.
  • Visual inspection and neutral red assay were used to measure cytopathic effect and toxicity.
  • EC50 and CC50 values were extracted from primary data.
  • EC50 refers to the compound concentration that reduces viral replication by 50%
  • CC50 refers to the compound concentration that reduces cell viability by 50%
  • SI50 is calculated as CC50/EC50. Compounds with SI values >10 are considered active and merit further investigation.
  • This example demonstrates synergy in the combination of TEMPOL with an iron chelator on CARS-CoV-2 replicase. Briefly, the activity of the anoxically purified RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 was assessed in a primer extension assay in the presence of TEMPOL, deferiprone (“DFP”), or a combination of TEMPOL and deferiprone.
  • RdRp anoxically purified RNA-dependent RNA polymerase
  • the primer/template duplex used in the RNA extension assay consisted of the 13-mer primer 5'-rArGrGrUrArArUrArArArUrU-3' (SEQ ID NO: 16 and the 29-mer template 5'- rUrUrUrUrArArUrCrCrUrArArArCrGrArArArUrUrUrUrArCrCrU-3 ' (SEQ ID NO: 17). (Note - these are the same sequences previously identified (see paragraph [0152]).
  • oligonucleotides were mixed at equal molar ratios in annealing buffer (50 mM NaCl and 10 mM Na-HEPES pH 7.5), denatured by heating to 75°C for 5 min and then slowly cooled to 4°C.
  • annealing buffer 50 mM NaCl and 10 mM Na-HEPES pH 7.5
  • the SARS-CoV-2 RdRp complex anoxically purified from Expi293F mammalian cells co-expressing 3xFLAG-nspl2 and nsp7/nsp8-Strep II at a final concentration of 1 mM was incubated with 3 mM dsRNA in the presence of 1.2 U/pl RNase inhibitor in reaction buffer containing in 100 mM NaCl, 20 mM Na-HEPES pH 7.5, 5% (v/v) glycerol, 10 mM MgCF and 0.5 mM TCEP, which were prepared in DEPC-treated water. Reactions were incubated at 37 °C for 15 min and the RNA extension was initiated by addition of NTPs (300 pM UTP, GTP and CTP, and 100 pM [y- 32 P]ATP (Perkin Elmer)).
  • the total reaction volume was 10 pi.
  • the reaction mix containing all components except the RdRp was also loaded on the gel (no RdRp lane) to show migration pattern of the labeled primer alone, along with the control mix (CTRL) in which DMSO, which was used to dissolve TEMPOL, was added.
  • CTRL control mix
  • DMSO which was used to dissolve TEMPOL

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Abstract

The invention comprises methods and compositions for treating or preventing coronavirus infections in subjects using nitroxide radicals as antiviral agents. In certain embodiments, the inventive methods and compositions further comprise the use of an iron chelator.

Description

NITROXIDE RADICALS FOR USE AS ANTIVIRAL TREATMENT FOR CORONAVIRUS INFECTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 60/193,656, filed May 27, 2021, which is incorporated by reference.
STATEMENT REGARDING
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Project Numbers ZIAHD008814 by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and ZIA BC011038 by the National Cancer Institute. The Government has certain rights in this invention.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED
ELECTRONICALLY
[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 21,563 Byte ASCII (Text) file named " 763144_ST25.TXT," created on May 27, 2022.
BACKGROUND OF THE INVENTION
[0004] Coronaviruses are a group of RNA viruses known to cause disease in mammals and birds. The novel coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS- CoV-2), has caused a global pandemic known as coronavirus disease (COVID-19). Other disease caused by coronaviruses include, e.g., SARS, MERS, and the common cold.
Vaccines are available for some coronaviruses (e.g., SARS-CoV2); however, a need remains for antiviral treatments for patients that are infected with coronaviruses, at risk for infection with coronaviruses, and/or patients that have been exposed to coronaviruses. BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides a method of treating a subject, the method comprising administering to the subject an effective amount of an anti -viral agent, wherein the anti -viral agent comprises a nitroxide radical, and wherein the antiviral agent treats or prevents infection by a coronavirus in the subject. In certain embodiments, the inventive method further comprises the administration of an iron chelator to the subject.
[0006] The invention also provides a composition for use in treating a subject, the composition comprising an effective amount of an anti-viral agent and a pharmaceutically acceptable carrier, wherein the anti-viral agent comprises a nitroxide radical, and wherein the composition treats or prevents infection by a coronavirus when administered to the subject.
In certain embodiments, the inventive composition further comprises an iron chelator.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0007] Figure 1 A-E collectively show that Fe-S cluster incorporation into nspl2 occurs through its interactions with components of the Fe-S biogenesis machinery.
[0008] Figure 1 A depicts representative Coomassie blue staining of pull-down assays performed with purified proteins. 0.25 pg of purified nspl2-FLAG or the variants wherein either or both LYR motifs were replaced by alanines (VYR-AAA, LYR-AAA and VYR/LYR-AAA, respectively) were combined with 0.25 pg of HSC20, as indicated. Immunoprecipitations (IPs) were performed with anti-FLAG antibody to immunocapture nspl2 proteins. The presence of HSC20 (aka HSCB) in the eluates after IPs of nspl2 proteins was analyzed by SDS-PAGE and Coomassie staining. Aliquots corresponding to 20% of the inputs were run on the gel for comparison (n= 5).
[0009] Figure IB depicts Eluates after IPs of nspl2 WT or variants recombinantly expressed in Vero E6 cells, as indicated, were probed with antibodies against FLAG to verify the efficiency of IP, and to components of the Fe-S cluster (HSC20, HSPA9, NFSl) and of the cytoplasmic Fe-S (CIA) assembly machinery (CIAOl, MMS19, FAM96B) (n= 6).
[0010] Figure 1C depicts mass spectrometry identification of affinity purified interacting partners of nspl2 that are components of the Fe-S cluster biogenesis pathway (see Table SI for a complete list). The protein ratios were calculated as reported in the methods (n= 6). The maximum allowed fold change value was set to 100. In the instances in which the interacting partner was detected in the ‘nspl2’ only samples and not in the negative controls, the nspl2/control ratios were set to 100 and reported without p-values.
[0011] Figure ID depicts levels of radiolabeled iron (55Fe) incorporated into nspl2 WT or the variants in control cells transfected with non-targeting siRNAs (NT-siRNAs) and in cells transfected with siRNAs directed against the main scaffold protein ISCU (si-ISCU). Levels of iron stochastically associated with the beads in lysates from cells transfected with the backbone plasmid (empty-vector, p3XFLAG-CMV-14) are also reported (accounting for 587 ± 292.62 cpm/mg of cytosolic proteins) and were not subtracted from measurements of radiolabeled iron incorporated into nspl2 WT or variants in the chart (n= 4). Significance was determined by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Mean ± 95% confidence interval (Cl). ***P<0.001. In the figure, for each bracketed set of bars, the order is, from left to right: WT, VYR-AAA, LYR-AAA, VYR.LYR-AAA, and the unbracketed bar on the right represents an empty vector control.
[0012] Figure IE depicts representative Coomassie staining showing levels of nspl2 WT or variants in control and ISCU-depleted cells that were quantified in (ID) for their iron content. Immunoblots to ISCU, showing the efficiency of its silencing (knock-down), and to a-tubulin, used as a loading control, are also presented.
[0013] Figure 2A-F collectively show evidence for ligation of two Fe-S metal cofactors by nspl2.
[0014] Figure 2A depicts UV-vis spectra of nspl2 WT or variants of the cysteine residues in the two metal ligating centers. In the figure, the order of traces/lines is, from top to bottom, WT, C301S-C306S-C310S, C847S-C645S-C646S, and C301/306/645/646-S.
[0015] Figure 2B depicts representative Coomassie blue staining of purified nspl2 WT or variants analyzed in Figure 2A.
[0016] Figure 2C depicts Mossbauer spectra of nspl2 WT and variants exhibited the parameters typical of [Fe4S4] clusters. For each of the two nspl2 cys-to-ser variants, approximately 95% of iron was still associated with a quadrupole doublet that matched parameters of WT nspl2.
[0017] Figure 2D depicts RNA polymerase activity of anoxically purified RdRp ([Fe-S]- RdRp at 1 mM) and aerobically purified RdRp reconstituted with zinc and containing 2 zinc ions/protomer (Zn-RdRp at 1 mM) (n= 4).
[0018] Figure 2E depicts conserved zinc-binding motifs in SARS-CoV-2 nspl2 (PDB ID: 7BTF) rendered in the ribbon representation. H295-C301-C306-C310 ligate zinc at the interface between the NiRAN and the catalytic domain, whereas the C487-H642-C645-C646 residues ligate zinc in the catalytic domain.
[0019] Figure 2F depicts levels of radiolabeled iron (55Fe) incorporated into nspl2 WT or variants, as indicated. 55Fe content of nspl2 treated with the chelator EDTA is also reported to provide a control for the complete loss of 55Fe in the protein (n= 4). Significance was determined by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Mean ± 95% CF ***/><o.001. In the figure the order of the bars is, from left to right: WT, LYR-AAA, C301S-C306S-C310S, C847S-C645S-C646S, C301-306-645-646-S, and EDTA.
[0020] Figure 3A-C collectively show that Fe-S cluster sites in nspl2 are important for activity and interactions with nspl3.
[0021] Figure 3 A depicts RNA polymerase activity of anoxically purified RdRp (all lanes except Zn-nspl2) (RdRp at ImM) and of aerobically purified and Zn-reconstituted RdRp containing 2 zinc ions/ protomer (3 technical replicates are shown, n= 4).
[0022] Figure 3B depicts schematic of the complex required for coronaviral replication in which the two Fe-S clusters and their coordination spheres are highlighted.
[0023] Figure 3C depicts co-IP of nspl2 WT or variants recombinantly expressed in Vero E6 cells co-transfected with helicase nspl3 and accessory factors nsp7 and nsp8 (Strep II tagged) probed with antibodies against FLAG, Strep II or nspl3 (3 technical replicates are shown, n= 4).
[0024] Figure 4A-F collectively show that the stable nitroxide TEMPOL potently inhibits the RdRp by causing disassembly of its Fe-S clusters and blocked viral replication in cell culture models of SARS-CoV-2 infection.
[0025] Figure 4A depicts UV-vis spectra of nspl2 anoxically purified from Expi293F control cells and from cells treated with TEMPOL. In the figure, the order of lines (traces) is, from top to bottom: nspl2, npl2 + Tempol 0.2 mM, and nspl2 + Tempol 0.5 mM.
[0026] Figure 4B depicts UV-vis spectra of as purified nspl2 and of purified nspl2 incubated with TEMPOL (1: 2 ratio nspl2: TEMPOL) for 10 min.
[0027] Figure 4C depicts RNA polymerase activity of the RdRp complexes anoxically purified from control and TEMPOL-treated Expi293F cells.
[0028] Figure 4D depicts representative Coomassie staining of the RdRp complexes analyzed for the activity in Fig. 4C. [0029] Figure 4E depicts RNA polymerase assay of the RdRp complexes (at 1 mM) anoxically purified from control, DEA/NO- or TEMPOL-treated Vero E6 cells, as indicated (n= 4).
[0030] Figure 4F depicts titer of infectious virus produced at 48 hours measured by TCID50 assay in Vero E6 cells infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.1 or 0.01 (n= 3).
[0031] Figure 5 provides multiple sequence alignments of the residues ligating zinc in the crystal structure of the RdRp of SARS-CoV-2. Multiple sequence alignments of the catalytic subunit of the RdRp, nspl2, from SARS-CoV-2 and other coronaviruses, as indicated, showing conservation of the residues ligating 2 zinc ions in the crystal structure. The conserved zinc-binding motifs in SARS-CoV (PDB ID: 6NUR) and SARS-CoV-2 (PDB ID: 7BTF) nspl2 are rendered in the ribbon representation. The coordinate details of the zinc binding residues are shown in stick representation. In the figure, the SEQ ID NOs are, from top to bottom, for the upper grouping SEQ ID Nos: 27-40 and for the lower grouping, SEQ ID Nos: 41-47.
[0032] Figure 6A-D collectively show that Fe-S cluster incorporation into nspl2 requires its interactions at LYR motifs with the Fe-S cluster biogenesis machinery.
[0033] Figure 6A depicts multiple sequence alignments of nspl2 from SARS-CoV-2 and other coronaviruses showing conservation of the VYR and LYR motifs that are present in the NiRAN and in the catalytic domains of the polymerase, respectively (UniProt entries are indicated). In the figure, the SEQ ID Nos are, from top to bottom, SEQ ID Nos: 48-62.
[0034] Figure 6B depicts proposed model of the chaperone/cochaperone-mediated transfer of nascent Fe-S clusters, assembled on the main scaffold protein, ISCU, through the direct binding of the cochaperone HSC20 to LYR motifs present in recipient apoproteins.
ATP hydrolysis by the HSC20-cognate chaperone, HSPA9, is proposed to facilitate cluster transfer to recipient proteins, while concomitantly driving folding of the recipient protein into its final conformation.
[0035] Figure 6C depicts Coomassie blue staining of 3xFLAG-nspl2 proteins recombinantly expressed and anoxically purified from Expi293F mammalian cells at different time points after transfection, as indicated. Optimal expression was achieved 48h post transfection.
[0036] Figure 6D depicts eluates after IPs of nspl2 wild type or variants, as indicated, recombinantly expressed in VeroE6 cells were probed with antibodies against FLAG to verify the efficiency of IP, and to components of the Fe-S cluster (HSC20, HSPA9, NFS1) and of the cytoplasmic Fe-S (CIA) assembly machinery (CIAOl, MMS19, FAM96B) (n= 4). [0037] Figure 7A-D collectively show that ligation of two Fe-S metal cofactors by nspl2 in sites occupied by zinc in the structure of the protein purified aerobically.
