SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

Targeting CTP Synthetase 1 to Restore Interferon Induction and Impede Nucleotide Synthesis in SARS-CoV-2 Infection.

The newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.

Synthesis and Target Identification of a Novel Electrophilic Warhead, 2-Chloromethylquinoline.

Despite its power in identifying highly potent ligands for select protein targets, conventional medicinal chemistry is limited by its low throughput and lack of proteomic selectivity information. We seek to develop a chemoproteomic approach for discovering covalent ligands for protein targets in an unbiased, high-throughput manner. Tripartite probe compounds composed of a heterocyclic core, an electrophilic "warhead", and an alkyne tag have been designed and synthesized for covalently labeling and identifying targets in cells. We have developed a novel condensation reaction to prepare 2-chloromethylquinoline (2-CMQ), an electrophilic heterocycle. These chloromethylquinolines potently and covalently bind to a number of cellular protein targets, including prostaglandin E synthase 2 (PTGES2), a critical regulator of cell proliferation, apoptosis, angiogenesis, inflammation, and immune surveillance. The 2-CMQs that we have developed here are novel PTGES2 binders that have the potential to serve as therapies for the treatment of human diseases such as inflammation.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor.

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.