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WO2021173592A1 - Rocaglates synthétiques ayant des activités antivirales à large spectre et leurs utilisations - Google Patents

Rocaglates synthétiques ayant des activités antivirales à large spectre et leurs utilisations Download PDF

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WO2021173592A1
WO2021173592A1 PCT/US2021/019295 US2021019295W WO2021173592A1 WO 2021173592 A1 WO2021173592 A1 WO 2021173592A1 US 2021019295 W US2021019295 W US 2021019295W WO 2021173592 A1 WO2021173592 A1 WO 2021173592A1
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virus
family
eif4a
cov
enantiomer
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Hans-Guido Wendel
Arnold Grunweller
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Philipps Universitaet Marburg
Memorial Sloan Kettering Cancer Center
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Philipps Universitaet Marburg
Memorial Sloan Kettering Cancer Center
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    • 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
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This disclosure relates to synthetic rocaglate compositions, uses thereof, and methods for treating a viral infection in a host cell or organism infected by the virus, such as coronaviruses (e.g., severe acute respiratory syndrome coronavirus [SARS-CoV], severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2, the virus and its mutant forms that cause COVID-19], Middle East respiratory syndrome coronavirus [MERS-CoV]), Zika virus, Lassa virus, Crimean Congo hemorrhagic fever virus, and hepatitis E virus, and other RNA viruses.
  • mRNA messenger ribonucleic acid
  • Rocaglates a class of natural compounds isolated from plants of the genus Aglaia in the mahogany family ( Meliaceae ), are potent inhibitors of translation initiation. They are proposed to form stable stacking interactions with polypurine sequences in the 5'-UTR of selected mRNAs thereby clamping the RNA substrate onto eIF4A causing the inhibition of the translation initiation complex.
  • the DEAD-box RNA helicase eIF4A which is part of the heterotrimeric translation initiation complex eIF4F, unwinds RNA secondary structures in 5 '-untranslated regions (5'-UTRs) of selected mRNAs to enable binding of the 43 S preinitiation complex (PIC).
  • eIF4A has a critical role in the translation of protooncogenic mRNAs with complex structured 5 '-UTRs. Viral RNAs also contain highly structured 5’-UTRs, suggesting that viral protein synthesis may also be eIF4A-dependent.
  • the specific eIF4A inhibitor Silvestrol a plant-derived rocaglate, has broad-spectrum antiviral activity at non-cytotoxic concentrations in a low nanomolar range. Silvestrol inhibits the replication of RNA viruses representing different virus families, like Ebola- (EBOV), Corona- (CoV), Zika- (ZIKV), Chikungunya- (CHIKV), and hepatitis E (HEV) viruses.
  • Silvestrol showed good bioavailability, in vitro, ex vivo and in vivo antiviral activity and low cytotoxicity in primary cells.
  • synthesis of Silvestrol is sophisticated, difficult, and time-consuming, thus hampering its prospects for further antiviral clinical development.
  • compositions and methods for inhibiting the replication of pathogenic RNA viruses including coronaviruses. It would also be desirable to have compositions and methods for treating or preventing human and other animal infections by RNA viruses, including coronaviruses.
  • compositions and methods provided herein are directed to inhibiting the replication of RNA viruses, including coronaviruses, and to treating or preventing human or other animal infections by RNA viruses, including coronaviruses.
  • CR-31-B comprises a racemic mixture of:
  • the CR-31-B comprises at least 50% CR-31-B (-) enantiomer. In some embodiments, the CR-31-B is a CR-31-B (-) enantiomer. In some embodiments, the CR-31-B reduces or inhibits a eukaryotic initiation factor 4A (eIF4A) activity.
  • eIF4A eukaryotic initiation factor 4A
  • mRNA messenger ribonucleic acid
  • a synthetic rocaglate composition for reducing or inhibiting translation initiation of a messenger ribonucleic acid (mRNA) of a virus in a host cell or organism infected by the virus, the synthetic rocaglate composition comprising a therapeutically effective amount of CR-31-B or a pharmaceutically acceptable salt thereof.
  • FIGURE 1 is a schematic depicting a comparison between the chemical structures of the rocaglates Silvestrol, Episilvestrol, CR-31-B (-), CR-31-B (+), and rocaglamide A (RocA).
  • the characteristic cyclopenta[b]benzofurane structure found in all rocaglates is indicated in red in the Silvestrol structure.
  • the dioxane moiety that is only found in Silvestrol and Episilvestrol is shown on the left side in black.
  • Silvestrol and Episilvestrol are enantiomers that differ only in one -OH group in the dioxan ring (marked with a circle: (R) for Silvestrol and (S) for Episilvestrol). Both have comparable effects as antiviral compounds.
  • the blue ring indicates the variable region in rocaglates.
  • FIGURES 2A-2C depict antiviral activities of the synthetic rocaglate CR-31-B (-) against coronaviruses HCoV-229E and MERS-CoV.
  • FIGURE 2A shows photographs of the results of Western blot analysis of HCoV-229E N protein accumulation (top panel) in cells treated with different enantiomers of CR-31-B (CR-31-B (-), left; CR-31-B (+), right).
  • b-Actin (beta-actin; lower panel) was used as a loading control.
  • FIGURE 2B is a graph depicting total (genomic and subgenomic) viral RNA produced in HCoV-229E-infected MRC-5 cells treated with the two enantiomers of CR-31-B. Relative changes in viral RNA levels were determined by RT-qPCR. The data were normalized to infected but untreated cells as well as GAPDH mRNA using the comparative AACt (delta-delta-Ct) method.
  • FIGURE 3 is a series of photographs depicting immunofluorescence analysis to visualize the effects of CR-31-B (-) on viral dsRNA (center column) and nonstructural protein 8 (nsp8; left column) accumulation in HCoV-229E- infected MRC-5 cells.
  • Cells were infected with an MOI of 1 and incubated with the indicated CR-31-B (-) concentrations: 10 nM CR-31-B (-) (top row); 0.1 nM CR-31-B (-) (middle row); and 0 nM CR-31-B (-) (control cells treated with DMSO; bottom row).
  • Cells were fixed at 24 hpi and analyzed by confocal laser-scanning microscopy using antibodies specific for dsRNA (red) and nsp8 (green). The images were also merged as shown (right column).
  • FIGURES 4A-4C show a series of graphs depicting a comparison of CR-31-B (-) vs. CR-31-B (+) with respect to reduction of viral titer and cytotoxicity.
  • FIGURE 4A shows bar graphs demonstrating CR-31-B (-) inhibits the production of infectious virus progeny of HCoV-229E (left) and MERS-CoV (right) at low nanomolar concentrations.
  • FIGURE 4B shows graphs demonstrating that treatment of MRC-5 cells for 24 h with CR- 31-B (-) and CR-31-B (+) caused no major cytotoxicity at concentrations of up to 5mM (micromolar) measured via MTT assay.
  • FIGURE 4C shows graphs and data demonstrating CC 50 values determined via ATPlite assay for MRC-5 cells incubated with CR-31-B (-) or CR-31-B (+) for 24, 48 or 72 h as indicated.
  • FIGURE 5 is a graph depicting CC 50 values were determined for a range of human skin carcinoma and liver carcinoma cell lines treated with racemic (+/-) CR-31-B.
  • the CC 50 value of (+/-) CR-31-B was determined using primary human dermal fibroblasts (HDF). Data is representative of four experimental replicates. The average CC 50 across the two biological replicates is plotted.
  • FIGURES 6A-6C demonstrate a comparison of antiviral effects of CR-31-B (-) vs. Silvestrol using human bronchial epithelial cells infected with HCoV-229E.
  • FIGURE 6A is a schematic depicting the method used. Human bronchial epithelial cells were cultivated and differentiated at an air liquid interface into different airway epithelial cell types (basal, ciliated, clara, and goblet cells) and used to assess antiviral effects of the respective compounds.
  • FIGURE 6B and FIGURE 6C show graphs comparing the effects of treating cells from Donor 1 and Donor 2, respectively.