[0038] Figure 7A depicts UV-vis spectra of nspl2 wild type or variants, as indicated. The spectrum of nspl2 treated with the chelator EDTA was included to show complete loss of absorbance at 420nm, which was similar to the loss of absorbance of the variant in which cysteines in both metal-ligating centers were replaced by serines (nsp 12C’I) I S_C’I)6S_C645S_C646S) The order of lines (traces) in the figure are reflected (top to bottom) in the legend of the figure.
[0039] Figure 7B depicts Coomassie blue staining of nspl2 proteins analyzed in Fig. 7A. [0040] Figure 7C depicts X-bnd EPR spectra, recorded at 20 K, of as purified nspl2 wild type and Cys-to-Ser variants of the two metal-ligating sites.
[0041] Figure 7D depicts X-band EPR spectra, recorded at 20 K, of nspl2 WT and variants exhibited the parameters typical of [Fe4S4]+ clusters upon reduction with sodium dithionite. The parameters obtained by simulating these spectra are as follows: nspl2 WT - g = (2.07, 1.92, 1.90); nspl2C301-C306-C310/s - g = (2.08, 1.92, 1.90); nspl2C487-C645-C646/s - g = (2.08, 1.92, 1.90).
[0042] Figure 8A-E collectively show that markedly increased RNA-binding and polymerase activities of the RdRp complex containing the Fe-S clusters compared to the zinc-RdRp.
[0043] Figure 8A depicts polymerase activity of anoxically purified RdRp ([FeS] at ImM) and aerobically purified RdRp reconstituted with zinc and containing two Zn ions/ protomer (Zn enzyme at ImM) upon addition of radiolabeled ATP only ([a-32P]ATP) or a mix of [a- 32P]ATP and remaining nucleoside triphosphates (UTP, CTP and GTP) (n= 4). The assay performed upon addition of only [a-32P]ATP enabled extension of the primer of a single nucleotide, thereby providing a way to assess template binding affinities of the [FeS] versus the Zn-containing RdRp complex.
[0044] Figure 8B depicts quantification of n= 5 independent RdRp assays equivalent to the one presented in (A). Significance was determined by two-tailed unpaired t-test. Mean ± 95% confidence interval (Cl). ***/><o.001.
[0045] Figure 8C depicts representative Coomassie blue staining of the anoxically purified ([FeS]) and aerobically, zinc-reconstituted (Zn) RdRp complexes. [0046] Figure 8D depicts UV-vis spectra of the [FeS]-nspl2 alone and in complex with the accessory factors nsp7 and nsp8, required for the activity, showing that the presence of nsp8 and nsp7 did not perturb the [Fe4S4] cluster typical absorbance at 420nm. As expected, the Zn-containing RdRp complex lacked the absorbance at 420nm.
[0047] Figure 8E depicts gel mobility Vshift assay to detect RdRp binding to RNA. Either the Fe-S- or the zinc-RdRp complex (9pg) was incubated with increasing amounts of template/primer RNA (0, 0.3, 0.6, 1.2 and 2 pg). For the reaction with the Fe-S- or zinc- nspl2 protein alone, 9 pg of purified nspl2 were combined with 1.5 pg template-primer RNA. Strikingly, the Fe-S-nspl2 bound the template/primer complex even in the absence of nsp7/nsp8, although less efficiently than the full RdRp complex (nspl2/nsp7/nsp8). (n= 4). [0048] Figure 9A-F collectively show that the doses of TEMPOL that disabled the RdRp in HEK293 cells did not impair mitochondrial function or diminish activity of the cytosolic Fe-S enzyme DP YD.
[0049] Figure 9A presents, from top to bottom, immunoblots to FLAG, cytosolic IRPl and DPYD on lysates from control (CTRL) HEK293 cells transfected with the backbone vector (empty) or with 3xFLAG-nspl2 and from cells transfected with 3xFLAG-nspl2 and treated with the indicated doses of TEMPOL. In-gel activity assays of cytosolic (ACOl) and mitochondrial (AC02) aconitases showed loss of cytosolic, but not mitochondrial, aconitase activity, upon TEMPOL treatment. In gel, respiratory complexes I and II activity assays were unaffected by TEMPOL treatment. TOM20 was used as a loading control for the mitochondrial fractions.
[0050] Figure 9B depicts schematic representation of respiratory complexes I-IV showing the Fe-S clusters and heme centers required for their activities.
[0051] Figure 9C depicts oxygen consumption rates (OCRs) of cells treated as in Figure 9A.
[0052] Figure 9D depicts schematic representation of the reaction catalyzed by the cytosolic Fe-S enzyme DPYD. DPYD converts [4-14C]-thymine to [4-14C]-dihydrothymine. [0053] Figure 9E depicts ribbon representation of the crystal structure of DPYD, which assembles into a dimer, containing a total of 8 [Fe4S4] clusters.
[0054] Figure 9F depicts DPYD-mediated conversion of [4-14C]-thymine to [4-14C]- dihydrothymine in control and TEMPOL-treated HEPG2 or Expi293F cells, as indicated, assayed by thin layer chromatography (TLC) and autoradiography. The reaction mix containing the substrate of the reaction [4-14C]-T without cell extract was loaded as a negative control (no extract) to visualize the substrate (4-14C-thymine) by TLC.
[0055] Figure 10A-G collectively show that the doses of Tempol that disabled the RdRp in VeroE6 cells did not adversely affect mitochondrial function or impair activity of the cytosolic Fe-S enzyme DPYD.
[0056] Figure 10A provides, from top to bottom, immunoblots to FLAG, IRPl and TOM20 in cells transfected with 3xFLAG-nspl2 and treated with TEMPOL, as indicated. In gel activity assays of respiratory complexes I, II and IV showing intact mitochondrial respiration in cells treated with TEMPOL.
[0057] Figure 10B depicts OCR measurements confirming that TEMPOL treatment did not affect the activities of the mitochondrial respiratory chain complexes.
[0058] Figure IOC depicts immunoblots to the Fe-S subunit of complex I, NDUFS1, the core subunit of complex III (UQCRC1), the heme subunit of complex IV (MTCOl), and the Fe-S subunit of complex II, SDHB, showed that TEMPOL treatments did not affect protein levels of the indicated subunits of the respiratory complexes. TOM20 was used as a loading control.
[0059] Figure 10D depicts in-gel activity assay of cytosolic (ACOl) and mitochondrial (AC02) aconitases showed loss of cytosolic, but not mitochondrial, aconitase activity, upon TEMPOL treatment.
[0060] Figure 10E depicts levels of cytosolic IRPl, IRP2, transferrin receptor (TFR1), ferritin heavy chain (FTH), the CIA component, CIAOl, and the Fe-S enzyme DPYD in control and TEMPOL treated cells.
[0061] Figure 10F depicts DPYD-mediated conversion of [4-14C]-thymine to [4-14C]- dihydrothymine in control or TEMPOL treated VeroE6 cells for the indicated time points, assayed by thin layer chromatography (TLC) and autoradiography. The reaction mix containing the substrate of the reaction [4-14C]-T without cell extract was loaded as a negative control (no extract) to visualize the substrate (4-14C-thymine) by TLC (n= 3).
[0062] Figure 10G depicts viability of Vero E6 cells treated with increasing concentrations of TEMPOL, as indicated. As a control, viability of cells treated with DMSO alone, used to dissolve TEMPOL, are also reported (n= 4).
[0063] Figure 11 A-D collectively show that binding of nspl2 to the components of the Fe-S cluster biogenesis machinery was not affected by TEMPOL treatment. [0064] Figure 11 A depicts eluates after IPs of nspl2 wild type or variants, as indicated, recombinantly expressed in VeroE6 cells were probed with antibodies against FLAG to verify the efficiency of IP, and to components of the Fe-S cluster (HSC20, HSPA9, NFS1) and of the cytoplasmic Fe-S (CIA) assembly machinery (CIAOl, MMS19, FAM96B) (n= 6) (TEMPOL at 0.5mM and DFO at IOOmM). WT C; cells expressing wild type nspl2. WT DFO; cells expressing wild type nspl2 treated with IOOmM of the iron chelator DFO. WT_T; cells expressing wild type nspl2 treated with 0.5mM Tempol. VYR/AAA and C301- 306-310/S correspond to lysates from cells transfected with nsp l 2VYR/AAA or nspl2C301s C306S C310S variants that were either untreated (C) or treated with DFO (_DFO) or TEMPOL (_T), as indicated. (n= 4).
[0065] Figure 1 IB depicts representative Ponceau S staining of blotting membrane used in Fig. 11 A.
[0066] Figure 11C depicts eluates after IPs of nspl2 wild type or variants, as indicated, recombinantly expressed in VeroE6 cells were probed with antibodies against FLAG to verify the efficiency of the IPs, and to components of the Fe-S cluster (HSC20, HSPA9, NFS1) and of the cytoplasmic Fe-S (CIA) assembly machinery (CIAOl, MMS19, FAM96B) (n= 6) (TEMPOL at 0.5mM and DFO 100mM). WT C; cells expressing WT nspl2.
WT DFO; cells expressing WT nspl2 treated with 100mM of the iron chelator DFO. WT_T; cells expressing WT nspl2 treated with 0.5mM Tempol. LYR/AAA and C487-645-646/S correspond to lysates from cells transfected with nspl2LYR/AAA or nspl2C487S C645S C646S variant and either untreated (_C) or treated with DFO (_DFO) or TEMPOL (_T). (n= 4). [0067] Figure 1 ID depicts representative Ponceau S staining of blotting membrane used in (C) for western.
[0068] Figure 12A-B collectively show that the nitric oxide donor diethylamine nonoate (DEA/NO) at ImM inhibited the activity of the Fe-S-RdRp by 60%.
[0069] Figure 12A depicts representative polymerase activity assay of the RdRp complexes anoxically purified from control and DEA/NO-treated cells at the different doses indicated.
[0070] Figure 12B depicts quantification of n= 5 independent RdRp activity assays equivalent to the one presented in Fig. 1 A. Significance was determined by two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Mean ± 95% confidence interval (Cl). ***/><o.001. [0071] Figure 13 shows RNA polymerase activity of the RdRp complex. Activity of the RdRp complex anoxically purified from Expi293F cells and treated with the indicated doses of RDV-TP and TEMPOL. The RdRp concentration was ImM (n= 3).
[0072] Figure 14 depicts the Synergistic effect of a combination of Tempol and RDV-TP in inhibiting the activity of the SARS-CoV-2 RdRp complex. Activity of the RdRp complex anoxically purified from Expi293F cells and treated with the indicated doses of DEA/NO, RDV-TP, TEMPOL or a combination of RDV-TP and TEMPOL in vitro. The RdRp was at ImM (n=3).
[0073] Figure 15 is a comparison of remdesivir efficacy on the FeS RdRp vs. the Zn- containing RdRp complex in vitro. RNA polymerase activity of the RdRp complex anoxically purified from Expi293F cells and treated with the indicated doses of DEA/NO, RDV-TP and TEMPOL. The activity of the Fe-S-containing enzyme was compared to the activity of the RdRp purified aerobically and reconstituted with zinc. The RdRp complexes containing the Fe-S clusters or Zn were at ImM (n= 3).
[0074] Figure 16 depicts the results of gel electrophoresis concerning the in vitro primer extension assay discussed in Example 7. Briefly, the activity of the anoxically purified RNA- dependent RNA polymerase (RdRp) of SARS-CoV-2 was assessed in a primer extension assay in the presence of TEMPOL (TL), deferiprone (DFP), or a combination of TEMPOL and DFP, which were then loaded onto an acrylamide gel as indicated (n= 4 biological replicates). The reaction mix containing all components except the RdRp was also loaded on the gel (no RdRp lane) to show migration pattern of the labeled primer alone, along with the control mix (CTRL) in which DMSO, which was used to dissolve TEMPOL, was added. [0075] Figure 17 presents a plot representing the quantification of the RdRp assay intensities of the bands from the gel represented by Figure 16. In brief, band intensities of the products of the primer extension assay (Elongated RNA) in control (CTRL), TEMPOL-, DFP-, and TEMPOL/DFP- treated samples were assessed by IMAGE J and the results were plotted using PRISM 9. Multiple comparisons were performed using One-way Anova Sidak's multiple comparisons test. P values were as follows: CTRL vs TEMPOL 100 mM, pO.OOOl; CTRL vs DFP 25 mM, p=0.0104; CTRL vs TEMPOL+DFP, p<0.0001; TEMPOL vs TEMPOL+DFP, p<0.001. In the figure, the circle indications at the group in the bottom right represent data for Tempol 100 mM+DFP 25 mM, whereas the group of circles in the center-right above the line demarcated by *** represent data for DFP 25 mM alone. The other data are identified in the legend within the figure. DETAILED DESCRIPTION OF THE INVENTION
[0076] Aspects of the invention include the treatment or prevention of infection by a coronavirus in a subject, for example a human subject. Coronaviruses are a group of RNA viruses known to cause disease in mammals and birds. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirale s, and realm Riboviria. Exemplary coronaviruses include, e.g., SARS-CoV2, SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63. Diseases caused by coronavirus infection include, e.g., SARS, MERS, COVID-19 and the common cold.
[0077] In an aspect of the invention, the coronavirus is SARS-CoV2, which causes COVID-19. In other aspects of the invention, the coronavirus is other than a SARS-CoV2 coronavirus, for example SARS-CoV, or MERS-CoV. The coronavirus may be human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63, which are known to cause the common cold.
