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WO2022232161A1 - Inhibiteurs de nsp1 pour le traitement de sars-cov-2 - Google Patents

Inhibiteurs de nsp1 pour le traitement de sars-cov-2 Download PDF

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Publication number
WO2022232161A1
WO2022232161A1 PCT/US2022/026374 US2022026374W WO2022232161A1 WO 2022232161 A1 WO2022232161 A1 WO 2022232161A1 US 2022026374 W US2022026374 W US 2022026374W WO 2022232161 A1 WO2022232161 A1 WO 2022232161A1
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Prior art keywords
nspl
pharmaceutical composition
cov
sars
montelukast
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English (en)
Inventor
Hung-Teh Kao
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Sypherion LLC
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Sypherion LLC
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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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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

Definitions

  • SARS-CoV-2 is the vims causative for COVID-19 [1], which in most infected individuals begins as a respiratory illness, but is capable of infecting other organs and causing an array of clinical symptoms.
  • SARS-CoV-2 is a member of the beta-coronavirus family, which contain related viruses known to cause human disease, including SARS, MERS, bronchitis, and the common cold.
  • WHO World Health Organization
  • compositions comprising a compound listed in any one of Tables 1-8, or a derivative thereof, and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides pharmaceutical compositions comprising at least two compounds listed in any one of Tables 1-8 and a pharmaceutically acceptable carrier or diluent. In some embodiments, the present disclosure provides pharmaceutical compositions which comprises three or more, four or more, or five or more compounds listed in any one of Tables 1-8 and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides pharmaceutical compositions which comprises Conivaptan, Montelukast, Pazopanib, Ponatinib, Rilpivirine, Tirilazad, and/or a derivative thereof.
  • the pharmaceutical compositions comprise Conivaptan or a derivative thereof.
  • the pharmaceutical compositions comprise Montelukast or a derivative thereof.
  • the pharmaceutical compositions comprise Pazopanib or a derivative thereof.
  • the pharmaceutical compositions comprise Ponatinib or a derivative thereof.
  • the pharmaceutical compositions comprise Rilpivirine or a derivative thereof.
  • the pharmaceutical compositions comprise Tirilazad or a derivative thereof.
  • the pharmaceutical compositions comprise (i) Ponatinib and Rilpivirine; (ii) Ponatinib and Montelukast; (iii) Montelukast and Rilpivirine; (iv) Montelukast, Ponatinib, and Rilpivirine; (v) Montelukast and Tirilazad; (vi) Ponatinib and Tirilazad; (vii) Conivaptan, Montelukast, and Ponatinib; (viii) Tirilazad, Montelukast, and Ponatinib; or (ix) Pazopanib, Montelukast, and Ponatinib.
  • the pharmaceutical compositions comprises Ponatinib and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Ponatinib and Montelukast. In some embodiments, the pharmaceutical compositions comprises Montelukast and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Montelukast, Ponatinib, and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Montelukast and Tirilazad. In some embodiments, the pharmaceutical compositions comprises Ponatinib and Tirilazad. In some embodiments, the pharmaceutical compositions comprises Conivaptan, Montelukast, and Ponatinib. In some embodiments, the pharmaceutical compositions comprises Tirilazad, Montelukast, and Ponatinib. In some embodiments, the pharmaceutical compositions comprises Pazopanib, Montelukast, and Ponatinib.
  • the present disclosure provides pharmaceutical compositions which can be effective to achieve an additive effect of inhibiting Nspl to achieve a greater therapeutic effect. In some embodiments, the present disclosure provides pharmaceutical compositions which can be effective to achieve a synergistic effect of inhibiting Nspl to achieve a greater therapeutic effect. In some embodiments, the present disclosure provides pharmaceutical compositions, further comprising an additional therapeutic agent.
  • the present disclosure provides pharmaceutical compositions, wherein the compound specifically binds to a Nspl (Non-Structural Protein 1) molecule.
  • Nspl Non-Structural Protein 1
  • the present disclosure provides pharmaceutical compositions, wherein the compound specifically binds to the N-terminal domain of an Nspl molecule. In some embodiments, the compound specifically binds within residues 1-120 of the Nspl molecule. In some embodiments, the compound specifically binds to an RNA groove in the N-terminal domain of the Nspl molecule. In some embodiments, the present disclosure provides pharmaceutical compositions, wherein the compound specifically binds to the C- terminal domain of an Nspl molecule. In some embodiments, the compound specifically binds within residues 121-180 of the Nspl molecule. In some embodiments, the compound specifically binds to a helix-loop-helix region in the C-terminal domain of the Nspl molecule.
  • the present disclosure provides pharmaceutical compositions, wherein the at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more) compounds specifically binds to non-competing epitopes on the same or different Nspl molecules.
  • pharmaceutical compositions comprise at least three compounds that specifically bind to non-competing epitopes on the same or different Nspl molecules.
  • the present disclosure provides pharmaceutical compositions, wherein the at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more) compounds independently bind to non-competing epitopes on the same or different Nspl molecules.
  • pharmaceutical compositions comprise at least three compounds that independently bind to non-competing epitopes on the same or different Nspl molecules.
  • the present disclosure provides pharmaceutical compositions, wherein the at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more) compounds independently bind to the N-terminal domain and the C-terminal domain on the same or different Nspl molecules.
  • pharmaceutical compositions comprise at least three compounds that independently bind to the N-terminal domain and the C-terminal domain on the same or different Nspl molecules.
  • the present disclosure provides pharmaceutical compositions, which may be effective to inhibit the activity of the N-terminal domain and the C-terminal domain on the same or different Nspl molecules to achieve a synergistic effect of inhibiting Nspl to achieve a greater therapeutic effect.
  • the present disclosure provides pharmaceutical compositions, wherein the Nspl molecule is derived from a virus.
  • the present disclosure provides pharmaceutical compositions, wherein the virus is a coronavims, optionally, selected from a severe acute respiratory syndrome coronavims (SARS-CoV), a severe acute respiratory syndrome coronavims 2 (SARS-CoV-2), a Middle East respiratory syndrome coronavims (MERS-CoV), a human coronavims OC43 (HCoV-OC43), a human coronavims HKU1 (HCoV-HKUl), a human coronavims 229E (HCoV-229E), a human coronavims NL63 (HCoV-NL63), and variants thereof.
