Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors.

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly "druggable" or relatively easy-to-drug target is the coronavirus Main Protease (3CLpro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.

SILAC-based chemoproteomics reveals a neoligan analogue as an anti-inflammatory agent targeting IRGM to ameliorate cytokine storm.

Cytokine storm is a key feature of sepsis and severe stage of COVID-19, and the immunosuppression after excessive immune activation is a substantial hazard to human life. Both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are recognized by various pattern recognition receptors (PRRs), which lead to the immune response. A number of neolignan analogues were synthesized in this work and showed powerful anti-inflammation properties linked to the response to innate and adaptive immunity, as well as NP-7 showed considerable anti-inflammatory activity at 100 nM. On the sepsis model caused by cecum ligation and puncture (CLP) in C57BL/6J mice, NP-7 displayed a strong regulatory influence on cytokine release. Then a photo-affinity probe of NP-7 was synthesized and chemoproteomics based on stable isotope labeling with amino acids in cell cultures (SILAC) identified Immunity-related GTPase M (IRGM) as a target suppressing cytokine storm, which was verified by competitive pull-down, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) and molecular dynamics simulations.

Selectivity aspects of activity-based (chemical) probes.

Selective chemical modulators are ideal tools to study the function of a protein. Yet, the poor ligandability of many proteins has hampered the development of specific chemical probes for numerous protein classes. Tools, such as covalent inhibitors and activity-based protein profiling, have enhanced our understanding of thus-far difficult-to-target proteins and have enabled correct assessment of the selectivity of small-molecule modulators. This also requires deeper knowledge of compound and target site reactivity, evaluation of binding to noncovalent targets and protein turnover. The availability of highly selective chemical probes, the evolution of activity-based probes, and the development of profiling methods will open a new era of drugging the undruggable proteome.