[0078] Coronaviruses employ a multi-subunit machinery for replication and transcription. A set of nonstructural proteins (nsps) produced as cleavage products of the ORFla and ORFlab polyproteins assembles to facilitate viral replication and transcription. The core component of this complex is the catalytic subunit (nspl2) of an RNA-dependent RNA polymerase (RdRp), which catalyzes the synthesis of viral RNA and thus plays a central role in the replication and transcription cycle of SARS-CoV-2, with the assistance of nsp7 and nsp8 as accessory factors. Structures of the RdRp (nspl2-nsp7-nsp8 complex) alone and in complex with the helicase have been determined by cryo-electron microscopy; in all of them the RdRp of SARS-CoV-2 was proposed to contain zinc ions ligated in the same locations as those observed in SARS-CoV in highly conserved metal binding motifs composed of H295- C301-C306-C310 and C487-H642-C645-C646 (see Fig. 5).
[0079] However, the inventors have surprisingly discovered that the catalytic subunit of the RdRp, nspl2, ligates two iron-sulfur metal cofactors in the sites that were previously thought to contain zinc ions. These metal binding sites are essential for replication and for interaction with the viral helicase. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.
[0080] Without wishing to be bound by a particular theory or mechanism of action, it is proposed that oxidation of the iron sulfur (Fe-S) metal cofactors by an anti-viral agent comprising a nitroxide radical, (for example, TEMPOL) causes disassembly of the clusters, potently inhibiting the RdRp, and preventing or reducing SARS-CoV-2 replication. Further, it is proposed that the highly conserved metal binding motifs found in RdRp in other coronaviruses also bind iron sulfur (Fe-S) metal cofactors. Accordingly, an anti-viral agent comprising a nitroxide radical can be used to inhibit RdRp, and, thus, can be used to treat coronaviruses that rely on RdRp, including coronaviruses other than SARS-CoV2. In aspects of the invention wherein the coronavirus is not SARS-CoV2, the coronavirus may be a coronavirus having a metal binding motif in the nspl2 subunit of RdRp. For example, coronaviruses having the highly conserved metal binding motif include SARS-CoV, MERS- CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63 as illustrated in sequence alignments presented in Fig. 5.
[0081] In one aspect, the present invention relates to antiviral agents. Antiviral activity refers to the ability of a compound or composition to prevent, inhibit, or lessen replication of a virus. In one aspect, the antiviral agent prevents, inhibits or lessens the replication of a coronavirus in a host, for example a human subject. Without wishing to be bound by a particular theory or mechanism of action, in aspects of the present invention, the anti-viral agents according to the present invention may block viral replication by oxidizing iron sulfur (Fe-S) metal cofactors ligated by the catalytic sub-unit of RNA-dependent RNA polymerase (RdRp), thereby inhibiting the RdRp and preventing, inhibiting and or lessening viral replication.
[0082] Any nitroxide radical may be used. Nitroxide radicals include compounds having the general formula R2NO, wherein the R groups may be the same or different. In aspects of the invention, the nitroxide radical is a cyclic nitroxide. In aspects of the present invention, the antiviral agent may comprise, consist of, or consist essentially of a nitroxide radical. Various nitroxide radicals are known in the art. Nitroxide radicals include, for example, piperidine nitroxide derivatives such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol); 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l- piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo- Tempo). Nitroxide radical compositions can also include other substituted variants of Tempo (typically in the 4 position) such as, for example, 4-(2-bromoacetamido)-2, 2,6,6- tetramethylpiperidine- 1 -oxyl; 4-ethoxyfluorophosphonyloxy-2,2,6,6-tetramethylpiperidine- 1 - oxyl; 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl, 4-isothiocyanato-2, 2,6,6- tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(4- nitrobenzoyloxyl)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-phosphonooxy-2,2,6,6- tetramethylpiperidine-l-oxyl; and the like. The nitroxide radical also includes other compounds known in the art, such as those disclosed in US20100179188A1 or US20120295937A1, the disclosures of which are hereby incorporated by reference.
[0083] In certain aspects of the invention, the nitroxide radical is 4-hydroxy-2, 2,6,6- tetramethylpiperidine- 1 -oxyl (Tempol).
[0084] Other suitable nitroxide radical compounds include 2-ethyl-2,5,5-trimethyl-3- oxazolidine-l-oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3- aminomethyl-Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-cyano-Proxyl); 3-Carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3 -Carbarn oyl-Proxyl); and 3- Carboxy-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy-Proxyl).
[0085] In certain aspects of the invention, (a) when the coronavirus is SARS-CoV2, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) or (b) when the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol), the coronavirus is other than SARS-CoV2.
[0086] In certain aspects of the invention, the activity of the nitroxide radical in treating or prophylaxis of a coronavirus infection can be enhanced by an iron chelator. Indeed, Example 7 herein demonstrates that a synergistic effect is realized when the nitroxide radical, tempol, is employed in conjunction with the iron chelator, deferiprone. However, any suitable iron chelator can be used, and the iron chelator can be administered with the nitroxide radical either within the same formulation (composition) or in a separate formulation, which can be administered prior to, concurrently with, or subsequent to the nitroxide radical.
[0087] An exemplary iron chelator contemplated for use in the present invention comprises deferiprone (DFP), which is currently approved by the United States Food and Drug Administration as a solution comprising either 80 or 100 mg/ml deferiprone or as a tablet comprising either 1 g or 500 mg deferiprone as an active agent, both formulations for oral administration. In the United States of America, deferiprone is marketed for pharmaceutical use under the tradename FERRIPROX®.
[0088] Another exemplary iron chelator contemplated for use in the present invention comprises deferasirox, which is currently approved by the United States Food and Drug Administration as orally-administrable granules (approved dosages being 90 mg, 180 mg, or 360 mg) or tablets (approved dosages being 90 mg, 180 mg, or 360 mg). In the United States of America, a granule formulation of deferasirox is marketed for pharmaceutical use under the tradenames JADENU® and JADENU® SPRINKLE.
[0089] Another exemplary iron chelator contemplated for use in the present invention comprises desferrioxamine, which is administered intravenously. Another orally active investigational iron chelator contemplated for use in the present invention comprises CN128 (see Chen et al., “CN128: A New Orally Active Hydroxypyridinone Iron Chelator,” ./. Med. Chem ., 63, 4215-4226 (2020), which is incorporated herein in its entirety).
[0090] In embodiments in which an iron chelator is employed, preferably one is selected that is orally active. Furthermore, a dosage preferably is selected to minimize the risks of side effects associated with the use of such agents. Commonly, iron chelators are administered to treat patients with iron overload. For use in the present invention, however, lower dosages than those approved to treat iron overload are preferred. Thus, for example, a suitable dosage of the iron chelator, DFP, contemplated for antiviral use in conjunction with Tempol, in accordance with the present invention, can be lower than the approximately 75 mg/kg dosage currently employed for DFP when it is employed to treat patients with iron overload. Exemplary dosages of DFP for use in the context of the present invention, thus, include less than or equal to 70 mg/kg, such as less than or equal to 60 mg/kg, such as less than or equal to 50 mg/kg, such as less than or equal to 40 mg/kg, such as less than or equal to 30 mg/kg, such as less than or equal to 20 mg/kg, and even lower dosages may be appropriate. Ultimately, it is within the skill of the treating physician to select an appropriate dosage of iron chelator to be used in conjunction with the nitroxide radical in accordance with the present invention.
[0091] The term “subject,” as used herein refers to an animal, e.g., preferably, a mammal and, more preferably, a human.
[0092] The invention provides a method for treating a subject, e.g., a human, that has, is suspected to have, or is at risk for contracting a viral infection. In aspects of the invention, the subject has been exposed to a viral infection. In aspects of the invention, the viral infection is due to a coronavirus, for example SARS-CoV2.
[0093] The terms “preventing” and “treating,” “treatment,” and the like are used herein to refer to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom, or condition associated with viral infection and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect associated with viral infection. The terms “preventing” and “treating,” as used herein, covers any prevention or treatment of a disease in a mammal, such as a human, and includes: (a) preventing the disease from occurring in a subject which may be at risk for contracting the disease but has not yet been diagnosed as having it, i.e., causing the clinical symptoms of the disease not to develop in a subject that may be at risk for contracting the disease but does not yet experience or display symptoms of the disease; (b) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and (c) relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions.
[0094] The terms “treat” and “prevent,” and the like, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential prophylactic benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention in a subject. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the diseases described herein being treated or prevented. Also, for purposes herein, “prevention” can encompass delaying the onset of the disease, or a symptom or condition thereof.
[0095] An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response, e.g., treating the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.
[0096] A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. [0097] Aspects of the invention include the administration of the antiviral agent prophylactically. “Prophylactically” means that the antiviral agent is administered to prevent a condition or to prevent one or more symptoms associated with a condition. A “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
[0098] A subject may be considered to have been exposed to coronavirus infection if the subject has recently (e.g. within a week or two weeks) been in contact with individuals known or suspected to have a coronavirus infection. Such contact will be of sufficient duration and under conditions sufficient to allow the coronavirus to spread from one subject to another. Exposure may be determined via techniques known in the epidemiology field, for example contact tracing. For example, a human subject may be considered to have been exposed to coronavirus infection if one or more members of the subject’s family or members of the subject’s household have recently been diagnosed with a coronavirus infection. The human subject may also have been exposed to coronavirus at a place of employment. For example, the human subject may be considered to have been exposed to coronavirus infection if the subject is a health care provider who recently treated or examined an individual or individuals known to have a coronavirus infection. In aspects of the invention, the exposed subject has not been tested for coronavirus infection. In some aspects of the invention, the antiviral agent is administered to a subject that has been exposed to a coronavirus infection. [0099] A subject may be considered at risk for coronavirus infection when the individual is likely to have come into contact with one or more individuals having a coronavirus infection. Risk may be assessed via techniques known in the epidemiology field, e.g. statistical methods and/or models of coronavirus infection. For example, the at-risk subject may live and/or work in an area in which a coronavirus infection is prevalent. A human subject may be considered at risk for coronavirus infection if one or more members of the subject’s family or members of the subject’s household have recently exhibited symptoms consistent with a coronavirus infection. As an additional example, the at-risk subject may be a health care provider that frequently examines or treats patients that may have a coronavirus infection. An at-risk subject may be a health care provider who recently treated or examined an individual or individuals presenting with symptoms consistent with a coronavirus infection. As an additional example, a human subject may be considered at risk if the subject regularly comes into contact with numerous individuals in a region where coronavirus infections are known to be present. In some aspects of the invention, the antiviral agent is administered prophylactically to a subject that is at risk for coronavirus infection.
[0100] Various methods for testing subjects for coronavirus infection will be known to those of skill in the art. In general tests will detect either the presence of viral genetic material in a subject, the presence of viral protein in a subject, or the presence of antibodies to the virus in the subject. Test types include, for example, reverse transcriptase polymerase chain reaction tests, antigen tests, and antibody tests. Techniques, material and procedures used to perform these tests are known in the art. In aspects of the invention, the subject has tested positive for a coronavirus infection. In other aspects of the invention, the subject has not tested positive for a coronavirus infection.
[0101] Coronavirus infection may cause various symptoms in a subject. Symptoms may range from mild to critical. For example, mild symptoms may include symptoms generally associated with respiratory infection, and include nasal congestion, runny nose, cough, muscle pain, sore throat, headache, fever, breathing difficulties, mild pneumonia, loss of smell, and loss of taste. Severe symptoms of coronavirus infection may include, for example, dyspnea and hypoxia. Critical symptoms may include, for example, respiratory failure (e.g., acute respiratory distress syndrome (ARDS)), shock and organ failure.
[0102] In aspects of the present invention, the subject to be treated may have mild or severe symptoms, but not critical symptoms. In other aspects of the present invention, the subject to be treated may have mild symptoms, but no severe or critical symptoms. For example, the subject may not be suffering from ARDS at the time of treatment.
[0103] In still other aspects of the invention, the subject, e.g. the human subject, may be asymptomatic. According to the present invention, a subject is considered asymptomatic if the subject does not exhibit clinical symptoms of an infection sufficient to lead one skilled in the art to conclude that an infection may be present, although infection might be confirmed by serological examination or other biological test for infection. In aspects of the invention, the subject may present with no symptoms associated with respiratory infection. In aspects of the invention, a subject may present without nasal congestion, runny nose, cough, muscle pain, sore throat, headache, fever, breathing difficulties, mild pneumonia, loss of smell, or loss of taste. In aspects of the invention wherein the subject is asymptomatic, the asymptomatic subject may have been exposed to coronavirus or may be at risk for coronavirus infection. In aspects of the invention wherein the subject is asymptomatic, the antiviral agent may be administered prophylactically to a patient who has not yet been infected with a coronavirus, or administered to treat an asymptomatic coronavirus infection. The effective amount (i.e., dose) of an anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) to be administered to a subject can be determined depending upon, for example, age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment. Exemplary doses can be from about 0.01 to about 1000 mg, by oral administration. Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.50, 1, 5, 10, 25, 50, 100, 125, 150, 200, or 250 mg to a maximum dose of about 300, 400, 500, 600, 700, 800, 900, or 1000 mg, wherein an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses. Specific examples of particular effective amounts contemplated via oral administration include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55,
0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,
340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425,
430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515,
520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,
610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,
700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785,
790, 795, 800, 805, 810, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880,
885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970,
975, 980, 985, 990, 995, 1000 mg or more. The oral dose can be administered once daily, twice daily, three times daily, or more frequently. In some embodiments, the method comprises administering Tempol in a dose of about 500-1000 mg (e.g., about 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg) once daily, twice daily, or three times daily over a suitable period to have the desired effect (e.g., 2, 3, 4, or 5 weeks). [0104] In some embodiments, the method comprises administering an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like), which, in some embodiments, can be administered using currently-approved dosages as noted above (for agents that are approved). However, as noted, such approved dosages are approved for treatment of patients with iron overload. For use in the context of the present invention, in combination with a nitroxide radical (such as Tempol), lower dosages of the iron chelator are preferred, to minimize potential side effects. Thus, for example, dosages of DFP suitable for use in the context of the present invention, thus, include less than or equal to 70 mg/kg, such as less than or equal to 60 mg/kg, such as less than or equal to 50 mg/kg, such as less than or equal to 40 mg/kg, such as less than or equal to 30 mg/kg, such as less than or equal to 20 mg/kg, and even lower dosages may be appropriate. Also, as with the nitroxide radical, an iron chelator can be administered in accordance with the present invention once daily, twice daily, or three times daily over a suitable period to have the desired effect (e.g., 2, 3, 4, or 5 weeks). One dosing regimen contemplated for application in the context of the present invention comprises administering Tempol (or another nitroxide radical) and deferiprone (or another iron chelator) in a single combined oral dosage form (e.g., a tablet or capsule) twice daily over a five to seven day course.
