Large Library Docking and Biophysical Analysis of Small-Molecule TMPRSS2 Inhibitors.

Area of Science:

• Biochemistry
• Drug Discovery
• Structural Biology

Background:

• Transmembrane protease serine-2 (TMPRSS2) is crucial for SARS-CoV-2 and influenza virus entry into human airways.
• TMPRSS2 is a promising antiviral drug target, but effective oral small-molecule inhibitors are lacking.

Purpose of the Study:

• To identify and characterize novel covalent and noncovalent small-molecule inhibitors of TMPRSS2.
• To establish a screening pipeline for rapid identification of TMPRSS2 inhibitors.
• To validate a new noncovalent inhibitor scaffold for potential therapeutic development.

Main Methods:

• Large-scale molecular docking using homology and crystal structures of TMPRSS2.
• Enzyme kinetics assays with peptide and protein substrates.
• Differential scanning fluorimetry to assess protein stability and binding.
• X-ray crystallography to determine inhibitor-bound structures.

Main Results:

• Identified potential covalent and noncovalent TMPRSS2 inhibitors through docking studies.
• Established a robust pipeline for screening TMPRSS2 inhibitors.
• Determined high-resolution crystal structures of TMPRSS2 with inhibitors like nafamostat, '157, and 6-amidino-2-naphthol.
• Biochemically validated a novel noncovalent inhibitor scaffold.

Conclusions:

• The developed screening pipeline enables rapid identification of TMPRSS2 inhibitors.
• Structural insights were gained into TMPRSS2 inhibition by various compounds.
• A novel noncovalent scaffold shows promise for developing selective TMPRSS2 inhibitors for antiviral therapies.