From crystallographic fragment screen to preclinical candidate: Open science discovery of SARS-CoV-2 antivirals

The development of novel, low cost and globally available antiviral therapeutics remains an essential goal for the current SARS-CoV-2 pandemic. Furthermore, future pandemics could be prevented with easily deployable broad-spectrum oral antivirals and open knowledge bases that de-risk and accelerate novel antiviral discovery and development. To identify starting points for the development of such therapeutics, the XChem team at Diamond Light Source, in collaboration with various international colleagues, performed large crystallographic fragment screens against 8 key SARS-CoV-2 protein targets including the Main protease1, the Nsp3 macrodomain2 and the helicase Nsp133. The expeditious collection and open dissemination of the data from these fragment screening campaigns was enabled by the well-established platform at Diamond Light Source and by the implementation of various experimental and computational tools. This work identified numerous starting points for the development of potent anti-viral therapeutics as exemplified by the COVID Moonshot - a fully open-science structure-enabled drug discovery campaign targeting the SARS-CoV-2 main protease. 4 By leveraging crowdsourced medicinal chemistry design, high throughput structural biology, machine learning and exascale molecular simulations, we discovered a novel chemical scaffold that is differentiated to current clinical candidates in terms of toxicity and pharmacokinetics liabilities, and developed it into orally-bioavailable inhibitors with clinical potential within 2 years. All compound designs, structural data, assay data and synthesized molecules have been shared rapidly and openly, creating a rich, IP-free knowledgebase for future anti-coronavirus drug discovery.

A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.