  • FIGURES 7A-7B are graphs demonstrating the potent antiviral activity of CR-31- B (-) and Silvestrol against the Zika virus (Uganda strain 976) in A549 cells. Cell were infected using a MOI of 0.1 for 16 hours and simultaneously treated with compounds in the concentrations as shown.
  • FIGURE 7A intracellular RNA was obtained, and after reverse transcription, the ZIKV genomes were quantified by qRT-PCR to determine the EC 50 .
  • cell viability of A549 cells was determined using the PRESTOBLUETM cell viability agent (THERMOFISHER SCIENTIFICTM) after treatment with the compounds in their respective concentrations for 72 hours.
  • CC 50 values are 9.42 nM for Silvestrol and 19.3 for CR-31-B (-).
  • FIGURES 8A-8C are graphs showing that CR-31-B (-) and Silvestrol inhibit LASV (left column) and CCHFV (right column) replication in primary murine hepatocytes with comparable efficiencies in a concentration range between 20 and 50 nM.
  • FIGURE 8A demonstrates potent antiviral activity of CR-31-B (-) against LASV and CCHFV without cytotoxicity in murine hepatocytes.
  • FIGURE 8B shows no antiviral effects of CR-31-B (+) up to a concentration of 5 mM.
  • FIGURE 8C demonstrates potent antiviral activity of Silvestrol against LASV and CCHFV without cytotoxicity in murine hepatocytes.
  • FIGURE 9 shows a graph demonstrating that CR-31 -B (-) and Silvestrol reduce the levels of extracellular HEV RNA at low nanomolar concentrations.
  • the graph depicts qRT- PCR measurement of extracellular HEV RNA of CR-31-B (+) (left), CR-31-B (-) (center), and Silvestrol (left) treated, persistently HEV infected cells. All data are referred to the DMSO control.
  • FIGURE 10 is a graph showing analysis of cytotoxicity of CR-31-B (+), CR-31-B (-), and Silvestrol in persistently HEV -infected A549 cells after treatment with the indicated compound for 72 h.
  • both CR-31-B enantiomers had no major cytotoxic effects, whereas the natural rocaglate Silvestrol reduced the cell viability by approximately 30%.
  • both CR-31-B (-) and Silvestrol caused a reduction of cell viability by approximately 40%.
  • FIGURE 11 is a schematic of the dual luciferase assay used to analyze the sensitivity of viral 5’-UTRs towards eIF4A inhibition.
  • FIGURE 12 is a graph showing the effects of 5 and 10 nM (nanomolar) Silvestrol on the translation efficiency of reporter gene expression constructs containing different 5’- untranslated regions (5’-UTRs) in the context of the following 5’-UTRs: (AC) 15 , polyAC- 5’-(AG) 2.5 , polyAC-5’-(AG) 5 , polyAC-5’-(AG) 7.5 , polyAC-5’-(AG) 10 , poly AC- mid(AG)2.5, polyAC-mid(AG)5, polyAC-mid-(AG)7.5, polyAC-mid(AG) 10 , and (AG)i5. Results were normalized to dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • FIGURES 13A-13B depict a comparison of the inhibitory effects of CR-31-B (-) and Silvestrol on reporter gene expression constructs containing different viral 5’-UTRs.
  • FIGURE 13A has a graph (above) showing the effects of 5 and 10 nM Silvestrol or CR- 31-B (-) on the translation efficiency of reporter gene expression in the context of 5’-UTRs from coronaviruses HCoV-229E and MERS-CoV, as well as EBOV VP30 and VP35.
  • the VP35 5’-terminal hairpin and the VP35 hairpin with (AG) 5 extensions were also analyzed. Results were normalized to DMSO.
  • FIGURE 13B has a graph (above) showing the analysis of the sensitivity of the 5’-UTR of HEV and derivatives thereof against 5 and 10 nM Silvestrol and CR-31-B (-) treatment in a dual luciferase assay.
  • AG 15 and AC 15 sequences were used as positive and negative controls, respectively.
  • Predicted RNA secondary structures of the HEV 5’-UTRs are shown (below). The reporter gene expression data were normalized to the transfection efficiencies and the corresponding DMSO controls.
  • FIGURE 14 is a schematic depicting a model of the activity of this class of drugs (rocaglates) on eIF4A and its ability to block unwinding of RNA with secondary structures by Silvestrol or CR-31-B (-) as exemplary inhibitors of eIF4A, which is shown as a model of the surface of human eIF4A with a bound poly AG sequence. Interaction of eIF4A with RNA unwinds the viral mRNA 5’-UTR hairpin structure upstream of the poly AG sequence to enable translation initiation at the AUG start codon downstream of the poly AG sequence, as depicted in the schematic.
  • This general viral mRNA structure is common to HCoV-229E, MERS-CoV, LASV, CCHFV, ZIKV, and HEV, as well as other RNA viruses, although there are differences in the nature of the 5’-UTRs.
  • FIGURES 15A-15E are models for the possible binding mode of Silvestrol and RNA clamping using structure-based comparative modeling of RocA or Silvestrol onto a 10-mer poly AG RNA bound to the surface of human eIF4A.
  • FIGURE 15A depicts the surface of human eIF4A with a bound poly AG 10-mer (adapted from Iwasaki et al. [2019] Mol. Cell 73: 738-748 e9) for comparison.
  • FIGURES 15B-15E zoom in to show the binding region of RocA and Silvestrol.
  • the dioxane moiety of Silvestrol is able to cross the RNA stretch to make additional contacts with proximal positioned arginine residues in eIF4A.
  • the PYMOLTM (SCHROEDINGER ® ) molecular graphics system was used for graphical illustration.
  • eIF4A grey; RNA: green; RocA: purple; Silvestrol: cyan.
  • FIGURES 16A-16B depict models for the possible binding mechanism of CR-31- B (-).
  • FIGURE 16A (upper panel) is a model for the predicted binding mode of CR-31- B (-) on a human eIF4A-RNA complex (red depicts negative charges; blue depicts positive charges; white depicts neutral amino acids with no charge or polar groups in the side chain; gold depicts the RNA structure). Binding of CR-31-B (-) leads to RNA clamping shown by structure-based comparative modeling.
  • FIGURE 16A (lower panel) is a schematic depicting SARS-CoV-2 infected cells, the predicted secondary structure of the SARS-CoV- 25 ’ -UTR with the 5 ' -cap bound translation initiation complex eIF4F (consisting of the cap- binding protein eIF4E, the bridging protein eIF4G and the DEAD-box RNA helicase eIF4A).
  • RNA clamping of CR-31-B (-) blocks translation and strongly reduces viral protein synthesis and as a consequence viral replication.
  • FIGURE 16B is a schematic depicting the RNA sequence (SEQ ID NO: 45; see Table 6) and predicted secondary structure of the SARS-CoV-25 ’UTR (identical with Group Ila). RNA secondary structures that form stem loops are abbreviated with SL. The sequence ends directly before the start codon AUG (marked in yellow) (... .AAG AUG... ) and is located between SL5c and SL5.
  • FIGURE 17 is a graph depicting the effect of CR-31-B (-) on reporter gene expression from constructs containing different 5’-UTRs using the dual luciferase reporter construct of FIGURE 11 to determine eIF4A-dependent translation of coronavirus 5’- UTRs.
  • the effects of 5 and 10 nM CR-31-B (-) on reporter gene expression are shown in the context of 5'-UTRs from three human coronaviruses, HCoV-229E, MERS-CoV, and SARS-CoV-2.
  • FIGURES 18A-18D are graphs, a Western blot, and an immunofluorescence depicting dose-dependent antiviral activity of the synthetic rocaglate CR-31-B (-) in SARS- CoV-2 infected Vero E6 cells.
  • FIGURE 18B shows two graphs.
  • FIGURE 18C depicts representative Western blot analysis of SARS-CoV-2 N protein accumulation (top panel) after treatment with the two enantiomers CR-31-B (-) and CR-31-B (+).
