The crowdsourcing initiative is producing powerful antivirals for SARS-CoV-2

The crowdsourcing initiative is producing powerful antivirals for SARS-CoV-2

A recent study published in the journal Sciences describe a crowdsourced, open, structure-based drug discovery program for the master protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Study: Open scientific discovery of key non-covalent SARS-CoV-2 protease inhibitors.  Image source: Corona Borealis Studio/ShutterstockStudy: Open scientific discovery of key non-covalent SARS-CoV-2 protease inhibitors. Image source: Corona Borealis Studio/Shutterstock

The importance of antiviral therapies in controlling COVID-19

Failure to mitigate coronavirus disease 2019 (COVID-19) will result in the virus becoming endemic unless an accessible treatment is available. Antiviral treatments are essential to control COVID-19, and several oral antiviral drugs have been approved, including molnupiravir, nirmatrivir, and ensetrelivir. SARS-CoV-2 Mpro has been an attractive target for drug development, given its role in replication, high degree of conservation across CoVs, and variation with human proteases.

Crowdsourcing drug discovery: the COVID-19 Moonshot initiative

In the current study, researchers report the open scientific discovery of powerful antivirals for SARS-CoV-2. This program, “COVID Moonshot,” was targeting SARS-CoV-2 Mpro, through a rapid screening of fragments that evaluated unique fragment-infused crystals and identified 71 hits that populated the active site. The noncovalent moieties lacked inhibitory activity in the enzymatic assay, but provided a high-resolution map of the interactions.

The team launched the online crowdsourcing platform in March 2020 and asked participants to submit compounds designed based on the fragment findings.

Synthesis and screening

Biochemical assays and X-ray crystallography were used to evaluate the compounds selected for synthesis, the results of which were also released on the same platform. Designs were contributed by the core group (from laboratories and medicinal chemists) and the community.

The contract research organization (CRO), Enamine, was tasked with synthesizing the compounds. The team calculated synthetic pathways for all submissions using CRO-specific elemental inventories and estimated synthetic complexity. The expected synthetic complexity is related to the actual time required to synthesize the target compound. Next, chemical free energy calculations were used to estimate the efficacy of the designs and their analogues from virtual synthetic libraries.

The researchers used a global distributed computing network (Folding@home) to estimate the free energy of binding for all applications submitted. Initially, a small study was conducted using data obtained from the first week of group designs. The results of these calculations correlate well with experimentally determined correlations. Subsequently, chemical free energy calculations were another criterion to guide the selection and iterative design.

Three temporally distinct design campaigns were observed – benzopyran ring decoration, benzopyran system replacement, and isoquinoline system replacement. These calculations helped select effective analogues from virtual libraries and highlighted areas where significant synthetic effort might be needed. As such, the team prioritized small libraries proposed for assembly.

Improving the effectiveness of antivirals through structure and activity analysis

Rapid structure-activity relationship (SAR) evaluation was completed using high-throughput chemistry (HTC) at the nanomole scale, as demonstrated by amide coupling optimizations to expand the MAT-POS-4223bc15-21 and Chan-Lam reaction to expand ADA-UCB-6c2cb422-1. . Seven and 20 compounds from the Chan-Lam and amide series, respectively, were selected for recombination.

The extended compounds had a similar association to the original compounds. One of the compounds in the Chan-Lam series had an inhibitory concentration slightly higher than the half-maximum (IC50) of the original compound. On the other hand, several compounds in the amide series showed up to 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 interaction and plasticity of binding pockets. Pockets P1 and P2 served as hotspots for interactions. Some notable interactions in the P1 pocket sampled by ligands included N145 (hydrophobic), H163 (hydrogen bond donor), and E166 (hydrogen bond acceptor).

In contrast, hydrophobic interactions with M165 and π-stacking interactions with H41 were dominant in P2. Next, the team explored P1, which has a sharp SAR due to its preference for directional hydrogen bond interactions and its rigidity. The potency was increased by replacing pyridine with isoquinoline, and introducing additional interactions with N142.

The SAR around P2 was fairly tolerant to change. The shift in effectiveness was possible by strengthening the scaffold. Specifically, a tetrahydropyran ring was introduced to convert the substituent to a chromane moiety. After that, the chromium was replaced with tetrahydroisoquinoline to maintain potency.

Finally, a library was generated through Schouten-Baumann sulfonamide conjugation that increased inhibitory activity and antiviral efficacy. Overall, this has resulted in a series of potent antivirals with low brain penetration, enhanced oral bioavailability, and a favorable but moderate safety profile. In vitro in vivo Link for clearance.

Promising results: Lead compounds show high efficacy

One of the final strands, MAT-POS-e194df51-1, was evaluated in antiviral assays in different cell lines and was not cytotoxic, showing a mean effective concentration of 126 nM in HeLa-ACE2 cells and 64 nM in A549-ACE2-TMPRSS2 cells. This compound was 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 approved Mpro inhibitors.

Implications and future prospects of the Covid Moonshot findings

Overall, the study demonstrated the success of an open-science, patent-free antiviral discovery program in developing differentiated leadership. Notably, the drug ensetrelvir approved in Japan was identified, in part, based on crystallographic data shared by the Covid Moonshot platform. This project and the COVID-19 lead series have been approved by the Drugs for Neglected Diseases Initiative to further optimize leads and preclinical development.

    (Tags for translation)Coronavirus diseaseCOVID-19

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