Authors

D Whalley¹; A Arduin¹; S Ward¹; R Shannon¹; R Clark¹; V Lazari¹; 
¹ Discovery from Charles River, UK

Discussion

A promising anti-viral drug target is the Main Protease (M^pro or 3CL protease) of SARS-CoV-2 which is a critical component of viral replication due to its ability to cleave polyproteins pp1a and pp1ab and yield various non-structural proteins with functions such as viral mRNA methylation and host DNA unwinding. Proof of concept that modulation of M^pro activity protects against infection has been demonstrated by Pfizer with PAXLOVID™ (PF-07321332; ritonavir) recently entering Phase 2/3 clinical trials (1).

At Charles River Early Discovery, we have developed a fluorogenic M^pro biochemical assay that detects the cleavage of a peptide substrate, derived from the N-terminal autocleavage sequence of the protease (TSAVLQ-|-SGFRK). The assay has been robustly optimised in terms of protein concentration, steady-state kinetic parameters and linearity.

Influenced by the achievements of the COVID Moonshot project (2), and associated Diamond Light Source X-Chem fragment-screen (3), we performed a fragment-screen composed of diverse fluorinated fragments. Conventionally, ¹⁹F fragments provide a benefit to NMR as the fluorine atom is not present within proteins, resulting in low background, however screening using a functional biochemical platform is possible if assay DMSO tolerance is good and the technology employed is not particularly sensitive to compound-mediated interference.

Here we present the results of this screen that yielded thirty reproducibly active compounds with IC₅₀ values down to 300 µM, some of which could offer suitable starting points for further SARS-CoV-2 antiviral drug discovery efforts. Furthermore, we describe the rapid generation of apo SARS-CoV M^pro crystals and introduce our internal crystal structure which will be a key and versatile tool to understand the structure activity relationship (