  • Protein accumulation was analyzed by Western blotting using polyclonal rabbit anti-SARS nucleocapsid protein antibody (ROCKLANDTM) and mouse anti-actin antibody (ABCAMTM), respectively, each diluted 1:500 in PBS containing 1% bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • FIGURE 18D depicts representative immunofluorescence analysis to determine the effects of CR-31-B (+) and CR-31-B (-) on viral double-stranded RNA (dsRNA) accumulation in SARS-CoV-2-infected Vero E6 cells.
  • RNA replication intermediate a viral RNA replication intermediate (red).
  • DAPI 4',6-diamidino-2-phenylindole
  • FIGURES 19A-19D are a schematic and graphs depicting a comparison of antiviral effects of CR-31-B (-) and Silvestrol using differentiated normal human bronchial epithelial (NHBE) cells infected with SARS-CoV-2.
  • FIGURE 19A is a schematic depicting how NHBE cells were cultivated and differentiated into different cell types (Clara, ciliated, goblet, and basal cells) under air-liquid interface conditions.
  • NHBE cells obtained from a 13-year-old Caucasian boy (Donor 1) and a 36-year-old Caucasian man (Donor 2), who were both non-smoking and lacking respiratory pathology, were seeded on collagen IV-coated transwell plates (CORNING COSTARTM, CLS3470-48EA) and grown in a mixture of DMEM (Invitrogen) and bronchial epithelial cell growth medium (BEGM) (LONZATM; CC-4175) supplemented with retinoic acid (75 nM).
  • DMEM Invitrogen
  • BEGM bronchial epithelial cell growth medium
  • FIGURES 19B, 19C pseudostratified human airway epithelia
  • the apical surface of the cells was incubated with 100 microliters (100 ⁇ L) PBS for 15 min, and SARS-CoV-2 titers in the cell culture supernatants were determined by virus plaque assay.
  • the CR-31-B comprises a racemic mixture of: 1. enantiomer having the formula n. a CR-31-B (+) enantiomer having the formula
  • the CR-31-B comprises at least 50% CR-31-B (-) enantiomer. In other embodiments, the CR-31-B is a CR-31-B (-) enantiomer.
  • the CR-31 -B reduces or inhibits a eukaryotic initiation factor 4A (eIF4A) activity. In some embodiments, the CR-31-B reduces or inhibits an eIF4A helicase activity. In some embodiments, the CR-31-B reduces or inhibits eIF4A clamping to a 5 ’-untranslated region (5’-UTR) of the mRNA of the virus. In some embodiments, the 5’-UTR comprises a hairpin structure. In some embodiments, the 5’-UTR comprises a polypurine sequence element comprising at least 5 purine nucleotides. In some embodiments, the polypurine sequence element comprises at least 20 purine nucleotides. In some embodiments, the polypurine sequence element comprises at least 30 purine nucleotides.
  • eIF4A eukaryotic initiation factor 4A
  • the CR-31-B reduces or inhibits an eIF4A helicase activity.
  • the virus is an RNA virus.
  • the virus comprises a virus from the Coronaviridae family, the Arenaviridae family, the Nairoviridae family, the Flaviviridae family, the Hepeviridae family, the Filoviridae family, or the Togaviridae family.
  • the virus from the Coronaviridae family comprises human coronavirus 229E (HCoV-229E) (human common cold coronavirus), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV - 2, COVID-19 virus), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV -NL63), or human coronavirus HKU 1 (HCoV -HKU 1 ). Since 2000, there have been three documented cases of a coronavirus outbreak of zoonotic origin to reach epidemic or pandemic scale.
  • HKU 1 human coronavirus HKU 1
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS Middle East respiratory syndrome
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV -2 severe acute respiratory syndrome coronavirus
  • Betacoronavirus in the subgenus Sarbecovirus, which is of zoonotic origin, causes a potentially severe respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic starting in early 2020.
  • the virus from the Arenaviridae family comprises Lassa mammarenavirus (LASV), Guanarito mammarenavirus, Junin mammarenavirus, Lujo mammarenavirus, Machupo mammarenavirus, Sabia mammarenavirus, or Whitewater Arroyo mammarenavirus.
  • the virus from the Nairoviridae family comprises Crimean- Congo hemorrhagic fever virus (CCHFV).
  • CHFV Crimean- Congo hemorrhagic fever virus
  • the virus from the Flaviviridae family comprises Zika virus (ZIKV), hepacivirus C (hepatitis C virus, HepC), dengue fever virus, yellow fever virus, Japanese encephalitis virus, or West Nile virus.
  • ZIKV Zika virus
  • hepacivirus C hepatitis C virus, HepC
  • dengue fever virus yellow fever virus
  • Japanese encephalitis virus or West Nile virus.
  • the virus from the Hepeviridae family comprises hepatitis E virus (HEV) or hepatitis B virus.
  • HEV hepatitis E virus
  • B virus hepatitis B virus
  • the virus from the Filoviridae family comprises Ebolavirus, Marburgvirus, Dianlovirus, Cuevavirus, Striavirus, or Thamnovirus.
  • the virus from the Togaviridae family comprises an Alphavirus.
  • the virus from the Alphavirus comprises Chikungunya virus, Eastern equine encephalitis virus, Western equine encephalitis virus, Barmah Forest virus, Mayaro virus, O’nyong’nyong virus, Ross river virus, Semliki Forest virus, Sindbis virus, Una virus, Tonate virus, or Venezuelan equine encephalitis.
  • the CR-31-B may be administered prophylactically before infection, may be administered after suspected or known virus exposure but prior to the appearance of symptoms of infection, administered during an incubation period of a virus, or any combination thereof.
  • the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • mRNA messenger ribonucleic acid
  • the virus is an RNA virus.
  • a synthetic rocaglate composition for reducing or inhibiting translation initiation of a messenger ribonucleic acid (mRNA) of a virus in a host cell or organism infected by the virus, the synthetic rocaglate composition comprising a therapeutically effective amount of CR-31-B or a pharmaceutically acceptable salt thereof.
  • the virus is an RNA virus.
  • Gene expression in prokaryotic and eukaryotic cells includes the steps of transcription of deoxyribonucleic acid (DNA) into ribonucleic acid (RNA). Transcription and subsequent processing of messenger RNA (mRNA) results in a template for protein synthesis via translation of the mRNA into protein, which is then further processed.
  • mRNA messenger RNA
  • protein synthesis includes initiation, elongation, and termination steps. Part of the initiation phase includes the binding and subsequent activity of initiation factors.
  • eIF4A DEAD-box RNA helicase eukaryotic initiation factor 4A
  • eIF4F heterotrimeric translation initiation complex eukaryotic initiation factor 4F
  • RNA ribonucleic acid
  • 5’-UTRs 5 ’-untranslated repeats
  • mRNAs messenger ribonucleic acids
  • PIC 43S preinitiation complex
  • eIF4A plays a role in the translation of protooncogenic messenger ribonucleic acids (mRNAs) with complex-structured 5’-UTRs.
  • RNAs contain 5’-UTRs with stable RNA structures (Madhugiri et al. [2016] Adv. Virus Res. 96: 127-163; Schlereth et al. [2016] RNA Biol. 13: 783-798) and are thus dependent on eIF4A for translation.
  • Viral protein synthesis is a host function critical to viral proliferation, and inhibition of viral protein synthesis can inhibit viral proliferation in the host.
  • Hallmark features of eIF4A-dependent translation define specific 5’-UTR elements that confer a requirement for the eIF4A RNA helicase.
  • the key features are longer 5 ’ -UTRs, a 12-mer (GGC)4 motif, and related 9-mer variant motifs.
  • GGC 12-mer
  • the 12-mer and 9- mer motifs precisely localize to between 53% and 65% of all predicted RNA G-quadruplex structures (depending on the analysis tool).
  • the 9-mer sequences require neighboring nucleotides to complete the structure as the minimal number is 12 nucleotides, and it was frequently observed that more than 12 nucleotides contribute to the G-quadruplex.
  • most of the remaining G-quadruplexes are based on highly similar sequence elements.