  • SARS-CoV severe acute respiratory syndrome coronavims
  • SARS-CoV-2 severe acute respiratory syndrome coronavims 2
  • MERS-CoV Middle East respiratory syndrome coronavims
  • HKU1 HoV-HKUl
  • HoV-229E human coronavims 229E
  • HCoV-NL63 human cor
  • the present disclosure provides pharmaceutical compositions, wherein the vims causes bronchitis and/or the common cold.
  • the present disclosure provides pharmaceutical compositions, which may be effective (i) to diminish the activity of Nspl in vivo , in vitro , and/or ex vivo ,
  • the present disclosure provides pharmaceutical compositions, wherein: (i) the compound has an EC 100 of between about 0.01 mM and about 100 pM; and/or (ii) the compound has a Safety Index of greater than about 5.
  • the compound may have an EC 100 of between about 0.01 pM and about 100 pM.
  • the compound may have an ECIOO of about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, about 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, about 0.09 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 60 pM, about 65 pM, about 70 pM
  • the compound may have a Safety Index of greater than about 5. In some embodiments, the compound may have a Safety Index of between about 5 and about 100. For example, the compound may have a Safety Index of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, or about 5000 or more.
  • the present disclosure provides pharmaceutical compositions, which can reverse Nspl toxicity to substantially the same extent as a null mutation in the Nspl gene itself, as determined by a cytopathic assay, optionally wherein the null mutation comprises N128S/K129E and/or K164A.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the viral infection is a coronavirus infection.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the coronavirus infection is an infection by a SARS-CoV-2 virus.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the subject has, or is at risk of having, COVID-19.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition is administered to the subject prior to onset of one or more manifestations of COVID-19.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition is administered to the subject after the subject exhibits one or more manifestations of COVID-19.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the method results in the amelioration of one or more manifestations of COVID-19.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition is administered by any suitable route, optionally, orally, intranasally, intravenously, intramuscularly, or subcutaneously.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition is administered before and/or after viral shedding is first detected in a sample from the subject.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the pharmaceutical composition is administered in combination with an additional therapeutic agent.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein, wherein the subject is human.
  • the present disclosure provides assays, which may be effective to identify a compound or combination of compounds that specifically bind to an Nspl molecule.
  • FIG. 1 depicts the secondary structure of Nspl (Non- Structural Protein 1) from SARS-CoV-2.
  • the N-terminal globular region consists of 7 beta-pleated sheets as shown. Juxtaposition of Beta- 1 with Beta-7 creates an RNA-binding groove that binds to the 5’ UTR of SARS-CoV-2 RNAs [9].
  • the N-globular region is connected via a disordered loop the C- domain, which is an alpha- loop-alpha (alpha-2 and alpha-3 helices as shown) [8].
  • the C- domain blocks the mRNA tunnel in human ribosomes to prevent host cell translation [2, 3, 12].
  • FIGS. 2A-2E depicts quantitation of the cytopathic effects of Nspl in H1299 cells.
  • H1299 cells were transfected with Nspl mRNA in 96-well plates.
  • FIG. 2A shows phase contrast images of non-transfected cells, and cells transfected with Tag-Red Fluorescent Protein (RFP) mRNA as a control or Nspl mRNA. Both RFP and Nspl mRNAs were flanked by viral UTRs, and cells were transfected under identical conditions on the same plate.
  • FIG. 2B shows control (non-transfected) and Nspl -transfected cells that were stained with Hoescht 33342 dye as a measure of cell attachment.
  • FIG. 2C shows Calcein-AM, as a measure of vitality or metabolic diversion.
  • FIG. 2E shows the Viability Index is the normalized product of quantitation using the latter three dyes. The images in FIG. 2E were pseudocolored blue (Hoescht), green (Calcein-AM) and red (TMRE).
  • FIG. 3 shows a schematic diagram depicting the secondary structure of Nspl represented as an unfolded chain (from N- to C-terminal) with numbered alpha helices (a), numbered beta sheets (b), and 3 io helices.
  • the C-terminal helix-loop-helix (oc2-oc3) is termed the ribosome gatekeeper [13] because of its role in blocking the RNA tunnel in the 40S ribosomal subunit.
  • the sequential order and beta sheet pairings were adapted from Semper et al [18] and Almeida et al [65].
  • the mutations depicted are A (L21M), B (D33R), C (L123S/R124E), D (N128S/K129E), and E(K164A).
  • FIG. 4 shows measurement of efficacy and relationship to Nspl mutants.
  • Nspl mRNA was also transfected into HI 299 cells in different contexts: wild-type Nspl flanked by the 5’UTR and 3’UTR corresponding to that of human alpha-globin mRNA (WT globin UTRs), or flanked by the viral UTRs corresponding to that of SARS-CoV-2 (WT Viral UTRs). All point mutations of Nspl (A through E, corresponding to the point mutations described in FIG.
  • FIGS. 5A-5B depicts the synergistic interactions among Ponatinib, Rilpivirine, and Montelukast. Combinations of drugs were applied to Nspl -transfected H1299 cells, and Efficacies were determined. The Efficacy at the indicated concentrations of each drug were used to visualize synergy using an online tool [35]. Areas demarcated in red represent synergistic combinations as shown in the scale quantitated by the ZIP method [35, 36].
  • FIG. 5A shows data pooled from triplicate experiments to generate the Efficacy table and Synergy plot of Ponatinib and Rilpivirine.
  • FIG. 5A shows data pooled from triplicate experiments to generate the Efficacy table and Synergy plot of Ponatinib and Rilpivirine.
  • 5B shows Efficacy table and Synergy plot between a combination of Ponatinib+Rilpivirine at a molar ratio of 2.5 to 1, and Montelukast.
  • the concentration for the Ponatinib+Rilpivirine combination reflects that of Ponatinib only.
  • FIGS. 7A-7B depicts dose-response of a Montelukast-Ponatinib-Rilpivirine (MPR) combination.