[0105] The dose of an anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or an iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) for use in parenteral administration (preferably intravenous administration) is generally from about 0.01 to about 300 mg/kg body weight. Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.50, 1, 5, 10, 25, 50, or 100 mg/kg body weight to a maximum dose of about 125, 150, 175, 200, 250, 275, or 300 mg/kg body weight, wherein an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses. Specific examples of particular effective amounts contemplated include about 0.02, 0.03, 0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125,
130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,
220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300 mg/kg body weight or more. Continuous intravenous administration is also contemplated for from 1 to 24 hours per day to achieve a target concentration from about 0.01 mg/L blood to about 100 mg/L blood. Exemplary dose ranges can include from a minimum dose of about 0.01, 0.10, 0.25, 0.50, 1, 5, 10, or 25 mg/L blood to a maximum dose of about 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, or 100 mg/L, wherein an exemplary dose range can include from any one of the foregoing minimum doses to any one of the foregoing maximum doses. Specific examples of particular effective amounts contemplated via this route include about 0.02, 0.03,
0.04, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100 mg/L blood or more. The dose to be used can depend upon various conditions, and there may be cases wherein doses lower than or greater than the ranges specified above are used.
[0106] In aspects of the invention, the nitroxide radical is administered in a dose sufficient to produce 200-400 micromolar concentration of the antiviral agent in tissues of the subject. For example, the dose may be sufficient to produce a 200-400 micromolar concentration in multiple tissues throughout the body of the subject, for example the pharynx, trachea, lungs, blood, heart, vessels, intestines, brain and/or kidneys.
[0107] Aspects of the present invention further provide a pharmaceutical composition comprising a compound as described above (nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like)) and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount, e.g., a therapeutically effective amount, including a prophylactically effective amount, of the compound of the present invention.
[0108] The pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemi co-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical compositions; the compound of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
[0109] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compound and one which has no detrimental side effects or toxicity under the conditions of use.
[0110] The choice of carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.
[0111] The anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) may be administered in the form of, for example, solid compositions, liquid compositions, or other compositions for oral administration, injections, liniments, or suppositories for parenteral administration. Solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules. Capsules include hard capsules and soft capsules. In such solid compositions, the nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be admixed with an excipient (e.g., lactose, mannitol, glucose, microcrystalline cellulose, or starch), combining agents (e.g., hydroxypropyl cellulose, polyvinyl pyrrolidone, or magnesium metasilicate aluminate), disintegrating agents (e.g., cellulose calcium glycolate), lubricating agents (e.g., magnesium stearate), stabilizing agents, agents to assist dissolution (e.g., glutamic acid or aspartic acid), or the like. The agents may, if desired, be coated with coating agents (e.g., sugar, gelatin, hydroxypropyl cellulose, or hydroxypropylmethyl cellulose phthalate), or be coated with two or more films. Further, coating may include containment within capsules of absorbable materials such as gelatin. A preferred composition for administering the nitroxide radical and the iron chelator comprises a combined oral dosage form (e.g., a tablet, capsule, syrup, and the like) comprising both agents formulated together in a single dosage form. [0112] Liquid compositions for oral administration include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs. In such compositions, the nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) is dissolved, suspended, or emulsified in a commonly used diluent (e.g. purified water, ethanol, or mixture thereof). Furthermore, such liquid compositions may also comprise wetting agents, suspending agents, emulsifying agents, flavoring agents (e.g., flavor-masking agents) sweetening agents, perfuming agents, preserving agents, buffer agents, or the like.
[0113] Injections for parenteral administration include solutions, suspensions, emulsions, and solids which are dissolved or suspended. For injections, the nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be dissolved, suspended, and/or emulsified in a solvent. The solvents are, for example, distilled water for injection, physiological salt solution, vegetable oil, propylene glycol, polyethylene glycol, alcohol such as ethanol, or a mixture thereof. Moreover, the injections also can include stabilizing agents, agents to assist dissolution (e.g., glutamic acid, aspartic acid, or POLYSORBATE 80™), suspending agents, emulsifying agents, soothing agents, buffer agents, preserving agents, etc. The compositions are sterilized in the final process or manufactured and prepared by sterile procedure. The compositions also can be manufactured in the form of sterile solid compositions, such as a freeze-dried composition, and can be sterilized or dissolved immediately before use in sterile distilled water for injection or some other solvent.
[0114] Other compositions for parenteral administration include liquids and ointments for external use, endermic liniments, compositions for inhalation, sprays, suppositories for rectal administration, and pessaries for vaginal administration, which compositions include a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) and are administered by methods known in the art. [0115] nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) compositions for inhalation or sprays may comprise additional substances other than diluents, such as, e.g., stabilizing agents (e.g. sodium sulfite hydride), isotonic buffers (e.g. sodium chloride, sodium citrate or citric acid). See, for example, the methods described in U.S. Patents. 2,868,691 and 3,095,355. The nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be effectively distributed by inhalation or spray using a self-propelling composition that includes a solution or dispersion of the nitroxide radical in micronized form. For example, an effective dispersion of finely divided drug particles can be accomplished with the use of very small quantities of a suspending agent, present as a coating on micronized drug particles. Evaporation of the propellant from the aerosol particles after spraying from the aerosol container leaves finely divided drug particles coated with a fine film of the suspending agent. In the micronized form, the average particle size can be less than about 5 microns. The propellant composition can employ, as the suspending agent, a fatty alcohol such as oleyl alcohol. Propellants that may be employed include hydrofluoroalkane propellants and chlorofluorocarbon propellants. Dry powder inhalation also can be employed.
[0116] Nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) compositions suitable for inhalation may be used, for example, in a method of the invention to treat or reduce the remodeling of the pulmonary vasculature associated with prolonged alveolar hypoxia, e.g., in subjects that have, are suspected to have, or are at risk for pulmonary arterial hypertension, and cor pulmonale. [0117] In some embodiments, the anti-viral agent comprising a nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be administered as a supplement with food or drink. Thus, the nitroxide radical (e.g., Tempol) and/or iron chelator (e.g., deferiprone, deferasirox, desferrioxamine, CN128, and the like) can be mixed into a food or drink composition, which, optionally, masks the flavor of the nitroxide. In aspects of the invention, the composition is administered in combination with one or more additional active agents. In aspects, the additional active agent or agents may be antiviral agents. The additional antiviral agent may be a nucleoside analog, for example remdesivir.
Aspects of the Invention
[0118] 1. A method of treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject an effective amount of an anti -viral agent comprising a nitroxide radical, and the antiviral agent treats or prevents the coronavirus infection in the subject. Optionally, (a) when the coronavirus is SARS-CoV2, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) or (b) when the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol), the coronavirus is other than SARS-CoV-2.
[0119] 2. The method of aspect 1 wherein the nitroxide radical is 2, 2,6,6- tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l-piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo-Tempo), 4-(2- bromoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-ethoxyfluorophosphonyloxy- 2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-l- oxyl, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6- tetramethylpiperidine-l-oxyl; 4-(4-nitrobenzoyloxyl)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4- phosphonooxy-2,2,6,6-tetramethylpiperidine-l-oxyl, 2-ethyl-2,5,5-trimethyl-3-oxazolidine-l- oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-aminomethyl- Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-cyano-Proxyl); 3-Carbamoyl-
2.2.5.5-tetramethyl-l-pyrrolidinyl-N-oxyl (3 -Carbarn oyl-Proxyl); or 3-Carboxy-2, 2,5,5- tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy-Proxyl).
[0120] 3. The method of aspect 1, wherein the nitroxide radical is 4-hydroxy -
2.2.6.6-tetramethylpiperidine-l-oxyl (Tempol). Optionally, in accordance with this aspect, the coronavirus is other than SARS-CoV-2
[0121] 4. The method of any one of aspects 1-3, wherein the coronavirus is
SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63.
[0122] 5. The method of any of one of aspects 1-3, especially aspect 1 and 2, wherein the coronavirus is SARS-CoV2. Optionally, in accordance with this aspect, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol). [0123] 6. The method of aspect 4 or 5, wherein the subject has not been tested for infection with the coronavirus.
[0124] 7. The method of aspect 4 or 5, wherein the subject has been exposed to the coronavirus.
[0125] 8. The method of aspect 4 or 5, wherein the anti-viral agent is administered prophylactically.
[0126] 9. The method of aspect 4 or 5, wherein the subject does not have acute respiratory distress syndrome (ARDS).
[0127] 10. The method of aspect 4 or 5, wherein the subject is asymptomatic.
[0128] 11. The method of any one of aspects 1-10, wherein the antiviral agent is administered orally, intravenously, subcutaneously, intradermally, via inhalation, or intramuscularly.
[0129] 12. The method of aspect 11, wherein the antiviral agent is administered orally.
[0130] 13. The method of any one of aspects 1-12, wherein the antiviral agent is administered in a dose sufficient to result in 200-400 micromolar concentration in tissues of the subject. [0131] 14. The method of any one of aspects 1-13, wherein the antiviral agent is administered either once or twice a day.
[0132] 15. The method of any one of aspects 1-14, further comprising administering an iron chelator to the subject.
[0133] 16. The method of aspect 15, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
[0134] 17. A composition for use in treating or preventing a coronavirus infection in a subject, the composition comprising an effective amount of an anti -viral agent comprising a nitroxide radical and a pharmaceutically acceptable carrier.
[0135] 18. The composition for use according to aspect 17, wherein the nitroxide radical is 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l- piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo- Tempo), 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4- ethoxyfluorophosphonyloxy-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(2-iodoacetamido)- 2,2,6,6-tetramethylpiperidine-l-oxyl, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(4-nitrobenzoyloxyl)-2, 2,6,6- tetramethylpiperidine-l-oxyl; 4-phosphonooxy-2,2,6,6-tetramethylpiperidine-l-oxyl, 2-ethyl- 2,5,5-trimethyl-3-oxazolidine-l-oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l- pyrrolidinyl-N-oxyl (3-aminomethyl-Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N- oxyl (3-cyano-Proxyl); 3-Carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3- Carbamoyl-Proxyl); or 3-Carboxy-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy- Proxyl).
[0136] 19. The composition for use according to aspect 17, wherein the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol). Optionally, in accordance with this aspect, the coronavirus is other than SARS-CoV-2 [0137] 20. The composition for use according to any one of aspects 17-19, wherein the coronavirus is SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63.
[0138] 21. The composition for use according to any one of aspects 17-19, wherein the coronavirus is SARS-CoV-2. Optionally, in accordance with this aspect, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol). [0139] 22. The composition for use according to aspect 20 or 21, wherein the subject has not been tested for infection with the coronavirus. [0140] 23. The composition for use according to aspect 20 or 21, wherein the subject has been exposed to the coronavirus.
[0141] 24. The composition for use of aspect 20 or 21, wherein the anti -viral agent is administered prophylactically.
[0142] 25. The composition for use of aspect 20 or 21, wherein the subject does not have acute respiratory distress syndrome (ARDS).
[0143] 26. The composition for use of aspect 20 or 21, wherein the subject is asymptomatic.
[0144] 27. The composition for use of any one of aspects 17-26, wherein the composition is formulated for administration orally, intravenously, subcutaneously, intradermally, via inhalation, or intramuscularly.
[0145] 28. The composition for use of aspect 27, wherein the antiviral agent is formulated for administration orally.
[0146] 29. The composition for use of any one of aspects 17-28, wherein the antiviral agent is formulated in a dose sufficient to result in a 200-400 micromolar concentration in tissues of the subject.
[0147] 30. The composition for use of any one of aspects 17-29, wherein the antiviral agent is formulated for administration either once or twice a day.
[0148] 31. The composition for use of any one of aspects 17-30, further comprising an iron chelator.
[0149] 32. The composition for use of aspect 31, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
[0150] 33. A method of treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject a combination comprising an effective amount of 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and an effective amount of an iron chelator, wherein the administration of the combination treats or prevents the coronavirus infection in the subject. Preferably, the coronavirus is SARS-CoV-2.
[0151] 34. The method of aspect 33, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
[0152] 35. One or more compositions for use in treating or preventing a coronavirus infection in a subject, the compositions comprising (a) an effective amount of 4- hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and a pharmaceutically acceptable carrier and (b) an effective amount of an iron chelator and a pharmaceutically acceptable carrier. Preferably, the coronavirus is SARS-CoV-2.
[0153] 36. The one or more compositions for use of aspect 35, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
[0154] 37. The one or more compositions for use of aspect 34, wherein the compositions comprise a single composition.
[0155] 38. The composition of aspect 37, which is suitable for oral administration.
EXAMPLES
[0156] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
[0157] The following materials and methods were utilized in the Examples 1-5, presented below.
Cell Lines and Cell Culture Conditions
[0158] HEK293, HEPG2 and VeroE6 cells were purchased from ATCC. Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5g/L glucose, supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C and 5% C02 in a humidified incubator. Expi293F cells used for mammalian cell expression of the RdRp of SARS-CoV-2 were purchased from ThermoFisher Scientific (catalog number: A14635).
Cells were propagated in suspension in chemically defined, serum-free, protein-free, animal origin-free Expi293 Expression Medium at 37°C and 8% C02 and subcultured according to the manufacturer’s instructions. All cell lines were subjected to mycoplasma testing.
