| |
Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing
|
| |
| |
Nature July 24 2020
Several major target classes were found to be
enriched for activity in this analysis, including ion channels, GPCRs, proteases, and kinases (Supplementary Table S3, Figure 3a).
We report the identification of 21 molecules, including remdesivir, which were confirmed to possess dose/activity relationships, and 13 of these compounds were found to harbor EC50 values <500 nM in at least one cell line (Figure 3b, 5a-b, ED6c, ED7).
Abstract
The emergence of the novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to require at least 12-18 months, and the typical timeline for approval of a novel antiviral therapeutic can exceed 10 years. Thus, repurposing of known drugs could significantly accelerate the deployment of novel therapies for COVID-19. Towards this end, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. We report the identification of 100 molecules that inhibit viral replication, including 21 known drugs that exhibit dose response relationships. Of these, thirteen were found to harbor effective concentrations likely commensurate with achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4, and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334. Notably, MDL-28170, ONO 5334, and apilimod were found to antagonize viral replication in human iPSC-derived pneumocyte-like cells, and the PIKfyve inhibitor also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, the known pharmacological and human safety profiles of these compounds will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.
Impact of Antivirals on the SARS-COV-2 Life Cycle
We next performed studies to evaluate if five of the most potent compounds identified in this study, apilimod, VBY-825, ONO 5334, Z LVG CHN2 and MDL 28170, act on entry or post-entry steps of the viral life cycle. We first conducted time of addition studies, where compared the effect of the compound either administered concurrently with viral infection, or two and five hours after viral challenge, to allow for viral entry (Figure 4a). To further corroborate these results, we also evaluated the impact of these molecules on infectivity of a VSV-based virus-like particles (VLPs) pseudotyped with SAR-CoV-2 S protein, MERS S protein, or VSV G protein (Figure 4b). Data from these two experiments indicate that these compounds inhibit the entry step of viral replication. Importantly, the protease inhibitors VBY-825, ONO 5334, Z LVG CHN2 and MDL 28170, were found to lack potent inhibitory activity upon 3C-like protease (3CLpro) and papain-like protease (PLpro) encoded by SARS-CoV-2 targeting, indicating that antiviral activities are based on inhibition of host proteases (Figure ED8). Z LVG CHN2 targets cysteine proteinases produced by group A streptococci, and has also been shown to suppress herpes simplex virus (HSV) replication by inhibiting the enzymatic activity of HSV-encoded cysteine protease42.
Thus, Z LVG CHN2 likely acts through inhibition of an endosomal protease, although its precise cellular target is unknown. MDL 28170 is a cathepsin B inhibitor that also impairs infection by SARS-COV-1 and Ebola virus (EBOV)29,43, ONO 5334 is a cathepsin K inhibitor, and VBY-825 acts as a reversible cathepsin protease inhibitor. Human cysteinyl cathepsins are required for the proteolytic processing of virally encoded proteins during infection44-46, and cathepsin activity is likely required for proper processing of the SARS-CoV-2 S protein within the endosome in order to activate its fusogenic acitivity45. Importantly, ONO 5334 was found to be well tolerated in phase II clinical trials for the treatment
of osteoporosis, and development was only discontinued due to an unfavorable competitive landscape47,48.
Dose response and synergy analysis
Although highly dependent on a compound’s pharmacokinetic properties, therapeutic dose ranges are typically expected to track to cellular EC50s below 1 μM concentrations. Therefore, we conducted a dose response analysis to determine the relationship between compound concentration and antiviral activity (Figure 3a-b and ED6b-c). In addition to remdesivir, 20 compounds harbored discernable dose-dependent antiviral activities, most of which could be segregated based on broad functional, structural, or target-based classes (Figure 3a-b, ED6b-c).
Importantly, we observed a significant divergence between cellular toxicity and antiviral activities (Figure 3a and ED6a), underscoring that the impact of these compounds on replication is well outside a range where cytotoxic or cytostatic activities of the molecules may be impacting measurements of viral growth (Supplementary Table S4).