  • FIG. 7A shows varying concentrations of MPR were added to Nspl -transfected H1299 cells and Efficacy determined and depicted. Cytotoxicity using the Viability Index in non- transfected H1299 were determined at various concentrations of MPR and depicted on the same graph.
  • FIG. 1 Montelukast-Ponatinib-Rilpivirine
  • FIG. 7B shows lx MPR was applied to H1299 cells transfected with wild-type (WT) Nspl or the indicated Nspl mutations (Mut A-C). Error bars represent ⁇ SEM. Statistical significance of treatment with lxMPR is indicated (n>5; p-value from a two-sided t-test).
  • FIGS. 8A-8C depicts synergistic interactions of potentially promising drug pairs.
  • Serial dilutions of the indicated drugs were applied to Nspl -transfected HI 299 cells in 96- well plates, in a 2x2 matrix, and Efficacies were determined.
  • the Efficacy at the indicated concentrations of each drug were inputted into SynergyFinder 2.0, an online tool for visualizing synergy [35].
  • Synergy is quantitated by the ZIP method [35, 36] and visualized on a red-green scale as indicated. Efficacy tables and Synergy plots are shown for each of the following drug pairs: (FIG.
  • FIG. 8A Montelukast versus Ponatinib (data were averaged or consolidated from 10 replicates);
  • FIG. 8B Montelukast versus Tirilazad (data were averaged or consolidated from 3 replicates); and
  • FIG. 8C Tirilazad versus Ponatinib (data were averaged or consolidated from 3 replicates).
  • FIGS. 9A-9C depicts synergistic interactions between selected drugs and Montelulast+Ponatinib (fixed molar ratio).
  • Serial dilutions of the indicated drugs and Montelukast+Ponatinib or MP (molar ratios fixed at 10:1) were applied to Nspl -transfected H1299 cells in 96-well plates, in a 2x2 matrix, and Efficacies was determined.
  • the concentration indicated for MP reflects that of Montelukast. Efficacy at the indicated concentrations of each drug were inputted into SynergyFinder 2.0, and synergy was quantitated and visualized as in FIGS. 8A-8C.
  • FIG. 9A Conivaptan versus MP
  • FIG. 9B Tirilazad versus MP
  • FIG. 9C Pazopanib versus MP.
  • the present invention provides compounds that specifically bind to a Nspl (Non- Stmctural Protein 1) molecule and methods of use to treat subjects having or at risk of having a viral, e.g., a coronavims, infection.
  • Nspl Non- Stmctural Protein 1
  • the present invention provides pharmaceutical compositions comprising compound(s) that specifically bind to a Nspl molecule derived from a severe acute respiratory syndrome coronavims 2 (SARS-CoV-2) and methods of use thereof to treat subjects having or at risk of having a SARS-CoV-2 infection (i.e ., COVID-19).
  • SARS-CoV-2 severe acute respiratory syndrome coronavims 2
  • the present invention provides a rapid multiplexed assay for detecting the action of Nspl in vitro (e.g., in cultured lung cells) and for identifying compounds that act as inhibitors of Nspl.
  • the present invention provides compounds that can be used to treat COVID-19.
  • the present invention provides compositions comprising synergistic combinations of compounds that significantly inhibit Nspl action.
  • the present invention provides compositions comprising Ponatinib, Rilpivirine, and/or Montelukast, which together, may reverse the toxic effects of Nspl, for example, to the same extent as null mutations in the Nspl gene.
  • compositions comprising inhibitors of Nspl (Non- Structural Protein 1) as well as methods for treating or preventing a viral infection in subjects, e.g., subjects susceptible to or diagnosed with a coronavims, e.g., SARS-CoV, e.g., SARS-CoV-2 infection (i.e., COVID-19).
  • a coronavims e.g., SARS-CoV, e.g., SARS-CoV-2 infection (i.e., COVID-19).
  • Additional embodiments contemplated by the invention include, without limitation, include:
  • a combination of a drug that inhibits the N-domain plus a drug that inhibits the C- domain has a synergistic effect by furthering the inhibition of Nspl, providing greater therapeutic benefit.
  • the assay described herein provides a useful index of the potential therapeutic effectiveness of a drug or drug combination.
  • the drugs in Tables 1-8 and related compounds could potentially be used to prevent COVID-19 in an endemic geographic region, or to prevent deleterious symptoms in individuals exposed to the virus.
  • the drugs indicated in Tables 1-8 have potential benefits in related human coronaviral diseases, such as MERS, bronchitis, and the common cold.
  • the drugs listed in Tables 1-8 may possess synergistic actions to drugs in development whose intended purpose is to treat COVID-19.
  • the drugs listed in Tables 1-8 could enhance the therapeutic effectiveness of protease inhibitors, polymerase inhibitors, or anti-helicase compounds that are in development as antiviral treatments for COVID-19. Accordingly, such drugs may constitute part of a cocktail for aggressive treatment of COVID-19.
  • an element means one element or more than one element, e.g., a plurality of elements.
  • nuclear number As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.
  • coronavirus refers to a group of highly diverse, enveloped, positive-sense, single- stranded RNA viruses that cause respiratory, enteric, hepatic and neurological diseases of varying severity in a broad range of animal species, including humans. Coronaviruses are subdivided into four genera: Alphacoronavirus, Betacoronavirus (13CoV), Gammacoronavirus and Deltacoronavirus .
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV represents the prototype of a new lineage of coronaviruses capable of causing outbreaks of clinically significant and frequently fatal human disease.
  • the complete genome of SARS-CoV has been identified, as well as common variants thereof.
  • the genome of SARS-CoV is a 29,727-nucleotide polyadenylated RNA, has 11 open reading frames, and 41% of the residues are G or C.
  • the genomic organization is typical of coronaviruses, with the characteristic gene order (5'-replicase (rep), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3' and short untranslated regions at both termini.
  • the SARS-CoV rep gene which comprises about two-thirds of the genome, is predicted to encode two polyproteins that undergo co-translational proteolytic processing.
  • ORFs open reading frames downstream of rep that are predicted to encode the structural proteins, S, E, M and N.
  • the hemagglutinin-esterase gene which is present between ORFlb and S in group 2 and some group 3 coronaviruses was not found.
