Biology subjects

Stewart, A. E.

Publications and source records attributed to Stewart, A. E..

3 recordsLinked to original sources

bioRxiv2024

Expanding the ligandable proteome by paralog hopping with covalent probes

More than half of the [~]20,000 protein-encoding human genes have at least one paralog. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to a subset of paralogous proteins. Here, we explore whether such covalent compound-cysteine interactions can be used to discover ligandable pockets in paralogs that lack the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we mutated the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling (ABPP) that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide-N112C-CCNE1 interaction into a NanoBRET-ABPP assay capable of identifying compounds that reversibly inhibit both N112C- and WT-CCNE1:CDK2 complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings thus provide a roadmap for leveraging electrophile-cysteine interactions to extend the ligandability of the proteome beyond covalent chemistry.

biochemistry↗

bioRxiv2022

Mediator Kinase Inhibition Impedes Transcriptional Plasticity and Prevents Resistance to ERK/MAPK-Targeted Therapy in KRAS-Mutant Cancers

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this novel class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and tumor-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved population-level process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, tumor-specific resistance programs.

cancer biology↗

bioRxiv2022

Structural basis for Nirmatrelvir in vitro efficacy against the Omicron variant of SARS-CoV-2

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (Mpro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). We evaluated the in vitro catalytic activity and in vitro potency of nirmatrelvir against the main protease (Mpro) of prevalent variants of concern (VOC) or variants of interest (VOI): Alpha (, B.1.1.7), Beta ({beta}, B.1.351), Delta ({delta}, B1.617.2), Gamma ({gamma}, P.1), Lambda ({lambda}, B.1.1.1.37/C37), Omicron (o, B.1.1.529) as well as the original Washington or wildtype strain. These VOC/VOI carry prevalent mutations at varying frequencies in the Mpro specifically for: , {beta},{gamma} (K90R), {lambda} (G15S) and o (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant Mpros demonstrate that they are catalytically comparable to wildtype. Nirmatrelvir has similar potency against each mutant Mpro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the o, {lambda} and {beta} Mpro at 1.63 - 2.09 [A] resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells (1).

biochemistry↗