We next evaluated potential synergies between known drugs confirmed in dose-response and remdesivir. Four compounds were observed to have notable levels of synergy with remdesivir, including hanfangchin A (also known as Tetrandrine), which was one of the two antimalarials validated in this study (Figure 3c). Hanfangchin A is a bis-benzylisoquinoline alkaloid that has been shown to inhibit multiple Ca+2 channels34, and has previously been reported to antagonize entry of Ebola virus in vitro and in vivo through the targeting of endosomal two pore channels (TPCs)35. However, the cellular antiviral activities of chloroquine derivatives have not been recapitulated in clinical trials36,37, possibly due to the inability to achieve a safe and efficacious concentration required for antiviral activities in patients38. These data suggest that a combinatorial approach should be further investigated to determine if in vivo and clinical synergies exist between the two drugs at acceptable safety margins.
Discussion
Since the beginning of January 2020, an extraordinary number of investigational programs and clinical trials has been initiated in a concerted effort to identify therapeutics against the rapidly growing COVID-19 pandemic. Critically, remdesivir was recently granted EUA for the treatment of COVID-19 based on data from a clinical trial conducted by NIAID which demonstrated significantly improved time to recovery (47%) in treated patients53. However, this therapeutic endpoint is far from optimal, and the elucidation of additional candidate therapies would enable the development of combinatorial regimens (“cocktails”), which would reflect the current treatment strategies for HIV-1 and hepatitis C virus (HCV)54-56.
Here, we report the high-throughput analysis of approximately 12,000 known drugs evaluated for activity against SARS-CoV-2 replication, revealing approximately 100 known drugs with antiviral activities (Figure ED10). Based on the known compound mechanisms of action, we extrapolated a cell map of druggable targets, pathways, biological processes and small molecules that modulate the SARS-CoV-2 replication cycle (Figure ED4). Several major target classes were found to be
enriched for activity in this analysis, including ion channels, GPCRs, proteases, and kinases (Supplementary Table S3, Figure 3a). It is important to note that selectivity and off-target activities of the identified compound can vary, and thus observed antiviral activities may derive from either modulation of the annotated drug target, or an off-target activity based on binding to a protein in the same or divergent family.
For example, while found that the activities of a RAR agonist could be reversed with the application of a RAR antagonist (Figure 2b), however similar relationships could not be established for several GPCR agonists that were evaluated (Figure ED3b). This is potentially suggestive of off-target activities underlying the antiviral effects of some of these molecules.
We report the identification of 21 molecules, including remdesivir, which were confirmed to possess dose/activity relationships, and 13 of these compounds were found to harbor EC50 values <500 nM in at least one cell line (Figure 3b, 5a-b, ED6c, ED7). The pharmacokinetic properties of each individual compound, including factors such as serum protein binding and bioavailability in the lung, will impact potential in vivo antiviral efficacy. However, in conjunction with safety data from phase I multiple ascending dose studies, as well as reported peak serum concentrations in humans (Cmax), these cellular potencies suggest that many of these known drugs may harbor sufficient antiviral activities during therapeutic administration. To enable prioritization of known drugs for in vivo preclinical and clinical evaluation for the treatment of
SARS-CoV-2, a summary of the publicly disclosed and relevant preclinical and clinical properties of the most advanced among these molecules are annotated in Supplementary Table S4. Thus, the availability of human safety and pharmacological data of clinical-stage molecules is expected to enable rapid preclinical and clinical assessment of these
compounds. However, expedited regulatory review under EUA guidelines also provides a rationale for the development of earlier stage candidate molecules that can be deployed for use during the current pandemic outbreak. It is critical that multiple therapeutic options that demonstrate efficacy against SARS-CoV-2 become available to mitigate potential emergence of drug resistance, as well as enable the evaluation of optimal therapeutic cocktails that are broadly curative for COVID-19 disease.
|
|
| |
| |
|
|
|
