Biology subjects

Gorham, L. J.

Publications and source records attributed to Gorham, L. J..

4 recordsLinked to original sources

bioRxiv2025

Chemoproteomic elucidation of β-lactam drug targets in Mycobacterium abscessus

The pathogen Mycobacterium abscessus (Mab) can cause severe and difficult to treat chronic lung infections. Despite the rising incidence and clinical concern of Mab infections, treatment options are limited and often ineffective. Treatment is complicated by Mabs ability to persist in a non-replicative, drug-resistant state. Several {beta}-lactam antibiotics are potently bactericidal against Mab but are underutilized because their molecular mechanisms of action against Mab are incompletely understood. In the current study, we used {beta}-lactam-derived activity-based probes and chemoproteomics to report the first comprehensive list of enzymes in Mab targeted by {beta}-lactams. We compared {beta}-lactam targets across two Mab subspecies in actively replicating and non-replicative cultures, using a new carbon starvation model of persistence. We identified 17 targets that were active in every condition tested, seven of which were previously unknown to bind {beta}-lactams. Lastly, we characterized the {beta}-lactamase activity and {beta}-lactam inhibition profiles of nine Mab enzymes, demonstrating that imipenem inhibits these targets more effectively than cefoxitin. These findings provide clarity on the mechanisms of action of clinically relevant {beta}-lactams in Mab, a crucial step toward fully realizing their potential for treating infections caused by this opportunistic pathogen.

microbiology↗

bioRxiv2025

Activity-targeted metaproteomics enhances the ecophysiological characterization of cryptic syntrophic metabolisms

Syntrophic microbial consortia can contribute significantly to the activity and function of anoxic ecosystems, yet are often too rare to study their in situ physiologies using traditional molecular methods. Here, we combined bioorthogonal non-canonical amino acid tagging (BONCAT), stable isotope probing, and metaproteomics to improve the recovery of proteins from active members and track isotope incorporation in an anaerobic digestion community. Both click chemistry-enabled cell-sorting and direct protein pulldown coupled to metaproteomics improved recovery of isotopically labeled proteins during anaerobic acetate oxidation. Resulting labeled protein expression profiles revealed elevated activity of a rare and so-far uncharacterized syntrophic bacterium belonging to the family Natronincolaceae. BONCAT-based capture of newly translated proteins provided direct molecular evidence for the expression of a previously hypothesized oxidative glycine pathway for syntrophic acetate oxidation by this microorganism, showcasing the potential of targeted metaproteomics to characterize rare and active cells central to community metabolism in natural and engineered ecosystems.

microbiology↗

bioRxiv2025

Comprehensive identification of β-lactam antibiotic polypharmacology in Mycobacterium tuberculosis

Infections with Mycobacterium tuberculosis (Mtb) cause tuberculosis (TB), which requires at least six months of treatment with multiple antibiotics. There is emergent interest in using {beta}-lactam antibiotics to improve treatment outcomes for patients. These drugs target cell wall biosynthesis, but a comprehensive list of enzymes inhibited by {beta}-lactams in Mtb is lacking. In the current study, we sought to identify and characterize Mtb enzymes inhibited by {beta}-lactam antibiotics using physiological conditions representative of both acute and chronic TB disease. We used new activity-based probes based on the {beta}-lactam antibiotic meropenem due to its approval by the World Health Organization for TB treatment. Activity-based probes label enzymes based on both substrate specificity and catalytic mechanism, enabling precise identification of drug targets. We identified previously undiscovered targets of meropenem in addition to known cell wall biosynthetic enzymes. We validated {beta}-lactam binding and hydrolysis for six newly identified targets: Rv1723, Rv2257c, Rv0309, DapE (Rv1202), MurI (Rv1338), and LipD (Rv1923). Our results demonstrate that there are at least 30 enzymes in Mtb vulnerable to inhibition by meropenem. This is many more {beta}-lactam targets than historically described, suggesting that efficacy in Mtb is a direct result of polypharmacology.

biochemistry↗

bioRxiv2025

Discovery of non-opioid receptor protein targets of fentanyl across tissues from animal models and humans using photoaffinity probes

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyls interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyls adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl. Affinity-based protein profiling using the FA-T1 probe in vitro in tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.

pharmacology and toxicology↗