Plasmids and Transfection of Mammalian Cells
[0159] Recombinant expression of C-terminally 3xFLAG-nspl2 in Expi293F, HEK293 and VeroE6 cells, was obtained from P3xFLAG-SARS-CoV-2-nspl2, a gift from Patrick Moore (Addgene plasmid # 154008; http://n2t.net/addgene: 154008; RRID: Addgene 154008). For assembling the stable nspl2-nsp7-nsp8 (RdRp) complex, the SARS-CoV-2 nsp7 and nsp8 open reading frames (ORFs), each engineered to harbor a Strep-tag II C- terminally fused to the recombinant protein, were subcloned on either side of the ECMV internal ribosome entry site (IREs) into the bicistronic pIRES expression vector (Takara; Cat. No. 631605) for the simultaneous translation of the two genes of interest from the same mRNA transcript. Expression of the SARS-CoV-2 helicase, nspl3, was obtained from pCMV3-nspl3 (Sino Biologicals, Cat. No. VG40596-UT).
[0160] Point mutations into nspl2 were introduced using the QuikChange II site- directed mutagenesis kit (Agilent Technologies), following the manufacturer’s instructions. All clones were verified for the insertion of the desired mutation(s) by Sanger sequencing at Eurofms USA. Plasmid transfections into mammalian cells, except Expi293F cells (see below), were performed with Lipofectamine 2000 (ThermoFisher Scientific), according to standard procedures.
Mutagenesis Primers
Figure imgf000029_0001
Figure imgf000030_0001
Protein Production and Purification
[0161] Expi293F cells at a final density of 3 c 106 viable cells/mL (2.25 x 109 cells at the time of transfection) were inoculated in 800 mL of fresh Expi293™ Expression Medium supplemented with 300 mM L-cysteine and 40 mM FeC13 or 57FeC13 (for Mossbauer and EPR analyses) and transfected with the following combinations of constructs: 840 pg P3xFLAG-SARS-CoV-2-nspl2 alone (for Mossbauer and EPR studies) or 420 pg P3xFLAG-SARS-CoV-2-nspl2 and 840 pg of pIRES-nsp7/nsp8-Strep II (for the RdRp activity assays), which were combined with 48 ml of Opti-MEM™ I Reduced Serum Medium (ThermoFisher Scientific) and incubated for 5 min at room temperature. The plasmid DNA mix was then combined with a mix containing 2.6 ml ExpiFectamine™ 293 Reagent and 45 ml Opti-MEM™ I Reduced Serum Medium and incubated at room temperature for additional 20 min prior to addition to the cell suspension. Forty-eight hours after transfection, cells were spun down and washed twice with cold PBS prior to being taken inside an argon recirculated glove box operated at <0.2ppm 02 for lysis. Cells were lysed in 100 ml of lysis buffer (150 mM NaCl, 50 mM Na-HEPES pH 7.4, 10% (v/v) glycerol, 3 mM MgCE, 2 mM TCEP, 0.5% NP-40, EDTA-free protease inhibitor cocktail) and homogenized by gently pipetting up and down until no cell clumps were visible. After clearing up the insoluble fractions by centrifuging the homogenate at 40,000 x g for 30 min, 4 ml of pre equilibrated FLAG-M2 beads were added to the lysate. Recombinant protein complexes were immunoprecipitated for 3 hours at room temperature, before being packed into a column. The resin was washed with 10 column volumes of lysis buffer, followed by 20 column volumes of lysis buffer supplemented with 300 mM NaCl. The FLAG-tagged nspl2 proteins or the 3xFLAG-nspl2/nsp7/nsp8-Strep tag II complexes were then eluted from the FLAG-M2 beads with 3xFLAG peptide dissolved in lysis buffer for 1 hour at room temperature. The eluates at ~50 mM were concentrated to -250 mM using a 30 kDa molecular weight cut-off filter.
Subcellular Fractionation and Immunoprecipitation (IP)
[0162] Subcellular fractionation into cytosol and intact mitochondria was done as previously described (see N. Maio et al., Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery. CellMetab 19, 445-457 (2014); N. Maio et al., Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB. Cell Metab 23, 292-302 (2016); N. Maio, K. S. Kim, A. Singh, T. A. Rouault, A Single Adaptable Cochaperone-Scaffold Complex Delivers Nascent Iron-Sulfur Clusters to Mammalian Respiratory Chain Complexes I-III. CellMetab 25, 945-953 e946 (2017)). Briefly, mitochondria from HEK293, Expi293F or VeroE6 cell pellets (-109 cells) were isolated from the cytosolic fractions after cell permeabilization with a buffer containing 0.1% digitonin in 210 mM mannitol, 20 mM sucrose, and 4 mM HEPES. The pellets after centrifugation at 700 x g for 5 min contained mitochondria, which were isolated by differential centrifugation and solubilized in lysis buffer I, containing 50 mM Bis-Tris, 50 mM NaCl, 10% w/v Glycerol, 0.001% Ponceau S, 1% lauryl maltoside, pH 7.2 and protease inhibitors.
[0163] The supernatants after the centrifugation at 700 x g containing soluble proteins were spun down at 21,000 x g for 20 min. The supernatant after the centrifugation was supplemented with a 1 : 1 volume (v/v) of a buffer containing 25 mM Tris, 200 mM NaCl,
1 mM EDTA, 1% NP-40 (pH 7.4) to obtain a final protein concentration of approximately 1 pg/pL. 500 pg of total cytosolic proteins were used for the IPs of 3XFLAG-nspl2 using M2 -FLAG beads (Sigma). Equilibrated FLAG M2 beads were added to the lysates and incubated for 2h at room temperature. Beads were recovered after extensive washing with lysis buffer, and proteins were eluted with Tris-Glycine pH 2.8 (150 pL/IP sample) for 10 min at RT, or for the native elution, protein complexes were competitively eluted with 100 pg/ml of 3xFLAG peptide (Sigma) for lh at RT. The eluates were analyzed by SDS PAGE and immunoblot. Pull Down Assays Performed with Purified Proteins
[0164] C-terminally 3xFLAG-nspl2 wild type or variants of the LYR motifs and C- terminally HA-tagged human HSC20 were individually expressed and purified from Expi293F cells. Affinity purification of 3xFLAG-nspl2 proteins was performed using M2- FLAG beads (Sigma), as indicated under “Protein Production and Purification”. Proteins were eluted, following extensive washing of the beads with lysis buffer supplemented with 100 pg/ml of FLAG peptide. Affinity purification of HA-tagged HSC20 was performed using anti -HA agarose beads (Pierce). HSC20 protein was eluted with 20 pg/ml of HA peptide. 0.25pg of purified nspl2-FLAG wild type or the variants of either one or both LYR motifs (VYR-AAA, LYR- AAA and VYR/LYR-AAA, respectively) were combined with 0.25 pg of HSC20. Immunoprecipitations (IPs) were performed with M2 -FLAG beads (Sigma) to immunocapture nspl2 proteins. The presence of HSC20 co-eluted with nspl2 proteins was analyzed by SDS-PAGE and Coomassie staining. Aliquots corresponding to 20% of the inputs were run on the gel for comparison.
Mass spectrometry analysis.
[0165] Proteins (~10 pg per sample) were run on SDS-PAGE and stained with Coomassie Blue G-250. The gel bands were excised and washed overnight in 50% methanol with 10% acetic acid. Proteins were reduced using 10 mM Tris(2-carboxy ethyl) phosphine hydrochloride at room temperature for 1 hour, then alkylated with 10 mM N-ethylmaleimide (NEM) for 10 minutes and digested with trypsin (Promega) 1:20 (w/w) at 37 °C for 18 hours. Tryptic digests were extracted from the gel and cleaned with an Oasis HLB microplate (Waters). The desalted peptides were injected into a Dionex UltiMate 3000 RSLCnano HPLC instrument (Thermo Fisher Scientific) with an ES802 nanocolumn (Thermo Fisher Scientific). The column temperature was set at 45 °C. Mobile phase A and B (MPA, MPB) contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The peptides were eluted at a flow rate of 300 nL/min using the following gradients: 3% to 22% MPB for 66 min, 22% to 33% MPB for 6 min, 33% to 80% MPB for 4 min. Thermo Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) was used for data acquisition. The LC- MS/MS data were acquired in data-dependent mode. The MSI scans were performed in orbitrap with a resolution of 120K at 200 m/z with a mass range of 375-1500 m/z and an automatic gain control (AGC) value of 2 x le5. The quadrupole isolation window was 1.6 m/z. Ions with an intensity greater than 1 x le4 were fragmentated by CID method with collision energy fixed at 30%. The MS2 scans were conducted in ion trap with and AGC target of 3 x le4.
[0166] The Proteome Discoverer software version 2.4 was used for protein identification and quantitation. Raw data were searched against Uniprot Human Database. The mass tolerances for precursor and fragment were set to 10 ppm and 0.6 Da, respectively. Up to 2 missed cleavages were allowed for trypsin digestion. NEM on cysteines was set as fixed modification. Variable modifications include Oxidation (M), Met-loss (Protein-N-term) and Acetyl (Protein N-term). Peptides were validated based on q-values using percolator algorithm. The search results were filtered by a false discovery rate (FDR) of 1% at the protein level. The protein abundances were calculated by summing the abundance of the connected peptides. The protein ratios were calculated by dividing the protein abundance values between the eluates after immunoprecipitation of 3xFLAG-nspl2 (nspl2, samples; n=6) and those of the IgG immunoprecipitations (control, samples; n=6). The maximum allowed fold change value was set to 100. In the instance in which the target protein was detected in the ‘nspl2’ only samples and not in negative ‘control’ samples, the nspl2/control ratios were set to 100. Vice versa, if the protein was detected in the negative ‘control’ samples only and not in ‘nspl2’ samples, the nspl2/control ratios were set to 0.01. ANOVA (Individual Proteins) method was used for statistical analyses. Because the ratios were calculated without normalization and imputation, if a target protein was not detected in either the ‘nspl2’ or ‘control’ group samples, a fold change value was calculated and reported without p-value.
RdRp activity assays (RNA extension assays)
[0167] All RNA oligonucleotides were purchased from Dharmacon (Horizon Discovery). RNA primers were radiolabeled at the 5' end with [g-32R] ATP (Perkin Elmer) using the T4 polynucleotide kinase from the KinaseMax 5' End-Labeling Kit (Thermo Fisher Scientific). Two distinct pairs of primer/template duplexes were used in the RNA extension assays. A shorter pair of primer/template oligonucleotides consisted of the 4-mer 5'- rArCrGrC-3' (SEQ ID NO: 26) and the 14-mer 5'-rUrUrUrUrUrUrGrUrCrUrGrCrGrU-3' template (SEQ ID NO: 15) and a longer pair consisted of the 13-mer primer 5'- rArGrGrUrArArUrArArArArUrU-3' (SEQ ID NO: 16), and the 29-mer template 5'- rUrUrUrUrArArUrC rCrUrArArArCrGrArArArUrUrUrUrArUrUrArCrCrU-3' (SEQ ID NO: 17). [0168] To anneal each primer to its complementary template, oligonucleotides were mixed at equal molar ratios in annealing buffer (50 mM NaCl and 10 mM Na-HEPES pH 7.5), denatured by heating to 75°C for 5 min and then slowly cooled to 4°C.
[0169] The SARS-CoV-2 RdRp complex anoxically purified from Expi293F mammalian cells co-expressing 3xFLAG-nspl2 and nsp7/nsp8-Strep II at a final concentration of 1 mM was incubated with 3 mM dsRNA in the presence of 1.2 U/pl RNase inhibitor in reaction buffer containing in 100 mM NaCl, 20 mM Na-HEPES pH 7.5, 5% (v/v) glycerol, 10 mM MgCF and 0.5 mM TCEP, which were prepared in DEPC-treated water. Reactions were incubated at 37 °C for 15 min and the RNA extension was initiated by addition of NTPs (300 pM UTP, GTP and CTP, and 100 pM [a-32P]ATP (Perkin Elmer)). The total reaction volume was 10 pi. Reactions were stopped by the addition of 2 c stop buffer (7 M urea, 50 mM EDTA pH 8.0, 1 c TBE buffer). Samples were digested with proteinase K (New England Biolabs) and RNA products were separated on 20% acrylamide gels in 1 x TBE buffer supplemented with 8M urea and imaged by autoradiography on Carestream BioMax Light films.
[0170] The inhibitory effects of TEMPOL (Sigma, Cat. No. 176141) or DEA/NO (Sigma, Cat. No. D5431) in vivo were assessed by treating Expi293F or VeroE6 cells co expressing 3xFLAG-nspl2 and nsp7/nsp8-Strep tag II, followed by anaerobic purification of the RdRp complex by anti-FLAG immunoprecipitations and RNA extension assays. The effect of remdesivir triphosphate (RDV-TP, aka GS-443902, MedChemExpress LLC, Cat. No. HY-126303), alone or in combination with TEMPOL, on the activity of the RdRp was assessed in vitro on the anoxically purified enzyme or on the enzyme purified aerobically and reconstituted with ZnCF.
Gel Mobility Shift Assay to Detect RNA-RdRp Binding
[0171] A gel mobility assay was performed to detect RNA binding by the RdRp complex. The binding reaction contained 25 mM HEPES pH 7.4, 100 mM sodium chloride, 2 mM magnesium chloride and 1 mM TCEP, 9 pg RdRp complex with increasing amounts of template-primer RNA (0, 0.3, 0.6, 0.9, 1.2 and 2 pg). For the assay employing only the nspl2 protein, which either contained the Fe-S clusters or zinc, the binding reaction was combined with 1.5 pg of template-primer RNA. Reactions were incubated for 1 hour at room temperature and resolved on 20% acrylamide gels in 1 x TBE buffer at 90 V for 1 hour on ice. The gel was stained with SYBR Safe RNA staining reagent (Thermo Scientific), according to the manufacturer’s instructions and visualized on a Bio-Rad Chemidoc imager.