  • IVS internal ribosome entry site
  • cis- regulatory elements such as 5’-terminal oligopyrimidine (TOP), 5-terminal oligopyrimidine-like (TOP-like), or pyrimidine-rich translational element (PRTE)
  • TOP 5’-terminal oligopyrimidine
  • TOP-like 5-terminal oligopyrimidine-like
  • PRTE pyrimidine-rich translational element
  • IRESs are 5’-UTR structural elements comprising stem-loop and pseudoknot structures that allow for an alternative method of cap- and 5 ’-end-independent translation initiation.
  • TOP mRNAs contain a 5'-terminal oligopyrimidine tract (5'-TOP), encode for ribosomal proteins and eukaryotic elongation factors 1 -alpha and 2 (eEF-1 A and eEF-2), and are candidates for growth-dependent translational control mediated through their 5'-TOP, a sequence of 6-12 pyrimidines at the 5’-end.
  • 5'-TOP 5'-terminal oligopyrimidine tract
  • eEF-1 A and eEF-2 eukaryotic elongation factors 1 -alpha and 2
  • the mTOR Complex 1 is a protein complex composed of mTOR itself, regulatory-associated protein of mTOR (commonly known as raptor), mammalian lethal with SEC 13 protein 8 (MLST8), PRAS40 and DEPTOR.
  • This complex embodies the classic functions of mTOR, namely as a nutrient/energy/redox sensor and controller of protein synthesis.
  • RNA G-quadruplex structures are typically made from at least two stacks of four guanosines exhibiting non-Watson-Crick interactions (e.g., hydrogen bonds) and connected by one or more linker nucleotides.
  • the linker is most often a cytosine and less frequently an adenosine.
  • the minimum requirement for the structure is a (GGC/A)4 sequence and neighboring nucleotides can complete the structure.
  • the cap-binding protein eIF4E is limiting for cap-dependent translation and its signaling is controlled by, e.g., mTORCl and eukaryotic translation initiation factor 4E- binding protein 1 (4E-BP).
  • mTORCl activates transcription and translation through its interactions with p70-S6 Kinase 1 (S6K1) and 4E-BP1, the eukaryotic initiation factor 4E (eIF4E) binding protein 1.
  • S6K1 p70-S6 Kinase 1
  • 4E-BP1 the eukaryotic initiation factor 4E binding protein 1.
  • Their signaling converges at the translation initiation complex on the 5' end of mRNA, and thus activates translation.
  • Activated mTORCl will phosphorylate translation inhibitor 4E-BP1, releasing it from eukaryotic translation initiation factor 4E (eIF4E).
  • eIF4E is now free to join the eukaryotic translation initiation factor 4G (eIF4G) and the eukaryotic translation initiation factor 4A (eIF4A). This complex then binds to the 5' cap of mRNA and recruits the helicase eukaryotic translation initiation factor A (eIF4A) and its cofactor eukaryotic translation initiation factor 4B (eIF4B).
  • eIF4A helicase eukaryotic translation initiation factor A
  • eIF4B cofactor eukaryotic translation initiation factor 4B
  • the initiation complex Once the initiation complex is assembled at the 5' cap of mRNA, it recruits the 40S small ribosomal subunit that is now capable of scanning for the AUG start codon start site, because the hairpin loop has been eradicated by the eIF4A helicase. Once the ribosome reaches the AUG codon, translation can begin. Hypophosphorylated S6K is located on the eIF3 scaffold complex. Active mTORCl is recruited to the scaffold, and once there, phosphorylates S6K activate it. mTORCl phosphorylates S6K1 on at least two residues, with the most critical modification occurring on a threonine residue (T389). This event stimulates the subsequent phosphorylation of S6K1 by PDPK1.
  • Active S6K1 can in turn stimulate the initiation of protein synthesis through activation of S6 Ribosomal protein (a component of the ribosome) and eIF4B, causing them to be recruited to the pre-initiation complex.
  • S6 Ribosomal protein a component of the ribosome
  • eIF4B a component of the ribosome
  • the eIF4A helicase activity is required and represents the point of attack for three natural compounds, Silvestrol, hippuristanol, and pateamine.
  • Regulatory interactions occur between eIF4A and the eIF4B, eIF4G, and eIF4H factors, and between S6 kinase in the phosphorylation and signaling control of eIF4B. These interactions define a broadly relevant layer of translational control that is distinct from the control of eIF4E by 4E-BP and mTORCl.
  • SF2 helicases DEAD-box and DEAH-box proteins
  • DEAD-box and DEAH-box proteins DEAD-box and DEAH-box proteins
  • a mechanism of translational control has been identified that is characterized by a requirement for eIF4A/DDX2 RNA helicase activity and underlies the antiviral effects of Silvestrol.
  • the eukaryotic initiation factor-4A (eIF4A) family consists of 3 closely related proteins eIF4Al, eIF4A2, and eIF4A3. These factors are required for the binding of mRNA to 40S ribosomal subunits.
  • these proteins are helicases that function to unwind double-stranded RNA.
  • RNA helicases are essential for most processes of RNA metabolism such as ribosome biogenesis, pre-mRNA splicing, and translation initiation. They also play an important role in sensing viral RNAs. RNA helicases are involved in the mediation of antiviral immune response because they can identify foreign RNAs in vertebrates. About 80% of all viruses are RNA viruses and they contain their own RNA helicases. Defective RNA helicases have been linked to cancers, infectious diseases, and neuro-degenerative disorders.
  • DEAD-box proteins named for the amino acid sequence of a highly conserved motif, which include, but are not limited to, eIF4Al, eIF4A2, and eIF4A3, function primarily as ATP-driven, non-processive helicases, binding and unwinding short, exposed RNA duplexes before releasing the RNA and repeating the process on another duplex segment.
  • DEAH-box proteins share many sequence and structural similarities with DEAD-box proteins, but have a different mechanism of duplex unwinding.
  • DEAH-box proteins use simple cycles of RNA duplex binding and are highly specific for dsRNA, unwinding, and release
  • DEAH-box proteins function as translocating helicases, advancing in the 3 '->5' direction to disrupt nucleic acid structures, and some members of the DEAH-box family can act on both DNA and RNA, leading to unwinding of helices and, for some DEAH-box proteins, four-stranded G- quadruplex structures.
  • DEAH- box helicases instead of binding directly to structured RNA elements, DEAH- box helicases require 3' single-stranded regions for unwinding activity.
  • DEAH-box proteins also lack specificity for ATP, binding and hydrolyzing all four NTPs to power cycles of directional movement.
  • DEAH box proteins 9 and 36 are cytosolic helicases.
  • DEAH-box protein helicases include, but are not limited to, DEAH box protein 9 (DHX9) and DEAH box protein 36 (DHX36).
  • RNA helicases include, but are not limited to, eIF4Al, eIF4A2, eIF4A3, DHX9 or DHX36.
  • eIF4A-dependent translation-controlling motifs are typically present in the 5’-UTR of the mRNA.
  • the eIF4A-dependent translation-controlling motif comprises a G-quadruplex structure.
  • Silvestrol or CR-31-B interferes with eIF4A activity. In one embodiment, Silvestrol or CR-31-B inhibits eIF4A helicase activity.
  • “Rocaglates” are a class of compounds that act as potent inhibitors of translation initiation. In some embodiments, they are proposed to form stacking interactions with polypurine sequences in the 5 ’-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex.
  • UTR 5 ’-untranslated region
  • Rocaglates include, but are not limited to, Silvestrol (methyl (lR,2R,3S,3aR,8bS)- 6-[[(2S,3R,6R)-6-[(lR)-l, 2-dihydroxy ethyl]-3-methoxy-l, 4-dioxan-2-yl]oxy]-l, 8b- dihydroxy-8-methoxy-3a-(4-methoxyphenyl)-3-phenyl-2,3-dihydro-lH- cyclopenta[b][l]benzofuran-2-carboxylate), ( ⁇ )-CR-31-B, among other rocaglamide ((lR,2R,3S,3aR,8bS)-l,8b-dihydroxy-6,8-dimethoxy-3a-(4-methoxyphenyl)-N,N- dimethyl-3-phenyl-2,3-dihydro-lH-cyclopenta[b]
  • Other compounds of interest include, but are not limited to, macrolides (e.g., pateamine A ((3S,6Z,8E,llS,15R,17S)-15-amino-3-[(lE,3E,5E)-7-(dimethylamino)-2, 5- dimethylhepta- 1 ,3,5-trienyl] -9, 11 , 17-trimethyl-4, 12-dioxa-20-thia-21 - azabicyclo[16.2.1]henicosa-l(21),6,8,18-tetraene-5, 13-dione)) and steroids (e.g., hippuristanol).