  • SARS- CoV-2 severe acute respiratory syndrome coronavirus 2
  • 2019-nCoV the terms “severe acute respiratory syndrome coronavirus 2,” “SARS- CoV-2,” “2019-nCoV,” refer to the novel coronavirus that caused a pneumonia outbreak first reported in Wuhan, China in December 2019 (“COVID-19”). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that SARS-CoV-2 was most closely related (89.1% nucleotide similarity similarity) to SARS-CoV.
  • terapéuticaally effective amount is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
  • treatment e.g., a coronavirus, e.g., SARS-CoV, e.g., SARS-CoV-2, infection is meant delaying or preventing the onset of such a disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or stopping the progression, aggravation or deterioration, the progression or severity of a condition associated with such a disease or disorder.
  • a coronavirus e.g., SARS-CoV, e.g., SARS-CoV-2
  • the symptoms of a disease or disorder, or pain and distress associated with an infection are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the transmission of a coronavirus infection is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • a “subject” means a human or an animal.
  • the animal may be a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, sheep, pigs, goats, birds, horses, pigs, deer, bison, buffalo, amphibians, reptiles, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is an embryo or a fetus, where a life-long protection is elicited after vaccination with the present invention.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • a primate e.g., a human.
  • patient and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, pig, sheep, goat, bird, reptile, amphibian, fish or cow. Mammals other than humans can be advantageously used as subjects that represent animal models of infectious diseases, or other related pathologies.
  • a subject can be male or female.
  • the subject can be an adult, an adolescent or a child.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a risk for developing a coronavirus, e.g., SARS-CoV, e.g., SARS-CoV-2, infection.
  • the subject is a human, such as a human being treated or assessed for a coronavirus, e.g., SARS-CoV, e.g., SARS-CoV-2, infection; a human at risk for a coronavirus, e.g., SARS-CoV, e.g., SARS-CoV-2, infection; a human having a coronavirus, e.g., SARS-CoV, e.g., SARS-CoV-2, infection.
  • the subject is a female human.
  • the subject is a male human.
  • the subject is an adult subject.
  • the subject is a pediatric subject.
  • vaccine includes any composition containing an immunogenic determinant which stimulates the immune system such that it can better respond to subsequent infections.
  • a vaccine usually contains an immunogenic determinant, e.g., an antigen, and an adjuvant, the adjuvant serving to non- specifically enhance the immune response to that immunogenic determinant.
  • an immunogenic determinant e.g., an antigen
  • an adjuvant the adjuvant serving to non- specifically enhance the immune response to that immunogenic determinant.
  • Currently produced vaccines predominantly activate the humoral immune system, i.e., the antibody dependent immune response.
  • Other vaccines focus on activating the cell-mediated immune system including cytotoxic T lymphocytes which are capable of killing targeted pathogens.
  • adjuvant refers to compounds that can be added to vaccines to stimulate immune responses against antigens.
  • Adjuvants may enhance the immunogenicity of highly purified or recombinant antigens.
  • Adjuvants may reduce the amount of antigen or the number of immunizations needed to protective immunity.
  • adjuvants may activate antibody- secreting B cells to produce a higher amount of antibodies.
  • adjuvants can act as a depot for an antigen, present the antigen over a longer period of time, which could help maximize the immune response and provide a longer-lasting protection.
  • Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells, for example, by activating T cells instead of antibody- secreting B cells depending on the purpose of the vaccine.
  • additive effect means that the activity of the combination of compounds is about equal to the sum of the individual activities of the compound in the combination when the activity of each compound is determined individually.
  • synergy or “synergistic” encompasses a more than additive effect of a combination of two or more agents compared to their individual effects.
  • synergy or synergistic effect refers to an advantageous effect of using two or more agents in combination, e.g., in a pharmaceutical composition, or in a method of treatment.
  • one or more advantageous effects is achieved by using one or more compounds that specifically binds to a Nspl (Non-Structural Protein 1) molecule in combination with a second therapeutic agent as described herein.
  • Nspl Non-Structural Protein 1
  • Nspl Non-Structural Protein 1
  • Nspl (Non-Structural Protein 1) is the first protein generated from the SARS-CoV-2 genome upon infection of human cells and is a major pathogenicity factor [7-9]. This protein plays a pivotal role in subverting the host cell’s translation machinery, by shutting down all host cell translation while allowing viral translation to occur [2-4]. Nspl has the distinction of being the only protein out of 30 proteins expressed from SARS-CoV-2 that is lethal to cells [5]. Previous studies in other beta-coronaviruses indicate that deletion of Nspl renders the virus nonpathogenic to the host [6], highlighting the importance of this protein in the life cycle of the virus.
  • Nspl is fundamentally an RNA binding protein that targets the 40S ribosomal subunit and the first stem-loop of the 5‘UTR of viral RNAs [8, 9]. Nspl also possesses significant protein-protein interactions that recruit translation factors and inhibit the export of mRNA from the nucleus [10]. Collectively, the outcome of these actions is the global inhibition of host cell mRNA translation while viral mRNA translation remains intact [7-13].
  • Nspl In addition to the key role played by Nspl during the initial stages of the viral life cycle, Nspl is the only protein expressed from the SARS-CoV-2 genome that leads to significant cell death [7]. The mechanism of cell death is apoptosis and occurs over the course of days after the expression of Nspl [7]. The inhibition of cell protein synthesis likely contributes to apoptosis as well as the severe respiratory symptoms associated with COVID- 19. Thus, therapeutics targeting Nspl could mitigate the symptoms of severe COVID-19 as well as slow the progression of the viral life cycle.
  • Nspl Another advantage to targeting Nspl is that the sequence of this protein has remained unaltered in all current SARS-CoV-2 variants of concern, including the alpha, beta, delta, epsilon, mu, and omicron variants. Therapeutics targeting Nspl may therefore target all such variants. Accordingly, targeting Nspl may have therapeutic benefits for patients stricken with COVID-19.
  • the 3-dimensional structure of Nspl has been solved using X-Ray crystallography and cryo-electron microscopy [2, 3, 7, 8].
  • the structure-function relation has also been deduced by comparison to SARS-CoV and by experimental methods.
