ABSTRACT
Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 M and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 [A], showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/422677v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17ca2aeorg.highwire.dtl.DTLVardef@19c5159org.highwire.dtl.DTLVardef@1a0adf6org.highwire.dtl.DTLVardef@1fd05cd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract Figure.C_FLOATNO Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray. C_FIG
ABSTRACT
SARS-CoV-2 rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus was able to infect millions of people. To date, close to half a million patients succumbed to the viral disease, COVID-19. The high need for treatment options, together with the lack of small animal models of infection has led to clinical trials with repurposed drugs before any preclinical in vivo evidence attesting their efficacy was available. We used Syrian hamsters to establish a model to evaluate antiviral activity of small molecules in both an infection and a transmission setting. Upon intranasal infection, the animals developed high titers of SARS-CoV-2 in the lungs and pathology similar to that observed in mild COVID-19 patients. Treatment of SARS-CoV-2-infected hamsters with favipiravir or hydroxychloroquine (with and without azithromycin) resulted in respectively a mild or no reduction in viral RNA and infectious virus. Micro-CT scan analysis of the lungs showed no improvement compared to non-treated animals, which was confirmed by histopathology. In addition, both compounds did not prevent virus transmission through direct contact and thus failed as prophylactic treatments. By modelling the PK profile of hydroxychloroquine based on the trough plasma concentrations, we show that the total lung exposure to the drug was not the limiting factor. In conclusion, we here characterized a hamster infection and transmission model to be a robust model for studying in vivo efficacy of antiviral compounds. The information acquired using hydroxychloroquine and favipiravir in this model is of critical value to those designing (current and) future clinical trials. At this point, the data here presented on hydroxychloroquine either alone or combined with azithromycin (together with previously reported in vivo data in macaques and ferrets) provide no scientific basis for further use of the drug in humans.
