Identification of Inhibitors of the TMPRss2 and SPIKE Interaction Through Novel uHTS Assays and Molecular Docking

Abstract

<b>Abstract ID 53428</b> <b>Poster Board 93</b> The few therapies available for SARS-CoV-2 have limited areas of effectiveness and resistance is inevitable. Identification of inhibitors of viral entry has been challenging and have focused on the strong interaction between ACE2 and SPIKE. However, the interaction between TMPRss2 and SPIKE is also critical for viral entry and identification of inhibitors of this interaction could be therapeutic starting points. Therefore, we seek to develop ultra high-throughput assays for identification of inhibitors of the TMPRss2 and SPIKE interaction and prioritize top-hit compounds based on biophysical and molecular docking. We first created a Time Resolved-Forster/Fluorescence Energy Transfer (TR-FRET) assay utilizing fluorescently labeled antibodies to detect interactions between overexpressed SPIKE and TMPRss2 proteins in cell lysates. To further narrow the hits from this TR-FRET screen, we developed an orthogonal uHTS Nanoluciferase Binary Technology (NanoBiT) assay to detect the interaction between tagged SPIKE and TMPRss2 in live cells. With these orthogonal assays, we expanded our TR-FRET screen to over 100,000 compounds. Several compounds were found to show activity in both the TR-FRET and the NanoBiT assay. Thermal shift assays further narrowed down compounds by identifying compounds that bound to either SPIKE or TMPRss2 purified recombinant proteins, prioritizing potential compounds of interest. Next, molecular docking was done to identify which of the top compounds may directly disrupt the SPIKE/TMPRss2 interaction. MM-GBSA binding energies were calculated to determine potential ligand binding sites for each compound on the interface of TMPRss2 and SPIKE. Binding energies were also calculated for compound binding to allosteric sites on SPIKE or TMPRss2, based on which protein they interacted with in thermal shift data. SiteScores were also calculated for each binding site of the compounds to identify at which binding site they were most likely to interact with the protein of interest. A detailed evaluation of the binding energies at each site revealed the contributions of various types of bonds contributing to the binding energy of the compounds at each binding site and the residues important for the ligand and protein interaction. This revealed a potential mechanism of action for these candidate TMPRss2/SPIKE interaction disruptors. For example, eltrombopag was found to have favorable energy for being an allosteric modulator of the interaction, but also may act as direct disruptor of the interaction. Thus, we have identified several potential inhibitors of the SPIKE/TMPRss2 interaction through the use of our newly developed orthogonal TR-FRET and NanoBiT uHTS assays and characterized the binding sites via molecular docking. These uHTS assays may be a tool for future TMPRss2/SPIKE disruptor discovery and the identified candidate compounds could provide promising groundwork for further development of inhibitors of SARS-CoV-2. This research was supported in part by The Emory School of Medicine COVID Catalyst-I3 award, the NCI Emory Lung Cancer SPORE (HF, P50CA217691) Career Enhancement Program (AAI, P50CA217691), Emory initiative on Biological Discovery through Chemical Innovation (AAI)<b>.</b>