Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline

Abstract

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC₅₀ values below 10 μM, while candesartan cilexetil had an IC₅₀ value of approximately 67 µM against M^pro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC₅₀) value was the only one greater than the limit of the assay (>100 μM).

Keywords: COVID-19; FRET; live virus; machine-learning; main protease; molecular simulations; protein assays; spike protein.

FIGURE 1

Panel (A) shows the docking site on the RBD of the spike protein in red (by residues 501–505) that are at the interface with ACE2 (show in green) and denoted spike1 in the text. A smaller secondary binding site (denoted spike2) in the spike protein in receptor binding motif domain was detected and used for docking studies (B). Panel (C) and (D) show the binding site of the M^pro with the N3 inhibitor removed (6LU7) is protease1 and the apo protein (6Y84) is protease2. The S2 binding pocket is below the sidechains of Met49 and Gln189 and is not visible in the picture.

FIGURE 2

Predicted M^pro drug inhibitors screened using a FRET-based protease assay with five down-selected hits. (A) A schematic of the FRET-based SARS-CoV-2 main protease assay is shown along with the hit identification overview. (B) Purified M^pro and FRET substrate proteins were incubated in the presence of 100 μM of drugs from a library of computationally predicted M^pro inhibitors. No drug, no protease, and Ebselen were used as controls to calculate the Z-factor for each plate and an average score is displayed above. Red dots indicated no drug (0% inhibition) or no protease (100% inhibition) conditions, while the black dots are the ordered percent inhibition values. (C) Identified hits from the primary screen were re-screened at 100 μM and the FRET values were normalized as percent inhibition values in the bar graph. Experiments were performed in duplicate and the presented results are the average values. (D) Verified compounds form rescreening were subjected to half-maximal inhibitory concentration (IC₅₀) analysis. Presented values are averaged from technical duplicate experiments. Black lines and values represent normalized data from FRET values while the red lines and values represent normalized data from gel electrophoresis (Supplementary Figure S1) and densitometry.

FIGURE 3

Molecular of structures for compounds that have a repressive effect on some aspect of the virus activity: (A) candesartan cilexetil, (B) flavin adenosine dinucleotide, (C) lapatinib, (D) tetracycline, (E) tigecycline, (F) imatinib, (G) icotinib, (H) adapalene, and (I) gestrinone.

FIGURE 4

Inhibition of ACE2-RBD binding after pre-treatment with 50 μM compound measured by Biolayer interferometry.

FIGURE 5

Predicted spike drug inhibitors screened using a VSV-SARS2 infection assay reveals two promising hits. (A) Individual drugs from the library set were used at 10 μM to treat GFP reporter viruses, VSV-SARS2 and VSV, for 30 min prior to infection of Vero cells at 0.5 MOI or 0.1 MOI respectively. The infection media was replaced with fresh media at 1 h post-infection and fluorescent reporter values were measured the next day. (B) Half-maximal inhibitory concentration (IC₅₀) curves and values were obtained for Imatinib (compound 20) and Lapatinib (compound 19) using the same VSV-SARS infection assay performed for library screening. All data were normalized as percent infection or inhibition for drug-treated conditions vs. no-treatment control. The values are means, with error bars displaying standard deviation between the triplicate wells.

FIGURE 6

Percentage inhibition and percentage cytotoxicity graphs form SARS-CoV-2 infection studies that show large therapeutic indexes in three hits. Varying concentrations of imatinib, lapatinib, and adapalene were used to treat virus for 30 min prior to infection in Vero cells, while candesartan cilexetil was added directly to cells without pre-treatment to virus. Infections were performed using SARS CoV-2mNeon at an MOI of 0.2. At 1 h post-infection, the media was removed and replaced with fresh media. Fluorescent reporter values were recorded 18 h post-infection. Similarly, Vero cells were treated with varying concentrations of indicated drugs, incubated for 18 h prior to analysis by Presto-Blue assays to assess cytopathic effect. Data were normalized to percent inhibition or percent cytotoxicity for drug-treated cells vs. no-treatment control. The values are means, with error bars displaying standard deviation between the triplicate wells. Half-maximal inhibitory concentration (IC₅₀) curves and values are represented in black while half-maximal cytotoxicity concentration 50 (CC₅₀) curves and values are represented in red.

FIGURE 7

Best-scoring pose from docking for (A) lapatinib, (B) imatinib, and (C) adapalene to the receptor binding domain of the spike protein (spike1 site). Panel (D) shows the best-scoring dock pose for candesartan cilexetil to M^pro. Labels identify protein residues neighboring the docked compounds.