Si-RNA-Mediated Knockdown of ISC U in Expi293F Cells
[0172] On-TARGET Plus siRNA pools against human ISCU (Cat. No. L-012837-01- 0005) and the control nontargeting (NT si-RNAs) pool (Cat. No. D-001810-10-05) were purchased from Dharmacon. Knockdown of ISCU in Expi293F cells was achieved by transfecting cells twice with siRNAs at a 48-hour interval using ExpiFectamine™ 293 Transfection Kit (ThermoFisher Scientific) according to manufacturer’s instructions. At the time of the second transfection with si-RNAs, cells were co-transfected with the constructs encoding the nspl2 WT or variants. Cell lysates were analyzed 48h after the second transfection by immunoblot to verify the efficiency of ISCU knockdown and by scintillation counting to measure 55Fe-incorporation into nspl2 WT or variants in control (NT si-RNAs) or ISCU depleted (si-ISCU) cells (for details on 55Fe incorporation see following paragraph). [0173] ON-TARGET Plus Human non targeting siRNA Target Sequences: UGGUUUACAUGUCGACUAA (SEQ ID NO: 18) UGGUUUACAUGUUGUGUGA (SEQ ID NO: 19)
U GGUUU AC AU GUUUU CU G A (SEQ ID NO: 20) UGGUUUACAUGUUUUCCUA (SEQ ID NO: 21)
[0174] ON-TARGET Plus Human siRNAs Targeting Sequences to ISCU: GAGCUAUGAGAUACGCACA (SEQ ID NO: 22) CAGCAUGUGGUGACGUAAU (SEQ ID NO: 23) CAUAAAACAGAUUGCGCAU (SEQ ID NO: 24) GGUCUGAAUAUUUGAUAGA (SEQ ID NO: 25)
Iron Incorporation Assay
[0175] The 55Fe incorporation assays into nspl2 WT or variants were performed as previously described (K. S. Kim, N. Maio, A. Singh, T. A. Rouault, Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAOl -mediated transfer to recipients. Hum Mol Genet, (2018); N. Maio et ak, Disease-Causing SDHAFl Mutations Impair Transfer of Fe-S Clusters to SDHB. Cell Metab 23, 292-302 (2016)) with some modifications. Expi293F cells were grown in expression medium in the presence of 1 mM 55Fe-Transferrin. Cytosolic extracts were subjected to immunoprecipitation with anti -FLAG to immunocapture 3XFLAG-nspl2 proteins 48h post-transfection. Samples collected after competitive elution (with 3X FLAG peptide at 100pg/ml) were analyzed by scintillation counting to assess 55Fe content. The background levels corresponding to 55Fe measurements on eluates after anti-FLAG immunoprecipitations on cytosolic extracts isolated from cells transfected with the empty vector, p3XFLAG-CMV-14 (Sigma, Cat. No.: E7908), were also included to account for nonspecific 55Fe amounts stochastically associated to the beads (corresponding to 587 ± 292.62 cpm/mg of cytosolic proteins). When 55Fe levels incorporated into nspl2 proteins were determined after knockdown of ISCU, cells were transfected twice with non-targeting or ISCU-directed siRNAs at a 48-h interval. At the time of the second transfection with si-RNAs, cells were co-transfected with the constructs encoding the nspl2 wild type or variants. Cell lysates were analyzed 48 h after the second transfection.
Ferrozine based colorimetric assay to determine iron stoichiometries of nspl2 WT and variants
[0176] The ferrozine based colorimetric assay (Sigma- Aldrich, Cat. No.: MAK025- 1KT) was used to determine the concentration of iron (Fe2+) in preparations of purified nspl2 WT and variants each at 250 mM protein concentration, according to the manufacturer’s instructions. In this assay, the iron released by the addition of an acidic buffer was reduced to measure both Fe2+ and Fe3+ and colorimetrically determined at 593 nm after reaction with a chromogen proportional to the iron present in the samples. To accurately determine protein concentrations for nspl2 WT and variants avoiding systematic over or under-estimations inherent to the routinely used colorimetric methods that are based on the absorbance of a standard protein (usually bovine serum albumin), protein concentrations determined with the method of Bradford were corrected with respect to the BSA standard by performing amino acid analysis (Alphalyse Inc.). Iron concentrations in nspl2 WT, nspl2C301S-C306S-C310S and nspl2C487S-C645S-C646S, each at 250 mM, were 1875 ± 87.5 mM, 950 ± 52.5 mM and 917.5 ± 82.5 mM (n=5), respectively, corresponding to 7.5 ± 0.35 iron/protomer for nspl2 WT, 3.8 ± 0.21 iron/protomer for nspl2C301S-C306S-C310S and 3.67 ± 0.33 iron/protomer for nspl2C487S-C645S-C646S. DPYD Activity Assay
[0177] The dihydropyrimidine dehydrogenase (DPYD) activity was determined by thin layer chromatography (TLC), following a previously described protocol (K. S. Kim, N. Maio, A. Singh, T. A. Rouault, Cytosolic HSC20 integrates de novo iron-sulfur cluster biogenesis with the CIAOl -mediated transfer to recipients. Hum Mol Genet , (2018)); O. Stehling et al., MMS19 assembles iron-sulfur proteins required for DNA metabolism and genomic integrity. Science 337, 195-199 (2012)), with the following modifications. Cell lysates containing 150 pg of proteins isolated from control or TEMPOL-treated cells, were applied to 50 pi of a reaction mix containing 25 mM Tris-HCl (pH 7.5), 0.1% digitonin, 2.5 mM MgCF, 2mM DTT, 10 mM [4- 14C]-thymine (O.lmCi/ml Moravek Inc. CA, USA), 10 mMNADPH. After 4 hours of incubation at 32 °C, the reaction was stopped by addition of 10 pi of perchloric acid (10% v/v). Reaction mixtures were centrifuged at 20000 x g for 5 minutes and the supernatants analyzed by TLC.
Native PAGE (BN-PAGE) Analyses of the Mitochondrial Respiratory Complexes [0178] The Native PAGE Novex Bis-Tris gel system (Thermo Fisher Scientific) was used for the analysis of native membrane respiratory chain protein complexes, with the following modifications: only the Light Blue Cathode Buffer was used; 20 pg of membrane protein extracts were loaded/well; the electrophoresis was performed at 150 V for 1 h and 250 V for 2 h.
Complex I, Complex II and Complex IV in-gel Activity Assays
[0179] In-gel Complex I, Complex II and Complex IV activity assays were performed as previously described ( N. Maio et al., Cochaperone binding to LYR motifs confers specificity of iron sulfur cluster delivery. Cell Metab 19, 445-457 (2014); N. Maio et al., Disease-Causing SDHAF1 Mutations Impair Transfer of Fe-S Clusters to SDHB. Cell Metab 23, 292-302 (2016), N. Maio, K. S. Kim, A. Singh, T. A. Rouault, A Single Adaptable Cochaperone-Scaffold Complex Delivers Nascent Iron-Sulfur Clusters to Mammalian Respiratory Chain Complexes I-III. Cell Metab 25, 945-953 e946 (2017)). For Complex I activity, after resolution of the respiratory complexes by BN-PAGE, the gel was incubated with 0.1 M TrisCl, pH 7.4, containing 1 mg/ml nitrobluetetrazolium chloride (NBT) and 0.14 mM NADH at room temperature for 30-60 min. For Complex II, detection of succinate CoQ- reductase activity (SQR) (CoQ-mediated NBT reduction) was performed by incubating the gel for 30 minutes with 84 mM succinate, 2 mg/ml NBT, 4.5 mM EDTA, 10 mM KCN, 1 mM sodium azide and 10 mM ubiquinone in 50 mM PBS, pH 7.4. For complex IV, the gel was incubated in 50 mM phosphate buffer pH 7.4 containing lmg/ml DAB (3,3’- diaminobenzidine) and lmg/ml cytochrome c at room temperature for 30-45 min.
[0180] Aconitase In-gel Activity Assay and Electrophoretic Mobility Shift Assay (EMSA)
[0181] Aconitase activity assay and IRP-IRE binding activities by EMSA were performed as previously described ( M. C. Ghosh et al., Tempol-mediated activation of latent iron regulatory protein activity prevents symptoms of neurodegenerative disease in IRP2 knockout mice. Proceedings of the National Academy of Sciences of the United States of America 105, 12028-12033 (2008)).
Ultraviolet-visible (UV-vis) Absorption Spectroscopy and Amino Acid Analysis (AAA)
[0182] Immunoprecipitations of recombinant 3xFLAG-nspl2 proteins were performed on lysates anoxically prepared in an argon recirculated glove box operated at <0.2 ppm 02 48h after transfection with plasmids encoding nspl2 wild type or variants. UV-vis spectra were acquired for anoxically purified nspl2 proteins at ~25 mM in sealed, air-tight cuvettes using aNanoDrop spectrophotometer (ThermoFisher Scientific) with the elution buffer containing 100 pg/ml 3 x FLAG peptide as blank.
[0183] AAA was performed by Alphalyse Inc. to precisely quantify the purified nspl2 wild type and variants.
EPR and Mossbauer Spectroscopies
[0184] To verify the presence and to identify the type and stoichiometry of iron-sulfur cluster(s) in wild type nspl2 and its variants, 57Fe enriched samples of these proteins were examined by EPR and Mossbauer spectroscopies. For EPR analyses, 150 pL aliquots of anoxically purified wild type nspl2 and its variants at a final concentration of 250 mM were transferred to separate EPR tubes, and rapidly frozen in liquid nitrogen. To test if the iron- sulfur cluster(s) could be reduced and to characterize the reduced iron-sulfur cluster(s), second set of EPR samples were prepared by incubating wild type nspl2 and its variants at a final concentration of 220 mM with 5 mM sodium dithionite for 15 min prior to freezing. Continuous-wave EPR (CW-EPR) spectra were acquired on an ESP300 Bruker X-band spectrometer equipped with an ER 410ST resonator. The temperature was held at 20 K by an ER 4112-HV Oxford Instruments (Concord, MA) variable-temperature helium flow cryostat. The38pectrumeter was controlled through EWIN 2012 software on an external personal computer with a GPIB interface. The modulation amplitude was 5 G, the microwave frequency was 9.439 GHz, and the microwave power was 20 mW. The “pepper” routine from the EasySpin package 5.2.30. was used to simulate the EPR spectra.
[0185] For Mossbauer analyses, 300 pL aliquots of 250 mM anoxically purified wild- type nspl2 or its variants were transferred into Mossbauer cups and froze them in liquid nitrogen. The Mossbauer spectra were recorded on an alternating constant acceleration Mossbauer spectrometer from SEE Co. (Edina, MN) equipped with a Janis SVT-400 variable-temperature cryostat. The external 53 mT magnetic field was oriented parallel to the direction of propagation of the g beam. All isomer shifts are quoted relative to the centroid of the spectrum of a-iron metal at room temperature. The Mossbauer spectra were simulated using the WMOSS spectral analysis software (wmossorg; Seeco Research, Edina, MN).
EDTA Treatment and Reconstitution with Zinc of Wild Type Nspl2
[0186] To obtain metal-free (apo-) nspl2, the protein purified aerobically was treated with 10 mM EDTA for 1 h and passed through a PD G-25 column (GE Healthcare).
[0187] For full reconstitution with zinc, which yielded a protein containing 2 zinc ions per protom er, aerobically purified nspl2 was incubated with 5 mM dithiothreitol and 10 equivalents of ZnCF at room temperature for 2 h and subsequently passed through a PD G-25 column (GE Healthcare).
Quantification of Zinc Content of Aerobically Purified and Reconstituted Nspl2 [0188] The QuantiChrom™ Zinc Assay Kit (BioAssay Systems) was used for quantitative determination of zinc content in nspl2 purified aerobically and reconstituted with ZnCF to promote full occupancy with zinc of all the metal ligating sites.
Oxygen Consumption Rates (OCRs)
[0189] OCRs were examined using the XF96 Seahorse Metabolic Analyzer from Seahorse Biosciences. Briefly, HEK293 or VeroE6 cells were plated at a seeding density of 3 x 104 cells/well in 200 pL of complete media in an extracellular flux tissue culture plate. Cells were incubated with or without the indicated doses of TEMPOL for 24h. Prior to metabolic test the media was removed and replaced with Seahorse XF assay media (Agilent; Cat. No. 102365-100) containing 25 mM glucose, 2 mM glutamine, ImM sodium pyruvate and the cell plates were incubated for 60 min in a non-C02, 37 °C incubator. For mitochondrial stress test, 1.5 mM oligomycin was injected in port A, 1 mM FCCP fluoro- carbonyl cyanide phenylhydrazone in port B, and 0.5 pM rotenone/1 pM antimycin A in port C. OCR measurements were normalized against the cell number using the Cytation 1 Cell Imaging Multi-Mode Reader (Biotek).
Antibodies
[0190] Antibodies in this study were as following: anti-HSC20 westerns were performed either with a custom-made antibody (Genscript) or with a commercial antibody (Sigma #HPA018447). Anti-CIAOl (sc-374498), NFS1 (sc-81107) and DPYD (sc-376681) were from Santa Cruz Biotechnology. Anti-nspl3 (NBP2-89168) was from Novus Biologicals. Anti-HSPA9 (HPA000898) and a-tubulin (T9026) were from Sigma. Anti- FAM96B (20108-1-AP), MMS19 (16015-1-AP), TOM20 (11802-1-AP) and ISCU (14812-1- AP) were from Proteintech. Anti-FLAG antibody was from Origene (TA50011). Mouse monoclonal antibody to TFR1 was from ThermoFisher Scientific (13-6800). Anti-Strep II (ab 184224), ferritin heavy chain (ab65080), NDUFSl (abl69540), UQCRC1 (abl97055), MTCOl (abl4705) and SDHB (abl4714) were from abeam.