  • macrolides e.g., pateamine A ((3S,6Z,8E,llS,15R,17S)-15-amino-3-[(lE,3E,5E)-7-(dimethylamino)-2, 5- dimethylhepta- 1 ,3,5-trienyl]
  • Synthetic rocaglates include, but are not limited to, CR-31-B (see FIGURE 1).
  • the CR-31-B comprises a racemic mixture of: i. a CR-31-B (-) enantiomer having the
  • the CR-31-B comprises at least 50% CR-31-B (-) enantiomer. In other embodiments, the CR-31-B is a CR-31-B (-) enantiomer.
  • Rocaglates inhibit translation by reducing or inhibiting eIF4A activity.
  • Reducing or inhibiting eIF4A activity can be achieved by reducing or inhibiting an eIF4A helicase activity and/or by reducing or inhibiting eIF4A clamping to a 5 ’-untranslated region (5’-UTR) of the mRNA of the virus.
  • the 5’- UTR comprises a hairpin structure.
  • the 5’-UTR comprises a polypurine sequence element comprising at least 20 purine nucleotides.
  • the polypurine sequence element comprises at least 30 purine nucleotides.
  • viruses are small infectious agent. While not inside an infected cell or in the process of infecting a cell, viruses exist in the form of independent particles, or virions, consisting of: (i) the genetic material (i.e., long molecules of DNA or RNA that encode the structure of the proteins by which the virus acts; non-coding regions e.g. 5'-UTRs, 3'-UTRs or intergenic regions have regulatory functions during the life cycle of a virus); (ii) a protein coat, the capsid, which surrounds and protects the genetic material; and in some cases (iii) an outside envelope of lipids.
  • the genetic material i.e., long molecules of DNA or RNA that encode the structure of the proteins by which the virus acts; non-coding regions e.g. 5'-UTRs, 3'-UTRs or intergenic regions have regulatory functions during the life cycle of a virus
  • non-coding regions e.g. 5'-UTRs, 3'-UTRs or intergenic regions have regulatory functions
  • a virus has either a DNA or an RNA genome and is called a “DNA virus” or an “RNA virus,” respectively.
  • the majority of viruses have RNA genomes. Different viruses can infect prokaryotes or eukaryotes.
  • An “RNA virus” usually has single- stranded RNA (ssRNA) as its genetic material, but may instead have double-stranded RNA (dsRNA) as its genetic material.
  • RNA viruses can have a (+)-strand RNA genome or a (-)- strand RNA genome. (-)-RNA has to be transcribed into a (+)-strand RNA that contains the information for the synthesis of virus proteins.
  • RNA viruses often have high mutation rates compared to DNA viruses, because viral RNA polymerases generally lack the proofreading ability of DNA polymerases. This high mutation rate often makes it difficult to construct effective vaccines against the diseases caused by RNA viruses.
  • Viruses cannot replicate on their own, but instead reproduce by infecting host cells and usurping the host cellular machinery, including the host transcription and/or translation machinery, to produce more virus particles. This property of viruses, as well as the ability of many viruses to mutate, makes treatment of viral infections difficult. Viral RNAs often contain highly structured 5’-UTRs, which may be eIF4A-dependent.
  • Viruses include, but are not limited to, coronaviruses, arenaviruses, bunyaviruses, flaviviruses, and orthohepeviruses.
  • Viruses include, but are not limited to, viruses from the Coronaviridae family, the Arenaviridae family, the Nairoviridae family, the Flaviviridae family, the Hepeviridae family, the Filoviridae family, or the Togaviridae family.
  • Viruses include, but are not limited to, RNA viruses for which viral protein synthesis is eIF4A- dependent.
  • the virus is from the Bunyavirales order, including, but not limited to the Arenaviridae family and/or the Nairoviridae family.
  • the virus from the Coronaviridae family comprises human coronavirus 229E (HCoV-229E) (human common cold coronavirus), Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2, COVID-19 virus), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), or human coronavirus HKU1 (HCoV-HKUl).
  • HCV-229E human common cold coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • SARS-CoV- 2 severe acute respiratory syndrome coronavirus 2
  • COVID-19 virus human coronavirus OC43
  • HoV-OC43 human coronavirus NL63
  • HKU1 HKU1
  • the virus from the Arenaviridae family comprises a mammarenavirus, including, but not limited to, Lassa mammarenavirus (LASV), Guanarito mammarenavirus, Junin mammarenavirus, Lujo mammarenavirus, Machupo mammarenavirus, Sabia mammarenavirus, and/or Whitewater Arroyo mammarenavirus.
  • LASV Lassa mammarenavirus
  • the virus from the Nairoviridae family comprises Crimean- Congo hemorrhagic fever virus (CCHFV).
  • the virus is from the Flaviviridae family, including, but not limited to, the Flavivirus genus (e.g., Zika virus (ZIKV), dengue fever virus, yellow fever virus, Japanese encephalitis virus, or West Nile virus) and/or the Hepacivirus genus (e.g., hepacivirus C).
  • the virus from the Flaviviridae family comprises Zika virus (ZIKV), hepacivirus C (hepatitis C virus, HepC), dengue fever virus, yellow fever virus, Japanese encephalitis virus, or West Nile virus.
  • the virus is from the Hepeviridae family, including, but not limited to, the Orthohepevirus genus.
  • the virus from the Hepeviridae family comprises hepatitis E virus (HEV) or hepatitis B virus.
  • the virus is from the Filoviridae family, including, but not limited to the Ebolavirus genus (Ebola virus disease; e.g., Zaire ebolavirus, Bombali ebolavirus, Bundabugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, and Tai Forest ebolavirus), the Marburgvirus genus (Marburg virus disease; e.g., Marburg mar bur gvirus [Marburg virus (MARV), Ravn virus (RAW)]), the Dianlovirus genus (Mengla virus disease; e.g., Mengla virus), the Cuevavirus genus (Lloviu virus disease; e.g., Lloviu cuevavirus), the Striavirus genus, and/or the Thamnovirus genus.
  • Ebolavirus genus Ebolavirus genus
  • Marburg virus disease Marburg virus disease
  • Marburg virus disease Marburg virus disease
  • MMV Marburg mar
  • the virus is from the Togaviridae family, including, but not limited to the Alphavirus genus (e.g., Chikungunya virus [Chikungunya virus disease], Eastern equine encephalitis virus [Eastern equine encephalitis], Western equine encephalitis virus [Western equine encephalitis], Barmah Forest virus, Mayaro virus, O’nyong’nyong virus, Ross River virus, Semliki Forest virus, Sindbis virus, Una virus, Tonate virus, Venezuelan equine encephalitis virus [Venezuelan equine encephalitis], and others).
  • Alphavirus genus e.g., Chikungunya virus [Chikungunya virus disease], Eastern equine encephalitis virus [Eastern equine encephalitis], Western equine encephalitis virus [Western equine encephalitis], Barmah Forest virus, Mayaro virus,
  • polynucleotide encompasses single-stranded or double- stranded nucleic acid polymers.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either general category of nucleotide (e.g., DNA or RNA).
  • operably linked encompasses components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.
  • a transcription control sequence "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.
  • control sequence encompasses polynucleotide sequences that can affect expression or processing of coding sequences to which they are ligated or operably linked.
  • compositions comprising a therapeutically effective amount of CR-31-B.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • “Pharmaceutically acceptable carriers” include any excipient which is nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed.
  • the pharmaceutical composition may include one or additional therapeutic agents.
  • Pharmaceutically acceptable carriers include solvents, dispersion media, buffers, coatings, antibacterial and antifungal agents, wetting agents, preservatives, buggers, chelating agents, antioxidants, isotonic agents and absorption delaying agents.
