A Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

Abstract

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allowed for rapid movement of existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.

Figure 1.

(A) Scheme displaying the enzymatic assay principle for the fluorogenic peptide substrate. The fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC has low fluorescence compared to the fluorescent 7-amino-4-methylcoumarin (AMC), which is released upon proteolytic cleavage. The scissile bond is indicated in red. (B) Schematic of the truncated yeast-expressed recombinant TMPRSS2 used in the fluorogenic assay, containing the low-density lipoprotein receptor A (LDLRA) domain, scavenger receptor cysteine-rich (SRCR) domain and protease domain.

Figure 2.

Assay performance for primary screening of the compound libraries in the TMPRSS2 fluorogenic biochemical activity assay. Z’ scores and signal-to-background values are plotted as data points for each plate. There were 32 positive and 64 negative control wells on each 1536-well plate. (A) NCATS Pharmaceutical Collection (NPC), 2,678 compounds, (B) Mechanism Interrogation Plate (MIPE) library, 2,480 compounds and (C) protease inhibitor library (PIL), 872 compounds. Black horizontal lines represent the mean values.

Figure 3.

Label-free mass spectrometry biochemical assay. (A) Peptide derived from the known S2’ cleavage site of SARS-CoV-2 Spike (S) protein. (B) Scheme displaying the enzymatic assay principle for the unlabeled peptide substrate. The unlabeled peptide substrate, Cbz-SKPSKRFIED, is cleaved by TMPRSS2 to create two cleavage products, Cbz-SKPSKR and SFIED. The scissile bond is indicated in red. (C) Initial velocity (V₀) was calculated for each substrate concentration by plotting product formation vs time. (D) The V₀ for each concentration were plotted against the various substrate concentrations to obtain the V_max and K_m. V_max: 1.95 μmol/min and K_m: estimated 2320 μM. (E) Comparison of TMPRSS2 fluorogenic detection and mass spectrometry detection assays by assessing dose-response inhibition by camostat and gabexate. (F) Mass spectrometry traces of the cleavage product (m/z: 418.8/702.4) showing that addition of camostat prevents product formation in dose-response. Each peak is labeled with the concentration of camostat (nM) for that condition. (G) Assay performance from the mass spectrometry detection assay when screening the hits identified from the primary screening. Z’ of 0.71 and S:B of 7.37. (H) Dose-response inhibition of 7-hydroxycoumarin against TMPRSS2 in both fluorogenic and mass spectrometry detection assays showing the fluorescent molecule as a false-positive hit in the fluorogenic assay, but not interfering with the mass spectrometry detection assay.

Figure 4.

Cell-based pseudotyped particle assay. (A) Pseudotyped particle production. Three plasmids (pCMV-MLVgag-pol, pcDNA-SARS-CoV-2 spike, and pTG-Luciferase) are co-transfected into HEK-293T/17 cells. The plasmids express MLV core gag-pol polyprotein, SARS-CoV-2 spike glycoproteins, and luciferase RNAs, which together assemble into pseudotyped particles. (B) Pseudotyped particle assay scheme. Order of addition of reagents to Calu-3 cells is 1. compounds, 2. pseudotyped particles and 3. the Promega reagent Bright-Glo. Once cell entry of pseudotyped particle occurs, RNAs of pseudotyped particles are released into the cell, where they are reverse transcribed into DNAs, integrated into the genome and express luciferase reporter enzyme. The amount of luminescence following addition of Bright-Glo is proportional to the amount of pseudotyped particle entry into cells. Illustration was made with BioRender. Activity of clinically-approved inhibitors in prevention of pseudotyped particle entry using the Spike sequence from the Delta variant (blue), WA1 + D614G variant (red) and cytotoxicity counter-assay (black). The molecular structures and dose-response inhibition of particle entry by (C) camostat, (D) nafamostat, (E) gabexate, (F) bortezomib. The calculated concentrations required for 50% inhibition (IC₅₀) are displayed in μM.