High throughput screening for SARS-CoV-2 helicase inhibitors

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC₅₀ <10 μM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

Keywords: Biochemical assay; COVID-19; HTS; Helicase; nsP13.

Figure 1:

SARS CoV-2 nsP13 assay principle. A schematic diagram of SARS CoV-2 nsP13 assay. The assay measures the fluorescence intensity of FMA that caused by unwinding of dsDNA substrate by SARS CoV-2 nsP13.

Figure 2:

Z-score analysis of SARS CoV-2 nsP13 primary HTS. Graphed is Z-score distribution across all 649,568 compounds tested. The red dots represent hit compound, black dots are non-hit compound, blue dots are low controls and orange dots are high controls.

Figure 3:

SARS CoV-2 nsP13 confirmation HTS result. Graphed is single point scatterplots of all compounds tested for each assay. Each dot graphed represents the activity result of a well containing test compound (black dots) or controls (red, pink and green dots). Overall screening statistics are described in the box below the graph. A. Summary of the result of SARS CoV-2 nsP13 confirmation screen including CRC data of the reference compound, SSYA10–001. B. Summary of the result of additional 3.6K compounds screening.

Figure 4:

Selected hit series studied in medchem round 1. Examples of purchased compounds subjected to the biochemical medchem assays utilizing either the FAM substrate (Red curve) or ATTO 647 substrate (Blue curve) and corresponding IC₅₀ are shown. The graphs display concentration response curves generated by 10pt 3-fold serial dilutions for each compound tested in triplicate. The X-axis is in log molar concentration and the Y-axis is normalized percent activity. Error bars are shown in SD and each point is an N=3 replicates.

Figure 5:

Selected most active hits identified in medchem round 2. Chemical structures, IC50, vendor and part number, and potential target class when known are shown for each molecule. The IC₅₀s shown are associated to the SCV2-NSP13 biochemical helicase assay.