  • Pharmaceutically acceptable carriers include water; saline; phosphate buffered saline; dextrose; glycerol; alcohols such as ethanol and isopropanol; phosphate, citrate and other organic acids; ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; EDTA; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN, polyethylene glycol (PEG), and PLURONICS; isotonic agents such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride; as well as combinations
  • compositions of the invention may be formulated in a variety of ways, including for example, solid, semi-solid (e.g., cream, ointment, and gel), and liquid dosage forms, such as liquid solutions (e.g., topical lotion or spray), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • the compositions are in the form of injectable or infusible solutions.
  • the composition is in a form suitable for oral, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topical administration.
  • the composition may be formulated as an immediate, controlled, extended or delayed release composition.
  • compositions suitable for use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).
  • the composition includes isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile solutions can be prepared by incorporating the molecule, by itself or in combination with other active agents, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions one method of preparation is vacuum drying and freeze- drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art.
  • preparations may be packaged and sold in the form of a kit.
  • Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering a viral infection as described herein.
  • Effective doses of the compositions of the present invention, for treatment of conditions or diseases as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human, but non-human organisms, including non-human mammals and birds, as well as transgenic organisms, can also be treated.
  • Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • compositions of the present invention may be administered prophylactically before infection, may be administered after suspected or known virus exposure but prior to the appearance of symptoms of infection, administered during an incubation period of a virus, or any combination thereof.
  • compositions of the invention may include a “therapeutically effective amount.”
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of a molecule may vary according to factors such as the disease state, species, age, sex, and weight of the individual, and the ability of the molecule to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.
  • modulating refers to “stimulating” or “inhibiting” an activity of a molecular target or pathway.
  • a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 10%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 90%, by at least about 95%, by at least about 98%, or by about 99% or more relative to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition.
  • a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100- fold relative to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition.
  • the activity of a molecular target or pathway may be measured by any reproducible means.
  • the activity of a molecular target or pathway may be measured in vitro or in vivo.
  • the activity of a molecular target or pathway may be measured in vitro or in vivo by an appropriate assay known in the art measuring the activity. Control samples (untreated with the composition) can be assigned a relative activity value of 100%. A change in activity caused by the composition can be measured in the assays.
  • the terms “treat” and “treatment” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.
  • a single bolus may be administered.
  • several divided doses may be administered over time.
  • a dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for treating mammalian subjects. Each unit may contain a predetermined quantity of active compound calculated to produce a desired therapeutic effect. In some embodiments, the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved.
  • composition of the invention may be administered only once, or it may be administered multiple times.
  • the composition may be, for example, administered three times a day, twice a day, once a day, once every two days, twice a week, weekly, once every two weeks, or monthly.
  • a compound “inhibits” an activity if the compound reduces the desired activity by at least 10% relative to the activity under the same conditions but lacking only the presence of the compound.
  • the activity may be measured by any reproducible means.
  • the activity may be measured in vitro or in vivo.
  • compounds used in the methods described herein inhibit a eIF4A activity by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 90%, by about 95%, by about 98%, or by about 99% or more.
  • dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • administering to a subject is not limited to any particular delivery system and may include, without limitation, topical, transdermal, oral (for example, in capsules, suspensions or tablets), parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), or rectal.
  • Administration to a subject may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition (described earlier).
  • physiologically acceptable salt forms and standard pharmaceutical formulation techniques are well known to persons skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co.).
  • the term “subject” includes mammals, e.g., humans, companion animals (e.g., dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, birds, and the like).
  • the subject is male human or a female human.
  • the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use.
  • a “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient.
  • prodrugs can also be prepared as prodrugs, for example pharmaceutically acceptable prodrugs.
  • pro-drug and “prodrug” are used interchangeably herein and may refer to any compound which releases an active parent drug in vivo. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) the compounds of the present invention can be delivered in prodrug form.
  • Consisting of shall thus mean excluding more than traces of other elements.
  • the term “comprising” is used, such a term may be replaced by the term “consisting of’, wherein such a replacement would narrow the scope of inclusion of elements not specifically recited.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined. In some embodiments, the term “about” refers to a deviance of between 0.0001-5% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviance of up to 25% from the indicated number or range of numbers. In some embodiments, the term “about” refers to ⁇ 10 %.
  • substantially means “being largely, but not wholly, that which is specified” (e.g., “substantially pure”).
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of certain embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Human dermal fibroblast (HDF) cells were cultured in Fibroblast Basal Medium (ATCC ® PCS-201-030TM) supplemented with Fibroblast Growth Kit - Low Serum (ATCC ® PCS-201-041TM).
  • Human dermal cancer cell lines (COLO-829, HS294T, and SK- MEL-31) and human liver cancer cell lines (HuH-1, SK-HEP-1, SNU-475, PLC/PRF/5, HuH-7, SNU-182, and HepG2) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • Vero E6 cells African green monkey kidney epithelial cells
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FCS micrograms/milliliter
  • Silvestrol was obtained from the Sarawak Biodiversity Centre (Kuching; North-Bomeo, Malaysia; purity >99 %). A 6 mM stock solution was prepared in DMSO (sterile-filtered; ROTHTM) and diluted in DMEM or IMDM. Control cells were treated with corresponding DMSO dilutions lacking Silvestrol.
  • CR-31-B (-) and/or CR-31- B (+) (Wolfe et al. [2014] Nature 513: 65-70) were dissolved in DMSO for a total concentration of 10 mM (individual enantiomer or total racemic mixture) and stored at - 20°C.
  • a sigmoidal dose-response curve was fitted to the data using PRISM GRAPHPADTM 6.0 (GRAPHPAD SOFTWARETM).
  • the inhibitory concentrations that reduced the virus titer by 50%, (IC 50 ,) were calculated from the sigmoidal functions.
  • Cell viability of A549 cells persistently infected with HEV was determined using the PRESTOBLUETM Cell Viability Reagent (THERMOFISHER SCIENTIFICTM) after treatment with the substances in the respective concentrations for 72 h.
  • CC 50 values for human dermal fibroblast cells human dermal cancer cell lines, and human liver cancer cell lines
  • cell lines were treated with racemic (+/-) CR-31-B for 48 h, and cell viability was measured by adenosine triphosphate (ATP) quantification using the CELLTITER-GLOTM Luminescent Cell Viability Assay (PROMEGATM G7571).
  • ATP adenosine triphosphate
  • NHBE normal human bronchial epithelial
  • Undifferentiated cells were seeded on transwell plates (CORNING COSTARTM) coated with Collagen IV (INVITROGENTM) and grown in a mixture of DMEM (INVITROGENTM) and bronchial epithelial cell growth medium (BEGM) (LONZATM) supplemented with retinoic acid (75 nM). Every other day fresh medium was added. After reaching confluence, the cells were cultivated under air- liquid conditions for 4 additional weeks to differentiate into pseudostratified human airway epithelia.
  • Viruses High-titer stocks of HCoV-229E (NCBI accession number NC_002645) and MERS-CoV (EMC/2012; NCBI accession number NC_019843) were produced using Huh-7 cells. High-titer stocks of CCHFV strain Afg-092990 (Olschlager et al. [2010] J. Clin. Virol. 50: 90-92) and LASV strain Ba366 (Lecompte et al. [2006] Emerg. Infect. Dis.
  • MRC5 cells or murine hepatocytes were inoculated with the respective virus at a multiplicity of infection (MOI) of 0.1 or 0.01 at 33°C (HCoV-229E) or 37°C (MERS-CoV, LASV, CCHFV). After 1 h, the inoculum was removed, and cells were incubated with fresh medium containing the inhibitor at increasing concentrations.
  • MOI multiplicity of infection
  • qRT-PCR quantitative reverse transcription-polymerase chain reaction
  • Vero E6 cells were inoculated with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 at 33°C. After 1 h, the inoculum was removed, and cells were incubated with fresh medium containing CR-31-B (-) at increasing concentrations. Virus-containing supernatants were collected at 24 hours post infection (hpi) and virus titers were analyzed via plaque assay. EC50 values were determined based on virus titers in supernatants of infected cells treated with solvent control (DMSO) compared to virus titers in supernatants of infected cells treated with the respective inhibitor concentration. EC 50 values were then calculated by non- linear regression analysis using GRAPHPAD PRISMTM 6.0 (GRAPHPAD SOFTWARETM).