  • the protein is 180 amino acids long and folds into an N-terminal globular domain, connected by a disordered loop to an alpha-loop-alpha region at the C-terminal domain (FIG. 1, FIG. 3).
  • the N-domain binds to the 5’UTR of viral RNAs via a groove created by juxtaposition of the first and last beta sheet of this region [9].
  • compositions and therapeutic formulations comprising any of the exemplary compounds described herein, for example, as listed in any one of Tables 1-8, in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof.
  • the present disclosure provides pharmaceutical compositions comprising a compound listed in any one of Tables 1-8, or a derivative thereof, and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides pharmaceutical compositions comprising at least two compounds (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more) listed in any one of Tables 1-8 and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides pharmaceutical compositions which comprises three or more, four or more, or five or more (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or more) compounds listed in any one of Tables 1-8 and a pharmaceutically acceptable carrier or diluent.
  • the present disclosure provides pharmaceutical compositions which comprises Conivaptan, Montelukast, Pazopanib, Ponatinib, Rilpivirine, Tirilazad, and/or a derivative thereof.
  • the pharmaceutical compositions comprise Conivaptan or a derivative thereof.
  • the pharmaceutical compositions comprise Montelukast or a derivative thereof.
  • the pharmaceutical compositions comprise Pazopanib or a derivative thereof.
  • the pharmaceutical compositions comprise Ponatinib or a derivative thereof.
  • the pharmaceutical compositions comprise Rilpivirine or a derivative thereof.
  • the pharmaceutical compositions comprise Tirilazad or a derivative thereof.
  • the pharmaceutical compositions comprise (i) Ponatinib and Rilpivirine; (ii) Ponatinib and Montelukast; (iii) Montelukast and Rilpivirine; (iv) Montelukast, Ponatinib, and Rilpivirine; (v) Montelukast and Tirilazad; (vi) Ponatinib and Tirilazad; (vii) Conivaptan, Montelukast, and Ponatinib; (viii) Tirilazad, Montelukast, and Ponatinib; or (ix) Pazopanib, Montelukast, and Ponatinib.
  • the pharmaceutical compositions comprises Ponatinib and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Ponatinib and Montelukast. In some embodiments, the pharmaceutical compositions comprises Montelukast and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Montelukast, Ponatinib, and Rilpivirine. In some embodiments, the pharmaceutical compositions comprises Montelukast and Tirilazad. In some embodiments, the pharmaceutical compositions comprises Ponatinib and Tirilazad. In some embodiments, the pharmaceutical compositions comprises Conivaptan, Montelukast, and Ponatinib. In some embodiments, the pharmaceutical compositions comprises Tirilazad, Montelukast, and Ponatinib. In some embodiments, the pharmaceutical compositions comprises Pazopanib, Montelukast, and Ponatinib.
  • Exemplary additional therapeutic agents include any therapeutic agents that may be used for the treatment of any disorders described herein.
  • the additional therapeutic agents may be used for the treatment of a coronavirus, e.g., SARS-CoV-2, infection in a subject.
  • the additional therapeutic agents may be used for the treatment of COVID-19.
  • the additional therapeutic agent(s) may be administered prior to, concurrent with, or after the administration of a composition of the present invention.
  • the present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising a one or more of the compounds and/or compositions described herein.
  • the therapeutic composition can comprise any of the compounds as disclosed herein and a pharmaceutically acceptable carrier or diluent.
  • a subject in need thereof means a human or non-human animal that exhibits one or more symptoms or indicia of a Nspl associated disorder or disease, and/or who otherwise would benefit from a decrease in Nspl activity.
  • the compounds of the invention (and therapeutic compositions comprising the same) are useful, inter alia, for treating any disease or disorder in which inhibition of Nspl is beneficial.
  • the present invention provides methods for treat various Nspl associated diseases.
  • Nspl associated disease is a disease or disorder that is caused by, or associated with, Nspl protein production or Nspl protein activity.
  • the term “Nspl associated disease” includes a disease, disorder or condition that would benefit from a decrease in Nspl protein activity.
  • Non-limiting examples of Nspl associated disease include, for example, a viral infection. Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.
  • the present disclosure provides methods of treating or preventing a viral infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described herein.
  • the viral infection may be a coronavirus infection. More particularly, the coronavirus infection may be an infection by a SARS-CoV-2 virus or variant thereof. Such method may result in the amelioration of one or more manifestations of COVID-19.
  • a pharmaceutical composition may be administered to the subject prior to onset of one or more manifestations of COVID-19. In some embodiments, a pharmaceutical composition may be administered to the subject after the subject exhibits one or more manifestations of COVID-19. In some embodiments, a pharmaceutical composition may be administered to the subject before and/or after viral shedding is first detected in a sample from the subject.
  • the pharmaceutical composition may be administered by any suitable route, including, for example, orally, intranasally, intravenously, intramuscularly, or subcutaneously.
  • compositions according to the methods of the invention may result in a reduction of the severity, signs, symptoms, or markers of a Nspl associated disease or disorder in a subject with a Nspl associated disease or disorder.
  • reduction in this context is meant a statistically significant decrease in such level.
  • the reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay used.
  • kits may further include reagents or instructions for using the compounds and/or compositions in a subject. It may also include one or more buffers.
  • AutoDock Vina was used to screen potential compounds that bind to the N-domain using the ZINC 15 database (Tables 2-3), and the eDmg3D compendium (Table 4). AutoDock Vina was also used to screen potential compounds that bind to the C-domain using 3 available structures, 6zlw [3], 6zok [2], 7k5i [12]. The list of C-domain binding candidates is shown in Table 5. Other compounds of theoretical interest (derived from literature searches) is shown in Table 6.
  • Nspl Non-Structural Protein 1 activity
  • SARS-CoV SARS-CoV
  • Nspl Non-Structural Protein 1 activity
  • Other assays depended on the ability of Nspl to inhibit translation in a cell-free system [2, 3]. These assays are cumbersome and not readily amenable for screening potential drugs that have an inhibitory effect on Nspl function.
  • mRNA encoding Nspl was transfected into H1299 at 75%-80% confluence in 96- well plates. Under these conditions, almost 100% of the cells are transfected, and expression of the protein occurs in 4-6 hours. This is contrast to the typical DNA transfection protocol used by most laboratories, which is generally inefficient in lung cells.
