Covalent Modification: Chemical Biology and Therapeutic Applications

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to directly target this oncogene have faced difficulties due to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. I will discuss the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras G12C. These compounds rely on the mutant cysteine for binding and therefore do not affect the wild type protein (WT). I will also discuss ways to leverage immune cell killing of K-Ras G12C cells treated by combining small molecule inhibitors and bi-specific T-Cell engagers.

Expanding the target landscape of covalent inhibitors requires engaging nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. I will discuss our efforts to develop reversible, lysine-targeted kinase inhibitors, with selectivity driven by differences in residence time.

Direct-acting antivirals for the treatment of COVID-19, which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), are needed to complement vaccination efforts. The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral proliferation and also dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. We have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophilic “warheads” onto analogs of the noncovalent PLpro inhibitor GRL0617. We show that the most promising PLpro inhibitor is potent and selective, with activity in cell-based antiviral assays rivaling that of the RNA-dependent RNA polymerase inhibitor remdesivir. An X-ray crystal structure of the most promising lead compound bound covalently to PLpro establishes the molecular basis for protease inhibition and selectivity against structurally similar human deubiquitinases. These findings present an opportunity for further development of potent and selective covalent PLpro inhibitors.

Bioorthogonal catalysis is an emerging field with applications in drug delivery and protein target identification. Most systems involve the expression of a non-native enzyme for the catalytic generation of biologically relevant compounds. However, targeted small molecule bioorthogonal catalysts would avoid the need for non-native expression. Furthermore, far-red photocatalysts allow for the use of long wavelength light as an external trigger. This work describes the localization of small molecule far-red photocatalysts for targeted uncaging of a biologically active compound. A dihydrotetrazine (DHTz) conjugated to a vinyl ether was used as a stable photocage for the tubulin disrupting compound nCA4. Once oxidized to tetrazine via far-red photocatalysis, an intramolecular Diels-Alder reaction occurs leading to uncaging of nCA4. In cellular studies, photocatalysts were localized to the nucleus or tubulin to observe uncaging. Cells treated with the DHTz photocage without photocatalyst or light demonstrated organized and elongated tubulin structures similar to the vehicle, whereas in the presence of both photocatalyst and light, cells exhibited disorganized tubulin structures similar to the free nCA4 control. Localized uncaging was further confirmed using ascorbate as a tool to quench extracellular photocatalysis. Overall, this work demonstrates the first photocatalytic reaction for directed uncaging at the organelle level in cells.

Activity-based probes (ABPs) are widely used to monitor the activity of enzyme families in biological systems. Inferring enzyme activity from probe reactivity requires that the probe reacts with the enzyme at its active site; however, probe-labeling sites are rarely verified. Here we present an enhanced chemoproteomic approach to evaluate the activity and probe reactivity of deubiquitinase enzymes, using bioorthogonally tagged ABPs and a sequential on-bead digestion protocol to enhance the identification of probe-labeling sites. We confirm probe labeling of deubiquitinase catalytic Cys residues and reveal unexpected labeling of deubiquitinases on non-catalytic Cys residues and of non-deubiquitinase proteins. In doing so, we identify ZUFSP (ZUP1) as a previously unannotated deubiquitinase with high selectivity toward cleaving K63-linked chains. ZUFSP interacts with and modulates ubiquitination of the replication protein A (RPA) complex. Our reactive-site-centric chemoproteomics method is broadly applicable for identifying the reaction sites of covalent molecules, which may expand our understanding of enzymatic mechanisms.

Proteases play a key role in health and disease through a wide range of functions, for example in regulation of paracrine signalling or protein deubiquitination. In my talk I will discuss our recent work on developing and applying novel activity-based probes (ABPs) and substrate-biased ABPs (sbABPs) for the analysis and discovery of known or novel secreted or intracellular protease activities involved in cancer and fibrosis, based on warhead classes with a strong preference for a specific target class. Our emerging data for the deubiquitinases (DUBs) suggest that small scaffold variations around privileged warheads enable in-cell probe or inhibitor specificity to be tuned either to single targets, or to encompass a remarkably broad spectrum of diverse family members, offering the promise of powerful new tools to interrogate DUB activity in intact cells or organisms.

The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19.

This presentation will describe the discovery and characterization of PF-07321332, an orally bioavailable, reversible covalent inhibitor of the SARS-CoV-2 main protease that is currently in clinical trials as a potential treatment for COVID-19. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved plasma concentrations exceeding the in vitro antiviral cell potency in a phase I clinical trial in healthy human participants following oral dosing.