  • the supernatant was replaced with fresh medium supplemented with the indicated concentrations of the respective CR-31-B enantiomer. After 24h, the medium was removed, the cells were washed with PBS and lysed using buffer containing 50 mM Tris-HCl (tris(hydroxymethyl)aminomethane-HCl), pH 7.5, 150 mM NaCl, 1% NP40 (nonyl phenoxypolyethoxylethanol), and lx protease inhibitor cocktail (P8340; SIGMA- ALDRICHTM).
  • the insoluble material was removed by centrifugation and the protein content in the supernatant was measured using a QUBITTM 3 fluorometer (INVITROGENTM) and equal amounts of proteins were separated in sodium dodecyl sulfate (SDS)-10% polyacrylamide gels and blotted onto a nitrocellulose membrane (AMERSHAMTM).
  • SDS sodium dodecyl sulfate
  • AMERSHAMTM nitrocellulose membrane
  • Membranes were incubated with polyclonal rabbit anti-SARS nucleocapsid protein antibody (ROCKLANDTM) and mouse-anti actin antibody (ABCAMTM), respectively, each diluted 1 :500 in PBS containing 1% bovine serum albumin (BSA).
  • qRT-PCR of HCoV-299E RNA, extracellular HEV RNA, or ZIKV RNA MRC-5 cells were infected with a MOI of 1 and incubated for 24h with the indicated inhibitor concentrations. Then, total cellular RNA was isolated using RNEASYTM kit (QIAGENTM), and quantitative RT-PCR was performed using 5 ng RNA and the LUNA UNIVERSAL PROBE ONE-STEPTM RT-qPCR Kit (NEW ENGLAND BIOLABSTM [NEB]). Sequences of primers used to amplify genomic and total viral RNA, respectively, and GAPDH mRNA are shown in Table 3.
  • Dual luciferase constructs All constructs are based on the commercially available plasmid pFR_ HCV_ xb (ADDGENETM) and were produced using PCR-based site-directed mutagenesis. Primers were designed using SNAPGENE 4.1.9TM (GSL BIOTECH LLCTM). Primer sequences are shown in Table 1 and Table 2. The respective 5 ’ -UTRs were cloned downstream of the HSV -TK promotor directly followed by the firefly luciferase gene, an HCV IRES, and the Renilla luciferase gene.
  • the total length of the analyzed 5 ’-UTRs ranges from 25 bp to 292 bp ((AG)i5/(AC)i5: 30 bp; poly(AC) 12.5 -5’-(AG)2.5/poly(AC) 12.5 -mid(AG) 2.5 : 30 bp; poly(AC) 10 -5’-(AG)5/poly(AC) 10 -mid-(AG) 5 : 30 bp; poly(AC) 7.5 -5’-(AG) 7.5 /poly(AC) 7 .
  • Dual luciferase reporter assay The dual luciferase reporter assay was performed as described previously (Muller et al. [2018a] Antivir. Res. 150: 123-129; Muller et al. [2020] Antivir. Res. 175: 104706). All experiments were performed in at least three independent replicates.
  • the total length of the analyzed 5 ’-UTRs ranges from 25 bp to 292 bp ((AG)i 5 /(AC)i 5 : 30 bp; poly(AC) 12.5 -5’-(AG)2.5/poly(AC) 12.5 -mid(AG)2.5: 30 bp; poly(AC) 10 -5’-(AG) 5 /poly(AC) 10 - mid-(AG)5: 30 bp; poly(AC)7 .5 -5’-(AG)7 .5 /poly(AC) 7 .
  • Example 1 Antiviral activity of CR-31-B (-) against Coronavirus in vitro.
  • Coronavirus N-protein levels were found to be strongly reduced in the presence of the (-)-enantiomer at concentrations > 10 nM, while CR-31-B (+) had no detectable effect (FIGURE 2A).
  • genomic and subgenomic RNA levels of HCoV-229E were reduced in the presence of sub- nanomolar concentrations of CR-31-B (-) (FIGURE 2B).
  • the formation of viral replication/transcription complexes was reduced in the presence of CR-31-B (-), as shown by immunofluorescence analysis using antibodies directed against nonstructural protein 8 (nsp8) and double-stranded RNA (dsRNA) (FIGURE 3).
  • Cytotoxicity tests using MRC-5 cells revealed that CR-31-B (-) and CR-31-B (+) caused a slight reduction of cell viability by 10-25 % and 10 %, respectively, if the cells were incubated for 24 h with concentrations of up to 5 mM of the respective compound (FIGURE 4B), indicating low cytotoxicities for both compounds with selectivity indices of > 1000 (see Table 4).
  • CC 50 and EC 50 values determined for Silvestrol and CR-31-B (-)- treated cells that were mock infected (CC 50 ) or infected with the indicated viruses (EC 50 ).
  • SI Selectivity Index. Experiments were done in biological triplicates.
  • Example 2 Antiviral activity of Silvestrol and CR-31-B (-) in a human bronchial epithelial cell system.
  • CR-31-B (-) and Silvestrol have potent antiviral activities with EC 50 values between approximately 20 and 50 nM with no detectable cytotoxicity at concentrations of up to 10 mM (FIGURES 8A-8C).
  • Virus titers showed an approximately 4-log drop for LASV- and a 3-log drop for CCHFV-infected cells (FIGURES 8A, 8C).
  • CR-31-B (-) inhibits HEV replication (FIGURE 9). Persistently HEV-infected A549 cells were treated with different concentrations of Silvestrol and the two CR-31-B enantiomers.
  • Example 4 Analyses of the 5’-UTR-mediated inhibitory activities of Silvestrol and CR-31-B (-).
  • Such a second purine stretch is absent in the VP35 5’-UTR.
  • the 5’- terminal alone is sufficient to mediate translation inhibition by Silvestrol.
  • this hairpin increased the reporter activity about twofold in the presence of CR-31-B (-) (FIGURE 13A, VP35-HP only).
  • This increase was reduced back to basic levels if an additional (AG)5-polypurine stretch was inserted at the 3 '-end of the VP35 hairpin.
  • this insertion resulted in a slightly increased translation inhibition by Silvestrol, indicating that polypurine sequences strengthen the inhibitory effects of rocaglates on eIF4A (FIGURE 13A, VP35-HP+(AG) 5 ).
  • HEVgt3c the 5'-UTR of HEV (HEVgt3c) lacks any polypurine sequence element, but it is predicted to form a stable RNA hairpin structure which, most likely, requires unwinding during translation initiation.
  • HEV replication can be inhibited by Silvestrol (Glitscher et al. [2018] pii. Viruses 10: E301) and, to a slightly less extent, by CR-31-B (-) (FIGURE 9). Therefore, we asked if the polypurine-free 5'- UTR of HEV is also sensitive towards Silvestrol or CR-31-B (-) treatment.
  • HEV 5' -UTR hairpin structure was thermodynamically destabilized by disrupting one or two base pairs in the stem (FIGURE 13B, HEVgt3c and HEVgt3C-G4CC6A). These changes led to a gradual loss of the inhibitory effect of Silvestrol.
  • the sequence in the HEV 5 -UTR was changed at the 5 '-end from 5 -GCAGACCA... (SEQ ID NO: 34) into 5’-GGAGAGGA... (SEQ ID NO: 35) (FIGURE 13B, HEVgt3c-Purine), thereby introducing a stretch of 8 consecutive purines.
  • thermodynamic stability of the hairpin structure was reduced by these sequence changes, the HEVgt3c-Purine 5’-UTR became sensitive now to CR-31-B (-) and Silvestrol treatment (FIGURE 13B, HEVgt3c-Purine).
  • Example 5 Modelling of Silvestrol onto the surface structure of the human eIF4A- polypurine RNA complex
  • Example 6 Inhibitory effect of CR-31-B (-) on elF 4A-dependent translation of viral 5’-UTRs.
