A recent study published in Science described an open science, structure-activated drug discovery program for the major protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Study: Open Science Discovery of Potent Noncovalent Major Protease Inhibitors of SARS-CoV-2. Image credit: Corona Borealis Studio / Shutterstock
The importance of antiviral therapy in the control of COVID-19
Failure to contain coronavirus disease 2019 (COVID-19) will result in the virus becoming endemic unless affordable treatment is available. Antiviral therapeutics are essential for the control of COVID-19, and several oral antiviral agents have been approved, including molnupiravir, nirmatrelvir, and ensitrelvir. SARS-CoV-2 Mpro is an attractive target for drug development given its role in replication, high degree of conservation in CoVs, and difference with human proteases.
Crowdsourcing Drug Discovery: The COVID Moonshot Initiative
In the present study, researchers report the open scientific discovery of potent antiviral agents for SARS-CoV-2. This program, “COVID Moonshot,” targeted the SARS-CoV-2 Mpro, building a rapid fragment screener that evaluated unique fragment-soaked crystals and identified 71 hits filling the active site. The hits of the non-covalent fragments have no inhibitory activity in an enzymatic assay, but offer a high-resolution map of the interactions.
The team initiated an online crowdsourcing platform in March 2020 and asked participants to submit compounds designed based on fragment hits.
Synthesis and screening
Biochemical analyzes and X-ray crystallography were used to evaluate the compounds selected for synthesis, and their results were also published on the same platform. Projects were provided by the core group (of laboratories and medicinal chemists) and the community.
A contract research organization (CRO), Enamine, was tasked with synthesizing compounds. The team calculated synthetic routes of all submissions using CRO’s building block inventories and estimated synthetic complexity. The predicted synthetic complexity correlates with the actual time required to synthesize a target compound. Alchemical free energy calculations were then used to evaluate the performance of designs and analogs from virtual synthetic libraries.
The researchers used a global distributed computing network (Folding@home) to estimate the binding free energy of all submissions. Initially, a small survey was undertaken using the data generated from the first week of crowdsourcing designs. The results of these calculations correlated well with experimentally determined affinities. Alchemical free energy calculations were then another criterion to guide selection and iterative design.
Three chronologically distinct design campaigns were observed – benzopyran ring decoration, benzopyran system replacement, and isoquinoline system replacement. These calculations helped select potent analogs from virtual libraries and highlighted where significant synthetic efforts would be needed. As such, the team prioritized small libraries proposed for synthesis.
Optimizing antiviral performance through structure-activity analysis
Rapid structure-activity relationship (SAR) evaluation was complemented using high-throughput chemistry (HTC) at the nanomole scale, as demonstrated by the amide coupling optimizations for the expansion of MAT-POS-4223bc15-21 and the Chan reaction -Lam to extend ADA- UCB-6c2cb422-1. Seven and 20 compounds of the Chan-Lam and amide series, respectively, were selected for resynthesis.
The extended compounds have similar binding as the parent compounds. One of the compounds in the Chan-Lam series has a slightly higher half-maximal inhibitory concentration (IC50) than the parent compound. On the other hand, several compounds in the amide series show up to a 300-fold improvement in IC50 relative to the parent compound.
Crystal soaking and X-ray diffraction yielded 587 structures. A subset of structures was analyzed that revealed hotspots of ligand engagement and plasticity of binding pockets. Pockets P1 and P2 were hotspots of interactions. Some notable interactions in the P1 pocket taken up by the ligands include N145 (hydrophobic), H163 (hydrogen bond donor), and E166 (hydrogen bond acceptor).
In contrast, hydrophobic interactions with M165 and π-stacking interactions with H41 are predominant in P2. Next, the team investigated P1, which has a steep SAR due to its preference for directional interactions and hydrogen bond rigidity. Efficacy is increased by replacing pyridine with isoquinoline, introducing additional interactions with N142.
The SAR around P2 was significantly tolerant to change. The change in potency was made possible by stiffening the scaffold. In particular, a tetrahydropyran ring was introduced to transform the substituent into a chromane moiety. The chroman is then replaced with tetrahydroisoquinoline to maintain potency.
Finally, a library was constructed by Schotten-Baumann sulfonamide coupling, which enhanced inhibitory activity and antiviral efficacy. Overall, this has resulted in a series of potent antivirals with low brain penetration, increased oral bioavailability, and a favorable safety profile, but moderate in vitro-in vivo correlation for clearance.
Promising results: Lead compounds show high efficacy
One of the terminal leads, MAT-POS-e194df51-1, was evaluated in antiviral assays in different cell lines and was not cytotoxic, showing an average effective concentration of 126 nM in HeLa-ACE2 cells and 64 nM in A549-ACE2-TMPRSS2 cells. This compound is also cross-reactive against SARS-CoV-2 variants. Crystallographic analysis showed that the interactions of this compound with the Mpro binding site were different from those of the approved Mpro inhibitors.
Implications and future perspectives of the findings of the COVID Moonshot
Overall, the study demonstrates the success of an open science off-patent antiviral discovery program in developing a differentiated lead. It should be noted that the ensitrelvir approved in Japan was identified in part based on crystallographic data shared by the COVID Moonshot. This project and the lead COVID-19 series were accepted into the Neglected Diseases Medicines Initiative for further lead optimization and preclinical development.