  • the mRNA contains the 5’UTR and 3’UTR elements of the SARS-Cov-2 genomic RNA. Inclusion of these elements in the mRNA significantly enhanced the toxicity of Nspl.
  • a compound, polyTC, which is double-stranded RNA, is included in the cell culture experiment.
  • PolyTC binds to the TL3 receptor, which potently induces interferon production [19].
  • PolyTC mimics infection by an RNA virus, and prompts cells to launch a robust innate cellular defense [19, 20].
  • Nspl blocks this defense from actually occurring by shutting down host cell translation. When this occurs, cells respond by committing suicide, that is, programmed cell death or apoptosis. It is thought that this curious response evolved to prevent the dissemination of virus [21], but it is also deleterious as observed during the clinical course of the disease.
  • This assay essentially magnifies the lethality of Nspl induction by simulating an infection with high viral load in cell culture.
  • Nspl is the only viral gene product that significantly promotes apoptosis in lung cells during COVID-19 infection [7]. This experiment exploited this property to design a cytopathic assay to quantitate the deleterious effects of Nspl.
  • a synthetic gene encoding Nspl was constructed using sequences obtained from the original SARS-CoV-2 strain [14]. Capped mRNA was transcribed from the Nspl synthetic gene for expression in cultured cells. mRNA transfection was used to transiently express Nspl in cultured adherent cells, in order to simulate the conditions of viral infection and to ensure rapid expression of Nspl in the majority of cells. The assay was conducted in H1299, a lung-derived adenocarcinoma cell line previously used in COVID-19 research [7, 15].
  • the three measurements of cell health are quantitated by fluorescent dyes with distinct excitation/emission spectra, permitting the simultaneous capture of multiplexed data (Fig. lb-e). Images of transfected cultured cells reveal that the 3 independent measures of cell health are not uniform throughout the population (FIGS. 2B-2E). Thus, combining the quantities should reduce the variability. Accordingly, this experiment defined a measure that incorporates all three quantities, which was termed the “Viability Index”.
  • the Viability Index is the product of all three measurements, normalized to 100 for healthy, non- transfected cells. As shown in FIG. 2E, the Viability Index shows a robust difference between Nspl -transfected cells and healthy non-transfected cells, with minimal quantitative variability.
  • Nspl is 180 amino acids long and folds into an N-terminal globular domain that contains 7 beta sheets (residues 1-120), connected by an unstructured linker to a helix-loop-helix region in the C-terminal domain (residues 121-180).
  • the globular N-domain contains an RNA groove that accommodates the 5’UTR of viral RNAs [9].
  • the RNA groove is created by juxtaposition of the first and last beta sheet of this region [9] (Fig. 2), and mutations within the groove almost always diminish the action of Nspl [19], strongly suggesting that the RNA-binding groove in the N-domain is a functional site.
  • the C-terminal helix-loop-helix domain fits into the RNA tunnel of ribosomes, thereby blocking host cell translation [8]. Mutations affecting this domain also attenuate the activity of Nspl [19, 20]. This region has been termed the “ribosome gatekeeper” because it binds to ribosomes and promotes translation of viral mRNA [13].
  • At least two functional sites are defined by mutational analysis within Nspl: the RNA groove in the N-domain and the C-terminal helix-loop-helix. Both sites are potential targets for drug development.
  • the Viability Index Enables Quantitative Evaluation of Nspl Context and Mutations. To validate the utility of the Viability Index as a quantitative measure of Nspl activity, this experiment measured the Viability Index of Nspl mRNA flanked by different untranslated regions, and Nspl mRNA containing different point mutations.
  • Nspl The context of the Nspl coding sequence impacts its toxicity.
  • Nspl is transcribed from mRNA consisting of the coding region flanked by the 5’UTR (untranslated region) and 3’UTR of the SARS-CoV-2 genomic RNA [21]. These UTRs are present in all viral mRNAs [21].
  • Nspl mRNA flanked by viral UTRs had greater toxicity compared to Nspl in which the UTRs are replaced with those corresponding to the human alpha- globin gene (Fig. 2a). This is consistent with the finding that Nspl binds to viral 5’UTRs and enhances the translation of the coding sequence [9].
  • Nspl also causes the degradation of host cell mRNAs through non-recognition of the 5’UTR [22, 23]. This likely explains why Nspl flanked by globin UTRs is less toxic that Nspl flanked by viral UTRs.
  • Point mutations within the Nspl coding region had either no effect, loss-of- function, or gain-of- function.
  • the relevant mutations are mapped to the 2D structure of Nspl (FIG. 3).
  • Mutation A (L21M) is an inadvertent mutation created outside of the RNA groove and had no apparent effect on Nspl activity as measured by the Viability Index (FIG. 4).
  • Mutation B (D33R) is an established gain-of-function mutation that was previously shown to potently block host cell mRNA translation, consequently inhibiting interferon production by the SARS-CoV Nspl [19].
  • the 3D model of Nspl indicates that D33 lies in close proximity to the RNA groove and may increase the binding affinity of the viral 5’UTR for this pocket. This mutation also led to a significant reduction of the Viability Index compared to wild-type Nspl.
  • C (L123A/R124E) and D (N128S/K129E) are neighboring mutations located within the RNA groove (FIG. 3).
  • D (N128S/K129E) is a well-characterized null mutation that blocks Nspl’s ability to suppress interferon activity in several studies [7, 8, 19].
  • the neighboring mutation had the opposite effect: a gain-of-function that increases Nspl toxicity.
  • Mutation E (K164A) is located in the C-terminal domain (FIG. 3) and was previously reported to abolish the ability of Nspl to suppress host cell defenses [20].
  • the function of the C-domain is to block the RNA tunnel of ribosomes [8, 11, 13].
  • K164A also led to a significant reduction of the Viability Index compared to wild-type Nspl (FIG. 4).
  • mutations define an essential role for this domain despite its small size (80 residues).