  • FIGURE 16A depicts a model for the predicted binding mode of CR-31-B (-) on a human eIF4A-RNA complex.
  • the RNA binds via its negative phosphate backbone onto eIF4A in a cavity formed by the two domains of the helicase that mainly consists of positively charged amino acids.
  • Silvestrol the arginines, which have a positive charge, can be reached by the dioxane moiety on the left side of the bound RNA.
  • CR-31- B (-) this is not particularly relevant.
  • White indicates neutral amino acids (no charge or polar groups in the side chain).
  • FIGURE 16A (lower panel) is a schematic depicting SARS-CoV-2 infected cells, the predicted secondary structure of the SARS-CoV- 25’-UTR with the 5'-cap bound translation initiation complex eIF4F (consisting of the cap- binding protein eIF4E, the bridging protein eIF4G and the DEAD-box RNA helicase eIF4A).
  • RNA clamping of CR-31-B (-) blocks translation and strongly reduces viral protein synthesis and as a consequence viral replication.
  • FIGURE 16B is a schematic depicting the RNA sequence and predicted secondary structure of the SARS-CoV-25’UTR (identical with Group Ila) (see SEQ ID NO: 45; Table 6). The sequence ends directly before the start codon AUG (marked in yellow) (... . AAG AUG... ), is located between SL5c and SL5 (SL means “stem loop”), and is shown in Table 6 (above).
  • the polypurine sequence (AG) 15 was used as a positive control since this sequence can be efficiently clamped onto the surface of eIF4A by different rocaglates due to p-p (pi-pi) stacking interactions (Müller et al. [2020] Antiviral. Res. 175: 104706; Iwasaki et al. [2019] Mol. Cell 73: 738-748 e9).
  • eIF4A-dependency was inferred from sensitivity of firefly luciferase mRNA translation to the presence of a specific eIF4A inhibitor.
  • Example 7 In vitro antiviral effect of CR-31-B (-) against SARS-CoV-2 in African green monkey Vero E6 cells.
  • CR-31-B (-) African green monkey Vero E6 cells were used (Ogando et al. [2020] J. Gen. Virol, doi: 10.1099/jgv.0.001453). Cytotoxicity of CR-31-B (-) was determined by MTT assay by treating Vero E6 cells with increasing concentrations of CR-31-B (-) for 24 h. No major cytotoxicity was detected for concentrations of up to 100 nM, with cell viability being reduced by about 10-25 % at the highest concentration tested (FIGURE 18A).
  • Vero E6 cells were infected with this virus at an MOI of 0.1 plaque forming units (pfu)/cell and incubated with medium containing the different concentrations of CR-31-B (-). At 24 h p.i., cell culture supernatants were collected, and virus titers were determined by plaque assay.
  • FIGURE 18D immunofluorescence analysis using antibodies specific for double-stranded RNA (dsRNA) (a mouse anti-dsRNA mAh (J2, SCICONS English & Scientific Consulting Kft.), representing a viral RNA replication intermediate, revealed a profound reduction of replicative organelles active in viral RNA synthesis (Müller et al. [2018a] J. Virol. 92: :(4): e01463-17. doi: 10.1128/JVI.01463-17).
  • dsRNA double-stranded RNA
  • Example 8 Antiviral activity of CR-31-B (-) and Silvestrol against SARS-CoV-2 in an ex vivo human bronchial epithelial cell system.
  • ALI air/liquid interface
  • CR-31-B reduced the production of infectious virus progeny by approximately 1.5 ( ⁇ 1.5) log steps at a concentration of 10 nM in differentiated NHBE cells obtained from two different donors.
  • CR-31-B reduced SARS-CoV-2 titers to undetectable levels, whereas the inactive enantiomer CR-31-B (+) did not affect viral replication compared to the solvent control (FIGURES 19C, 19D). No obvious cytotoxicity could be observed at this concentration using light microscopy.
  • This system provides a pseudostratified organization of basal, ciliated, goblet, and other less common types of cells and plays a crucial role in maintaining airway homeostasis by regulating innate and acquired immunity through the production of a wide range of cytokines as well as chemokines (Davies DE [2014] Ann. Am. Thorac. Soc. 11 (Supp. 5): S224-S251).
  • cytokines As well as chemokines
  • CR-31-B (-) has a potent antiviral activity similar to that of the more complex-structured Silvestrol. It is active at low nanomolar concentrations with low cytotoxicity in primary human cells, while it has a higher cytotoxicity in cancer cell lines (see FIGURE 5). Thus, CR-31-B (-) opens a broad therapeutic window for the treatment of viral infections and qualifies as an interesting synthetic rocaglate for further in vivo evaluations. Even though the antiviral potential of CR-31-B (-) is similar to Silvestrol, we identified substantial mechanistically differences between the two compounds as detailed below.
  • RNAs generally bind to eIF4A in a sequence- and structure-independent manner via their phosphate backbone. Thus, if no RNA clamping by rocaglates occurs, active eIF4A retains its ability to unwind secondary structures in the bound substrate RNA.
  • Silvestrol was able to clamp polypurine-free stable hairpin structures onto eIF4A, whereas CR-31-B (-) required an accessible polypurine-sequence in proximity to the 5 '-terminal hairpin. This conclusion is supported by data showing that translation of the reporter construct containing the EBOV VP35 5’-UTR is insensitive to CR-31-B (-), while it is sensitive to Silvestrol.
  • CR-31-B (-) may represent an interesting alternative to the broad-spectrum antiviral Silvestrol, which remains to be confirmed in appropriate in vivo studies to evaluate and compare the antiviral potential, toxicity, and pharmacokinetics profiles of the two compounds.
  • CR-31-B inhibits replication of Corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E viruses at non-cytotoxic low nanomolar concentrations. Since hepatitis E virus has a polypurine-free 5'-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5’-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (-).
  • CR-31-B (-) inhibited SARS-CoV-2 replication with an EC 50 of approximately 1.8 (-1.8) nM. In line with this, viral protein accumulation and repbcation/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels.
  • the data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating results obtained for other coronaviruses and supporting the concept that rocaglates may be used in first-line antiviral intervention strategies against RNA virus outbreaks.

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Abstract

L'invention concerne des compositions, des utilisations associées, et des méthodes de traitement d'une infection virale dans une cellule ou un organisme hôte infecté par le virus, tels que des coronavirus (par exemple, le coronavirus du syndrome respiratoire aigu sévère [SRAS-CoV], le coronavirus du syndrome respiratoire aigu sévère 2 [SRAS-CoV-2, le virus et ses formes mutantes provoquant COVID-19], le coronavirus du syndrome respiratoire du moyen-orient [MERS-CoV]), le virus Zika, le virus de Lassa, le virus de la fièvre hémorragique du Congo, le virus de l'hépatite E et d'autres virus à ARN. L'invention concerne également des compositions synthétiques de rocaglate, leurs utilisations, et des procédés de réduction ou d'inhibition de l'initiation de la traduction d'un acide ribonucléique messager (ARNm) d'un virus dans une cellule hôte ou un organisme infecté par le virus.
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WO2022221519A1 (fr) * 2021-04-14 2022-10-20 Memorial Sloan-Kettering Cancer Center Rocaglates synthétiques à activités antivirales à large spectre et leurs utilisations
EP4161646A4 (fr) * 2020-06-05 2024-07-24 Chan Zuckerberg Biohub, Inc. Compositions et procédés de traitement d'une infection virale

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WO2010011856A2 (fr) * 2008-07-23 2010-01-28 Board Of Regents Of The University Of Nebraska Stéréospécificité de la réduction du méthylsulfinyle
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WO2010011856A2 (fr) * 2008-07-23 2010-01-28 Board Of Regents Of The University Of Nebraska Stéréospécificité de la réduction du méthylsulfinyle
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4161646A4 (fr) * 2020-06-05 2024-07-24 Chan Zuckerberg Biohub, Inc. Compositions et procédés de traitement d'une infection virale
WO2022221519A1 (fr) * 2021-04-14 2022-10-20 Memorial Sloan-Kettering Cancer Center Rocaglates synthétiques à activités antivirales à large spectre et leurs utilisations

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