High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2

Abstract

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

Keywords: 3CL-PRO; COVID-19; Cell-based; HTS; M-PRO; PL-PRO.

Fig. 1

Schematic of Assay Design. A. Schematic of the HA-tagged 3CLpro protein encoded by SARS-CoV-2 nsp5 . B. Schematic of the FLuc reporter construct. C- and N-terminal portions of Firefly luciferase gene are separated by a target peptide containing the cleavage sequence from the junction of nsp4/5. N- and C-terminal DnaE inteins assist in dimerization once cleaved (Q is the cleavage site). C. Schematic of the assay. In the presence of 3CLpro, the target peptide is cleaved and FLuc domains dimerize and are catalytically active.

Fig. 2

3CLpro ReFRAME Primary HTS assay results. Graphed is a single point scatterplot of all 13,135 compounds tested. Each dot graphed represents the activity result of a well containing test compound (black dots) or controls (red, violet, and green dots).

Fig. 3

Downstream characterization of 4 HTS active inhibitors. The 4 compounds selected from the HTS campaign with their dose response results and structures are shown in A. The blue curves represent the 3CLpro response while the red is the cytotoxicity response. These 4 compounds were confirmed in the 96-well confirmation assays against 6 different viral constructs (B). Tranilast and analogs were tested in the enzymatic inhibition assay and a representative graph with the Disulfiram control, Tranilast, and the most active inhibitor 32229-1 dose responses is shown in C. The corresponding table of results from the different assays is displayed in D.