  • Efficacy as the ability of a compound to reverse the effects of Nspl to the level observed in the absence of Nspl under the same conditions (FIG. 4). Using this as a scale, a compound simulating the well-characterized null mutations D (N128S/K129E) and E (K164A) would have Efficacies of 60% and 54% respectively.
  • the assay for Nspl activity was designed to simulate the early phase of COVID-19 infection in lung cells.
  • Previous cell culture experiments utilize an MOI of 0-3, that is, up to 3 virions per adherent cell [24, 25]. It is estimated that during infection, about 10 3 infectious virions are eventually generated per cell [26].
  • the number of Nspl mRNA molecules introduced into each cell is about 5 x 10 6 copies, which is several orders of magnitude greater than the number of Nspl transcripts introduced per cell in live viral studies. This excessive Nspl expression accelerates H1299 cell death in this assay, but also ensures a stringent screen for potential Nspl inhibitors.
  • ICM Internal Coordinate Mechanics
  • AutoDock Vina determines the theoretical binding affinity of compounds but assumes the 3D structure of the protein receptor is fixed [28]. Data from both types of calculations differ, but results from both were used to guide subsequent experimental assays.
  • ICM was used to screen the DrugBank database [29], focusing on the RNA groove region represented in the crystal structure, 7k7p [17]. Due to the small size of the C-domain, it was not possible to use ICM to screen DrugBank for potential inhibitors.
  • Autodock Vina was used to virtually screen publicly available compound databases: FDA-approved and World- approved drugs in the ZINC15 database [30], eDrug-3D [31], and selected compounds from PubChem [32]. Almost all the compounds screened are also contained within DrugBank. Autodock Vina was applied to compound interactions with both the RNA groove in the N-domain and the helix-loop-helix region in the C-domain to stratify potential inhibitors.
  • Cyclo(L-His-L-Pro) is a natural compound already produced in the body, which may preclude its use as a drug.
  • Flufenoxuron is an insecticide and toxic, which may preclude its use as a drug.
  • Beta-Carotene is a food product, which creates obstacles to accurate dosing.
  • Milbemycin oxime is a veterinary antiparasitic and toxic, which may preclude its use as a drug.
  • Tirilazad is an investigational drug for strokes that failed Phase 3 trials and is not readily available for clinical use.
  • Golvatinib is an investigational drug that is not readily available for clinical use.
  • Glycyrrhizic acid is used in flavorings and found in licorice; as a food product there will be obstacles to dosing.
  • CC50 half-maximal cytotoxic concentration
  • Table 8 The Safety Index is defined as the ratio of CC50 over EC 100.
  • Bolded values were determined under serum-free conditions (i.e. DMEM-N2), while all other measurements were determined in cells incubated with fetal calf serum (Table 8).
  • Nspl-inhibitory Interactions among Compounds Two potentially functional sites within Nspl were used to screen for potential inhibitors of Nspl: the N- terminal RNA groove and the helix-loop-helix C-terminal region. Most of the compounds that are thought to bind to the N-terminal RNA groove also have high binding affinities for the C-terminal region (Table 7). However, synergy may exist between compounds that bind to either site preferentially . In addition, the RNA groove can accommodate several compounds, raising the possibility of synergistic binding to this region.
  • Tirilazad Another compound that consistently raised Efficacy in a synergistic pattern was Tirilazad, which was reported to bind tightly to the RNA groove in previous screens [9, 33].
  • MPR Montelukast+Ponatinib+Rilpivirine
  • the CC50 of MPR was 8X, providing a safety index of 16 (FIG. 7A).
  • lxMPR was applied to HI 299 cells transfected with the Nspl point mutations, Mut A, B and C (FIG. 7B).
  • MPR treatment of H1299 cells transfected with these Nspl mutations failed to rescue these cells from toxicity.
  • lxMPR raised Efficacy in the gain- of -function point mutant B (D33R), which lies outside of the RNA groove.
  • D33R gain- of -function point mutant B
  • lxMPR did not raise Efficacy to a statistically significant level with mutations A and C, which are located adjacent to or within the RNA groove.
  • Nspl is a promising molecular target because of its critical role during early SARS- CoV-2 pathogenesis.
  • SARS-CoV-2 [8, 11, 13], SARS-CoV [37], and MERS [38]
  • Nspl shuts down host protein synthesis but ribosomes remain permissive for viral protein synthesis.
  • Experimental deletion of Nspl in a highly virulent beta-coronavirus, murine hepatitis virus converts the virus from a lethal pathogen to a nonlethal one [39].
  • naturally occurring variants of SARS-CoV-2 containing deletions in the helix-loop-helix region of the C-domain of Nspl have been identified in China [40]. This variant renders the virus less severe clinically, with lower viral loads and smaller plaque size [40].
  • SARS-CoV-2 induces apoptosis in lung cells has not been fully elucidated, but targeting this process can attenuate disease severity [41].
  • SARS-CoV-2 infected alveolar epithelial cells include those affecting eukaryotic translation elongation and viral mRNA translation [42], consistent with Nspl’s primary action of subverting host protein synthesis [8, 11, 13]. Accordingly, Nspl blocks the production of interferon I [43-45] and interferon III [22], key players in the innate defense against viral infection.
  • Nspl contributes to the apoptotic process by inhibiting host protein translation and interferon action.
  • the Nspl assay described here is essentially a cytopathic assay that simulates the expression of Nspl mRNA during infection.
  • Nspl functions only when introduced inside cells, its actions are not dependent on viral tropism. Indeed, we observed similar actions of Nspl on HeLa cells (data not shown), a cell line that does not support SARS-CoV-2 replication [46]. SARS-CoV-2 infection can lead to multi-organ damage, and Nspl is a likely contender in this pathogenesis.
  • the design of the assay described herein can be adapted to investigate the role of Nspl in other tissues.
  • the steady state plasma concentration of Ponatinib in patients taking the standard oral dose of 15 mg was 43.6 ng/mF or 80 nM [51].
  • the standard oral dose of Rilpivirine is 25 mg daily, resulting in plasma levels of 30-70 nM [52].
  • All three drugs can be given orally and are expected to attain plasma concentrations that in our preclinical study, inhibits Nspl to the same extent as a null mutation.
  • Montelukast, Ponatinib, and Rilpivirine have been suggested as treatments for COVID-19 in other studies.