Statistical Analyses
[0191] Where applicable, pairwise comparisons between two groups were analyzed using the two-tailed unpaired Student’s t test. Significance for multi-group comparisons were analyzed with two-way ANOVA followed by Sidak’s multiple comparisons test. All tests were performed with GraphPad Prism 7, and data were expressed as mean ± 95% confidence interval (Cl) except where indicated otherwise.
Densitometry of Band Intensities
[0192] Where applicable, quantification of band intensities was performed using ImageJ and Chemidoc™ Image Lab Software (Bio-Rad).
SARS-CoV-2 virus stocks
[0193] The SARS-CoV-2 USA-WA1/2020 isolate, originally deposited by the CDC and obtained through BEI Resources (NIAID, NIH), was propagated in Vero cells (ATCC CCL81). Vero E6 cells were inoculated at a multiplicity of infection (moi) of 0.01 in DMEM supplemented with GlutaMAX, sodium pyruvate, 2% heat-inactivated fetal bovine serum,
100 El/ml Penicillin, 100 pg/ml Streptomycin (D2 medium) and cultivated at 37oC at 5% C02. After 48 hours, cell culture supernatant was collected, clarified by centrifugation at 4,000 x g for 10 minutes at 4 °C, aliquoted and frozen at -80 °C. Virus titer was measured in a TCID50 assay. Full genome sequencing was performed to assess genomic integrity of the viral stock.
Viral inhibition assay
[0194] Vero E6 cells for SARS-CoV-2 infection studies were maintained in complete DMEM supplemented with GlutaMAX, sodium pyruvate, 10% heat-inactivated fetal bovine serum, 100 U/ml Penicillin, 100 pg/ml Streptomycin (D10 media). The day before infection, cells were plated at a density of 2x 105 cells per well in 24-well plates (Coming) supplemented with increasing concentrations of TEMPOL (range 0.1 pM to lOOOpM). DMSO was used as a solvent and included as the negative control. The next day, plates were washed twice with D2 media. Cells were infected at an moi of 0.1 or 0.01 with SARS-CoV-2 in 400pl. After lh of absorption at 37oC, the inoculum was removed and cells were washed twice with D2 medium, and then cultivated at 37oC and 5% C02 in D2 medium supplemented with TEMPOL at the same concentration used prior to infection. Cultures were monitored daily for the appearance of cytopathic effect (CPE) using an EVOS 5000 digital microscope. After 48h, culture supernatants were collected, and vims titers were determined by TCID50 assay using Vero E6 cells. Vims titers were calculated using the method of Reed and Muench.
Cytotoxicity assay
[0195] Viability of Vero E6 cells treated with increasing concentrations of TEMPOL up to 5 mM was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat. No. G7570), according to the manufacturer’s instructions. As a control, cell viability upon treatment with DMSO alone, used to dissolve TEMPOL, was also determined. EXAMPLE 1
[0196] This example describes the analysis of the primary sequence of SARS-CoV-2 proteins.
[0197] The primary sequences of SARS-CoV-2 proteins were analyzed to investigate whether any of the proteins might incorporate Fe-S clusters. The presence of Fe-S cofactors in candidate proteins can be predicted based on the identification of specific amino acid sequence motifs. The analysis identified two highly conserved LYR (leucine-arginine- tyrosine)-like motifs (Fig. 6A) in the nspl2 subunit of RdRp, which are potential binding sites for the cochaperone HSC20 (aka HSCB) of the Fe-S biogenesis machinery, which facilitates Fe-S cluster transfer from the main scaffold protein, ISCU, to recipient proteins (Fig. 6B).
EXAMPLE 2
[0198] This example describes assays investigating the structure and function of nspl2. [0199] To assess whether the LYR-like motifs were involved in direct binding of nspl2 to HSC20, incubated full-length SARS-CoV-2 nspl2 wild type or variants where incubated wherein either or both LYR motifs were replaced by alanines (Fig. 6C) with purified HSC20. Nspl2 wild type (WT) bound HSC20, indicating that the RdRp subunit interacts directly with the cochaperone (Fig. 1 A). Substitution of either of the two LYR motifs with alanines decreased the amount of bound HSC20 (Fig. 1 A), which was even more profoundly diminished by loss of both motifs in nspl2VYR/LYR-AAA (Fig.lA). Co- immunoprecipitation (co-IP) experiments in Vero E6 cells and mass spectrometry analysis confirmed that nspl2 transiently interacted with HSC20 and with components of the de novo Fe-S cluster (the chaperone HSPA9, the cysteine desulfurase NFS1, the main scaffold ISCU) and cytoplasmic Fe-S (CIA) biogenesis (CIAOl, MMS19, FAM96B) machineries (Figs. IB, 1C, 6D and Table SI), suggesting that these interactions may be required for Fe-S cluster acquisition by nspl2. To investigate whether nspl2 coordinated a Fe-S cluster, 55Fe incorporation into the expressed protein was quantified in cells transfected with either a pool of nontargeting siRNAs (NT) or with si-RNAs against the initial Fe-S biogenesis scaffold, ISCU. In control cells (NT si-RNAs), nspl2 WT bound radiolabeled iron (8312±775 cpm/mg of cytosolic proteins, Figs. 1D-E), whereas nspl2 that lacked the LYR motifs, did not interact with HSC20 and bound significantly less iron (250±92 cpm/mg of cytosolic proteins, Figs. 1D-E). Nspl2 expressed in cells silenced for ISCU (si-ISCU) failed to incorporate iron (Figs. 1D-E).
[0200] These results demonstrate that nspl2 binds iron, likely in the form of a Fe-S cluster. Nspl2 expressed in Expi293F mammalian cells and purified anoxically exhibited a shoulder at -420 nm in its UV-vis absorption spectrum (Figs. 2A-B, 7A-B), suggesting that it harbored one or more Fe-S cluster. To determine the type and stoichiometry of Fe-S cluster(s), a 57Fe-enriched nspl2-FLAG sample was analyzed by Mossbauer spectroscopy (Fig. 2C). The 4.2-K Mossbauer spectrum collected in a 53-mT magnetic field applied parallel to the direction of g radiation (Fig. 2C) shows the presence of a single quadrupole doublet with parameters typical of [Fe4S4]2+ clusters [isomer shift (5) of 0.44 mm/s and quadrupole splitting parameter (AEQ) of 1.25 mm/s, blue line]. Wild type nspl2 bound 7.5 ± 0.35 iron atoms per monomer, and thus, the Mossbauer spectrum may be interpreted as two [Fe4-S4]2+ clusters. The X-band EPR spectrum, recorded at 20 K, showed no signal (Fig. 7C), ruling out the presence of Fe-S clusters with a half-integer spin ground state. However, upon reduction with dithionite, EPR signal characteristics of [Fe4S4]+ clusters were observed (Fig. 7D). Notably, the nspl2-nsp7-nsp8 complex anoxically purified with the Fe-S cluster(s) showed markedly increased binding to the template/RNA primer (Figs. 8A-E) and increased polymerase activity relative to the aerobically purified complex that contained two zinc ions per protomer (Figs. 2D, 8A-E). Co-expression of nspl2, nsp7 and nsp8 in Expi293F cells yielded the RdRp complex in its active form, used in the primer extension assays, that exhibited the same spectroscopic features typical of [Fe4-S4] clusters that were observed for nspl2 (Fig. 8D).
[0201] The previously available cryo-EM structures of the RdRp complex have assigned two chelated zinc ions in the highly conserved metal binding motifs of nspl2 composed of H295-C301-C306-C310 at the interface between the NiRAN (nidovirus RdRp- associated nucleotidyltransferase domain) and the catalytic domain and of C487-H642-C645- C646 in the fingers of the catalytic domain (Figs. 2E and 5). The hypothesis that it is actually two [Fe4-S4] clusters coordinated by these motifs was tested by replacing selected cysteines with serines and characterizing the variant nspl2 proteins. The two variants lacking any one of the set of three Cys residues of either the interfacial motif (nspl2C301S-C306S-C310S) or the catalytic domain (nspl2C487S-C645S-C646S) (replaced by Ser) contained 3.8 ± 0.21 and 3.67 ± 0.33 Fe/nspl2 protomer, respectively, and exhibited approximately half of the absorbance at 420 nm (Figs. 2A-B, 7A-B) and the 55Fe radiolabel seen for the WT nspl2 (Fig. 2F). The 4.2-K/53-mT Mossbauer spectra of these two variants revealed that -95% of Fe is associated with the quadrupole doublet with the same parameters deduced from the spectrum of WT nspl2, thus revealing the presence of one [Fe4S4]2+ cluster in the unmodified binding site (Fig. 2C). The 20 K, X-band EPR spectra of the variants after they were treated with sodium dithionite are also consistent with the presence of one [Fe4S4]2+ cluster (Figure 7D). A variant lacking a total of four cysteines from both motifs (nspl2C301S-C306S- C645S-C646S) did not bind Fe and had no absorbance at 420 nm, consistent with the notion that both [Fe4S4] cluster binding sites had been eliminated (Figs. 2A-B, 7A-B). The two [Fe4S4 ]2+ clusters incorporated in a mammalian over-expression system are thus ligated by cysteine residues located in the two zinc-binding sites identified in the cryo-EM structures.
EXAMPLE 3
[0202] This example describes the characterization of the function of the two Fe-S clusters in SARS-CoV2 RdRp.
[0203] Functional studies revealed that the [Fe4S4] cluster in the catalytic domain of nspl2 is required for the RNA polymerase activity of the nspl2-nsp7-nsp8 complex (Figs.
3 A-B), in addition to presumably maintaining structure. In fact, the absence of the cysteine ligands in the catalytic domain in the nspl2C487S-C645S-C646S variant caused a more profound decrease in the polymerase activity than was observed in the zinc complex (Fig.
3 A), suggesting that Zn, by coordinating the same cysteine residues, can partially fulfill the structural role of the Fe-S cluster, preserve the architecture of the fingers subdomain, and maintain some polymerase activity, which is strictly associated with the palm of the catalytic domain. Fe-S enzymes involved in DNA and RNA metabolism have often been mischaracterized as zinc-containing proteins, as Fe-S clusters readily undergo oxidative degradation during standard aerobic purification procedures of proteins, allowing zinc to coordinate the same cysteine residues. Moreover, zinc-containing enzymes have been shown to retain activity in vitro on short templates, which previously supported the conclusion that zinc was the physiological cofactor of these enzymes. Fe-S clusters in nucleic acid metabolism enzymes have not been thought to participate directly in catalysis, but rather in modulating binding of the enzyme to the template and/or to other components of the replication complex, as well as in increasing processivity and enabling repair through a proposed charge transfer mechanism. Consistent with the notion that zinc is likely not the physiological cofactor in several viral replicases that have so far been crystallized with chelated zinc ions, supplementation with zinc has been reported to inhibit replication in several cell culture models of viral infection. Loss of the [Fe4-S4] cluster ligated by H295- C301-C306-C310, which is located at the interface between the NiRAN and the catalytic domain of nspl2, had minimal effect on the RNA polymerase activity (Figs. 3A-B).
However, loss of this cluster profoundly diminished the interaction with the helicase nspl3 (Figs. 3B-C), which is an essential component of the replication complex.
EXAMPLE 4
[0204] This example describes the use of a nitroxide radical anti-viral to prevent coronavirus replication.
[0205] The sensitivity of Fe-S clusters to oxidative degradation was exploited to prevent coronavirus replication in cell culture models. RdRp isolated from Expi293F cells that had been treated with the nitroxide radical TEMPOL (Fig. 4A) had diminished absorbance at 420 nm relative to the complex isolated from untreated cells, indicative of loss of the Fe-S clusters of nspl2. Likewise, treatment with TEMPOL of the Fe-S cluster- containing protein in vitro caused loss of absorbance in the same region (Fig. 4B). Either treatment resulted in loss of polymerase activity (Figs. 4C-E). The TEMPOL treatment of cells did not impact the activities of several mitochondrial Fe-S enzymes, including the respiratory complexes and mitochondrial aconitase (AC02), and the cytosolic Fe-S enzyme, DP YD (Fig. 9A-F and 10A-F), nor it caused any cytotoxicity at doses up to 5 mM (Fig. 10G). TEMPOL treatment also did not affect the interactions of nspl2 with the components of the Fe-S and CIA biogenesis machinery from which nspl2 acquires its Fe-S clusters (Figs. 11 A- D). Accordingly, it may be inferred that TEMPOL directly reacts with Fe-S clusters in RdRp, leading to their degradation.
[0206] In support of this mechanism of action, DEA/NO, a nitric oxide donor that readily reacts with Fe-S clusters to form dinitrosyl complexes with diminished absorbance, also inhibited the RdRp (Figs. 4E, 12A-B), although less effectively than TEMPOL. TEMPOL was found to be both a more potent RdRp inhibitor (Fig. 13) and synergized with remdesivir (RDV) (Fig. 14), a nucleoside analog that has been used to target the replication of SARS-CoV-2. RDV was notably less effective against the Fe-S-RdRp than the zinc-RdRp (Fig. 15). EXAMPLE 5
[0207] This example describes the antiviral activity of TEMPOL against live SARS- CoV2 viral replication.
[0208] Having demonstrated a strong inhibitory effect of TEMPOL on the activity of the RdRp of SARS-CoV-2, it was investigated whether TEMPOL might exhibit antiviral activity against live virus replication. Vero E6 cells were infected with the SARS-CoV-2 USA-WA 1 /2020 isolate in the presence of increasing concentrations of TEMPOL (range 0.1 mM to 1 mM). TEMPOL exhibited a strong antiviral activity at concentrations above 0.2 mM. Viral titers were reduced by more than 5 loglO in the presence of 0.4 mM TEMPOL, which is reported to have a CC50 greater than 100 mM. Accordingly, these studies present a molecular basis for pursuing TEMPOL and other related nitroxide radicals as potential SARS-CoV-2 therapies during active viral infection.
EXAMPLE 6
[0209] This example demonstrates the activity of TEMPOL as an antiviral for SARS- CoV-2, SARS-CoV, and MERS-CoV. Briefly, the in vitro cellular antiviral activity of TEMPOL against three coronaviruses (SARS-CoV-2, SARS-CoV and MERS-CoV) was measured using cytopathic effect (CPE) assays using Vero 76 cells.
Methods
[0210] SARS coronavirus (strain: Urbani) was tested in Vero 76 cells (ATCC CRL- 1587™), with M128533 being used as a control drug at 0.1-100 pg/ml MERS coronavirus (strain: EMC) was tested in Vero 76 cells, with M128533 being used as a control drug at 0.1- 100 pg/ml. SARS-CoV-2 (strain: USA_WAl/2020) was tested in Vero 76 cells, with EIDD- 1931 being used as a control drug at 0.1-100 pg/ml. TEMPOL was dissolved in DMSO and tested in the concentration range of 25-1000 pM for SARS-CoV-2 and MERS-CoV and 0.1- 100 pM for SARS-CoV, with DMSO being used as a vehicle control. Visual inspection and neutral red assay were used to measure cytopathic effect and toxicity. EC50 and CC50 values were extracted from primary data. EC50 and CC50 results from visual inspection and neutral red assay were consistent. Results
[0211] The data demonstrate that TEMPOL demonstrated potent antiviral activity against SARS-CoV with EC50 values lower than 0.1 mM, and MERS-CoV with an EC50 of 73 mM, in their respective cellular assays. EC50 of control drugs on MERS-CoV, SARS-CoV and SARS-CoV-2 were 0.28 mM, 0.67 mM and 0.3 mM, respectively. The results are presented in the following table:
Figure imgf000047_0001
In this table, “EC50” refers to the compound concentration that reduces viral replication by 50%, “CC50” refers to the compound concentration that reduces cell viability by 50%, and “SI50” is calculated as CC50/EC50. Compounds with SI values >10 are considered active and merit further investigation.
EXAMPLE 7
[0212] This example demonstrates synergy in the combination of TEMPOL with an iron chelator on CARS-CoV-2 replicase. Briefly, the activity of the anoxically purified RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 was assessed in a primer extension assay in the presence of TEMPOL, deferiprone ("DFP”), or a combination of TEMPOL and deferiprone.
Methods
[0213] In vitro activity of SARS-CoV-2 RdRp was performed as previously described (Maio et al., “Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets.” Science 373 , 236-41 (2021), which is incorporated herein in its entirety). Briefly, all RNA oligonucleotides were purchased from Dharmacon (Horizon Discovery). RNA primers were radiolabeled at the 5' end with [y- ’2P]ATP (Perkin Elmer) using the T4 polynucleotide kinase from the KINASEMAX 5' END-LABELING KIT (Thermo Fisher Scientific). The primer/template duplex used in the RNA extension assay consisted of the 13-mer primer 5'-rArGrGrUrArArUrArArArArUrU-3' (SEQ ID NO: 16 and the 29-mer template 5'- rUrUrUrUrArArUrCrCrUrArArArCrGrArArArUrUrUrUrArUrUrArCrCrU-3 ' (SEQ ID NO: 17). (Note - these are the same sequences previously identified (see paragraph [0152]). [0214] To anneal the primer to its complementary template, oligonucleotides were mixed at equal molar ratios in annealing buffer (50 mM NaCl and 10 mM Na-HEPES pH 7.5), denatured by heating to 75°C for 5 min and then slowly cooled to 4°C.
[0215] The SARS-CoV-2 RdRp complex anoxically purified from Expi293F mammalian cells co-expressing 3xFLAG-nspl2 and nsp7/nsp8-Strep II at a final concentration of 1 mM was incubated with 3 mM dsRNA in the presence of 1.2 U/pl RNase inhibitor in reaction buffer containing in 100 mM NaCl, 20 mM Na-HEPES pH 7.5, 5% (v/v) glycerol, 10 mM MgCF and 0.5 mM TCEP, which were prepared in DEPC-treated water. Reactions were incubated at 37 °C for 15 min and the RNA extension was initiated by addition of NTPs (300 pM UTP, GTP and CTP, and 100 pM [y-32P]ATP (Perkin Elmer)).
The total reaction volume was 10 pi.
[0216] Reactions were stopped by the addition of 2x stop buffer (7 M urea, 50 mM EDTA pH 8.0, lx TBE buffer). Samples were digested with proteinase K (New England Biolabs) and RNA products were separated on 20% acrylamide gels in 1 c TBE buffer supplemented with 8M urea and imaged by autoradiography on CARESTREAM BIOMAX LIGHT films. The resulting images is presented at Figure 16.
[0217] The inhibitory effects on the activity of the RdRp of TEMPOL (Sigma, Cat. No. 176141) or DFP (Sigma, Cat. No. Y0001976) and the combination of TEMPOL and DFP in vitro at the indicated concentrations indicated were assessed on the anoxically purified enzyme complex at 1 pM.
[0218] Following gel electrophoresis, band intensities of the products of the primer extension assay (Elongated RNA) in control (CTRL), TEMPOL-, DFP-, and TEMPOL/DFP- treated samples were assessed by Image J and the results were plotted using Prism 9 (Figure 17). Multiple comparisons were performed using One-way Anova Sidak's multiple comparisons test. P values were as follows: CTRL vs TEMPOL 100 pM, p<0.0001; CTRL vs DFP 25 pM, p=0.0104; CTRL vs TEMPOL+DFP, pO.0001; TEMPOL vs TEMPOL+DFP, pO.OOl. Results
[0219] The results of the gel electrophoresis of the reaction mixtures representing anoxically purified RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 assessed in the primer extension assays in the presence of TEMPOL (TL), deferiprone (DFP), or a combination of TEMPOL and DFP are presented in Figure 16 (n= 4 biological replicates).
The reaction mix containing all components except the RdRp was also loaded on the gel (no RdRp lane) to show migration pattern of the labeled primer alone, along with the control mix (CTRL) in which DMSO, which was used to dissolve TEMPOL, was added. The plot of the quantification of the RdRp assay is presented in Figure 17.
[0220] Having demonstrated a strong inhibitory effect of TEMPOL on the activity of the RdRp of SARS-CoV-2, this Example studied whether a combination therapy of TEMPOL and the iron chelator deferiprone (DFP) might exhibit synergistic antiviral activity against the SARS-CoV-2 RdRp in a primer extension assay in vitro. The results, which are represented in Figures 16 and 17, reveal that a combination therapy of TEMPOL at 100 mM and the iron chelator deferiprone (DFP) at 25 pM indeed exhibits a synergistic inhibitory effect on the activity of the RdRp compared to TEMPOL alone at 100 pM or DFP alone at 25 pM.
[0221] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0222] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0223] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):
1. A method of treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject an effective amount of an anti -viral agent comprising a nitroxide radical, and the antiviral agent treats or prevents the coronavirus infection in the subject.
2. The method of claim 1, wherein (a) when the coronavirus is SARS-CoV-2, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) or (b) when the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol), the coronavirus is other than SARS-CoV-2.
3. The method of claim 2, wherein the nitroxide radical is 2, 2,6,6- tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l-piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo-Tempo), 4-(2- bromoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-ethoxyfluorophosphonyloxy- 2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-l- oxyl, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-mal eimido-2, 2,6,6- tetramethylpiperidine-l-oxyl; 4-(4-nitrobenzoyloxyl)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4- phosphonooxy-2,2,6,6-tetramethylpiperidine-l-oxyl, 2-ethyl-2,5,5-trimethyl-3-oxazolidine-l- oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-aminomethyl- Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-cyano-Proxyl); 3-Carbamoyl- 2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3 -Carbarn oyl-Proxyl); or 3-Carboxy-2, 2,5,5- tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy-Proxyl).
4. The method of claim 2, wherein the nitroxide radical is 4-hydroxy-2, 2,6,6- tetramethylpiperidine- 1 -oxyl (Tempol).
5. The method of any one of claims 1-4, wherein the coronavirus is SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63.
6. The method of claim 2 or 3, wherein the coronavirus is SARS-CoV2.
7. The method of claim 5 or 6, wherein the subject has not been tested for infection with the coronavirus.
8. The method of claim 5 or 6, wherein the subject has been exposed to the coronavirus.
9. The method of claim 5 or 6, wherein the anti-viral agent is administered prophylactically.
10. The method of claim 5 or 6, wherein the subject does not have acute respiratory distress syndrome (ARDS).
11. The method of claim 5 or 6, wherein the subject is asymptomatic.
12. The method of any one of claims 1-11, wherein the antiviral agent is administered orally, intravenously, subcutaneously, intradermally, via inhalation, or intramuscularly.
13. The method of claim 12, wherein the antiviral agent is administered orally.
14. The method of any one of claims 1-14, wherein the antiviral agent is administered in a dose sufficient to result in 200-400 micromolar concentration in tissues of the subject.
15. The method of any one of claims 1-14, wherein the antiviral agent is administered either once or twice a day.
16. The method of any one of claims 1-15, further comprising administering an iron chelator to the subject.
17. The method of claim 16, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
18. A composition for use in treating or preventing a coronavirus infection in a subject, the composition comprising an effective amount of an anti -viral agent comprising a nitroxide radical and a pharmaceutically acceptable carrier.
19. The composition for use according to claim 18, wherein (a) when the coronavirus is SARS-CoV-2, the nitroxide radical is other than 4-hydroxy-2,2,6,6-tetramethylpiperidine- 1-oxyl (Tempol) or (b) when the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine- 1-oxyl (Tempol), the coronavirus is other than SARS-CoV-2.
20. The composition for use according to claim 18 or 19, wherein the nitroxide radical is 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempo); 4-amino-2,2,6,6-tetramethyl-l- piperidinyloxy (Tempamine); and 4-oxo-2,2,6,6-tetramethyl-l-piperidinyloxy (4-oxo- Tempo), 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidine-l-oxyl; 4- ethoxyfluorophosphonyloxy-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(2-iodoacetamido)- 2,2,6,6-tetramethylpiperidine-l-oxyl, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-maleimido-2,2,6,6-tetramethylpiperidine-l-oxyl; 4-(4-nitrobenzoyloxyl)-2, 2,6,6- tetramethylpiperidine- 1-oxyl; 4-phosphonooxy-2,2,6,6-tetramethylpiperidine-l-oxyl, 2-ethyl- 2,5,5-trimethyl-3-oxazolidine-l-oxyl (Oxano); 3-aminomethyl-2,2,5,5-tetramethyl-l- pyrrolidinyl-N-oxyl (3-aminomethyl-Proxyl); 3-cyano-2,2,5,5-tetramethyl-l-pyrrolidinyl-N- oxyl (3-cyano-Proxyl); 3-Carbamoyl-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3- Carbamoyl-Proxyl); or 3-Carboxy-2,2,5,5-tetramethyl-l-pyrrolidinyl-N-oxyl (3-Carboxy- Proxyl).
21. The composition for use according to claim 18 or 19, wherein the nitroxide radical is 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol).
22. The composition for use according to any one of claims 18-21, wherein the coronavirus is SARS-CoV, MERS-CoV, human coronavirus HKU1, human coronavirus OC43, human coronavirus 229E, or human coronavirus NL63.
23. The composition for use according to any one of claims 19-20, wherein the coronavirus is SARS-CoV-2.
24. The composition for use according to claim 22 or 23, wherein the subject has not been tested for infection with the coronavirus.
25. The composition for use according to claim 22 or 23, wherein the subject has been exposed to the coronavirus.
26. The composition for use of claim 22 or 23, wherein the anti-viral agent is administered prophylactically.
27. The composition for use of claim 22 or 23, wherein the subject does not have acute respiratory distress syndrome (ARDS).
28. The composition for use of claim 22 or 23, wherein the subject is asymptomatic.
29. The composition for use of any one of claims 18-28, wherein the composition is formulated for administration orally, intravenously, subcutaneously, intradermally, via inhalation, or intramuscularly.
30. The composition for use of claim 29, wherein the antiviral agent is formulated for administration orally.
31. The composition for use of any one of claims 18-30, wherein the antiviral agent is formulated in a dose sufficient to result in a 200-400 micromolar concentration in tissues of the subject.
32. The composition for use of any one of claims 18-31, wherein the antiviral agent is formulated for administration either once or twice a day.
33. The composition for use of any one of claims 18-32, further comprising an iron chelator.
34. The composition for use of aspect 33, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
35. A method of treating or preventing a coronavirus infection in a subject, the method comprising administering to the subject a combination comprising an effective amount of 4-hydroxy-2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and an effective amount of an iron chelator, wherein the administration of the combination treats or prevents the coronavirus infection in the subject.
36. The method of claim 35, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
37. The method of claim 36, wherein the iron chelator comprises deferiprone.
38. The method of any one of claims 35-37, wherein the coronavirus is SARS-CoV-2.
39. One or more compositions for use in treating or preventing a coronavirus infection in a subject, the compositions comprising (a) an effective amount of 4-hydroxy - 2,2,6,6-tetramethylpiperidine-l-oxyl (Tempol) and a pharmaceutically acceptable carrier and (b) an effective amount of an iron chelator and a pharmaceutically acceptable carrier.
40. The one or more compositions for use of claim 39, wherein the iron chelator comprises deferiprone, deferasirox, desferrioxamine, or CN128.
41. The one or more compositions for use of claim 40, wherein the iron chelator comprises deferiprone.
42. The one or more compositions for use of any one of claims 39-41, wherein the coronavirus is SARS-CoV-2.
43. The one or more compositions for use of any one of claims 39-42, wherein the compositions comprise a single composition.
44. The composition of claim 43, which is suitable for oral administration.
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