  • Montelukast binds to the SARS-CoV-2 proteins Mpro [53], RdRp [53], and 3CL [54].
  • Montelukast inhibited SARS-CoV-2 replication, albeit at a high IC50 of 18.82 mM [55].
  • Montelukast inhibits the action of inflammatory cytokines, suggesting it could tame cytokine storms during severe COVID-19 infection [56].
  • No prospective trials of Montelukast have been performed, but a retrospective study suggests that COVID- 19- positive patients taking this drug (10 mg daily) had fewer deleterious symptoms compared to patients not taking the drug [57].
  • Recent in silico docking studies also suggest that Ponatinib binds to host factors that influence infection [58, 59], and that Rilpivirine can bind to Mpro, PLpro, Spro, ACE2, and RdRp [60].
  • H1299 cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% fetal calf serum (FCS) and 100 U/mF penicillin- streptomycin. Cells were grown in an incubator that maintained the temperature at 37°C, air humidity at 95%, and CO2 concentration at 5%, and passaged every 3-4 days with PBS and 0.05% Trypsin-EDTA. To prepare cells for transfection, they were plated at a density that would be predicted to reach 50% the next day in a volume of 70 pF per well on 96-well plates.
  • FCS fetal calf serum
  • RNA transfections were carried out in 96-well plates with H1299 cells plated at 50% density.
  • the equivalent of 0.05-0.2 pF of FipofectamineTM MessengerMaxTM was first diluted in Opti-MEMTM in a volume of 5 pF for 5 min, and then added to 50-200 ng RNA (diluted in Opti-MEM to a volume of 5 pF) in a total volume of 10 pF, and incubated for an additional 10 minutes at room temperature.
  • the mixture was then diluted to a volume of 50 pL with Opti-MEM and added to H1299 cells growing in a single well. This would be scaled depending on the number of wells to be transfected per plate.
  • Nspl Assay H1299 cells growing on 96-well plates were transfected with Nspl mRNA as described above and incubated with the lipofectamine-RNA mix for 3 hours. Little difference in gene expression was observed between 2 to 4 hours of incubation with the lipofectamine-RNA mix. Media was then replaced by addition of 80 pL DMEM-10% FCS- lOOU/mL Pen-Strep or 80 pL serum- free DMEM-1% N2 supplement- lOOU/mL Pen-Strep.
  • HI 299 cells were then returned in the CO2 incubator overnight.
  • Fluorescence from 96-well plates was measured using a Spectramax Microplate Gemini XPS reader with the following parameters: Hoechst 33342 staining — excitation-355 nm; emission-460 nm; calcein-AM — excitation-485 nm; emission-520 nm; TMRE — excitation- 544 nm; emission-590 nm. After normalizing values for each fluorescence reading to non-transfected controls, the product of all three readings represents the “Viability Index”.
  • Efficacy is quantified as the degree to which a drug or drug combination reverses all toxic effects of Nspl as determined by the Viability Index. The quantity is value between 0 and 100, where 0 represents no effect and 100 is complete reversal.
  • the ECIOO is the concentration of drug where maximum Efficacy is observed.
  • the CC50 is the half-maximal concentration of drug that produces death in H1299 cells. The half-maximal concentration was determined from dose response data fitted to a sigmoidal curve (www.aatbio.com/tools).
  • the ICM Pocket Finder method [61] in ICM-Pro v3.9-lc was used to define a dmggable pocket within the Nspl crystal structure, 7k7p [17].
  • the pocket is essentially identical to the RNA groove that accommodates the 5’UTR of viral mRNAs [9].
  • the ICM-VLS method [62, 63] (MolSoft LLC) was used to dock, score and rank chemicals from the Drugbank database [29] that are predicted bind to this pocket.
  • PyRx [64] is a user interface that assimilates Autodock Vina [28] with other programs, and was used to screen the compound libraries ZINC 15 [30] and eDrug-3D [31].
  • the molecular targets were the RNA groove pocket within the structures, 7k7p [17] and 7k7n [18], and the loop-helix-loop regions of the C-domain (structures 6zlw [8], 6zok [11], and 7k5i [12]).
  • H1299 cells were plates on 96-well plates and transfected with Nspl mRNA as described above. Lipofectamine-RNA mixtures were replaced with 80 pL serum-free DMEM-1% N2 supplement- lOOU/mL Pen-Strep. 20 pL of serial dilutions of each drug (diluted in DMEM-1% N2 supplement- lOOU/mL Pen- Strep) were added in a matrix configuration, and the cells were incubated for another 20 hours in the tissue culture incubator. Plates were then subjected to the multiplexed fluorescent assay described above, and the Variability Index and Efficacy over a range of drug concentrations were determined.
  • the viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation.
  • RNA 2020.
  • Coronavirus nonstructural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines. PLoS Pathog, 2007. 3(8): p. el09.
  • Abagyan, R., M. Totrov, and D. Kuznetsov, ICM A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation. Journal of Computational Chemistry, 1994. 15: p. 488-506.
  • the viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation.
  • RNA 2020.
  • Calcein-AM is a detector of intracellular oxidative activity. Histochem Cell Biol, 2004. 122(5): p. 499-505.
  • RNA 2021.
  • Coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines. PLoS Pathog, 2007. 3(8): p. el09.
  • Genomic monitoring ofSARS- CoV-2 uncovers an Nspl deletion variant that modulates type I interferon response.

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Abstract

La présente invention concerne des inhibiteurs de Nspl (protéine non-structurale 1), des compositions pharmaceutiques et des procédés d'utilisation de ceux-ci pour traiter des sujets ayant ou risquant d'avoir un coronavirus du syndrome respiratoire aigu (SARS-CoV), par exemple, une infection due au coronavirus du syndrome respiratoire aigu 2 (SARS-CoV-2).
PCT/US2022/026374 2021-04-26 2022-04-26 Inhibiteurs de nsp1 pour le traitement de sars-cov-2 Ceased WO2022232161A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118615290A (zh) * 2024-06-28 2024-09-10 兰州大学 甲磺酸二氢麦角碱在制备抗SARS-CoV-2感染药物中的应用

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