Biology subjects

Gomez, J. E.

Publications and source records attributed to Gomez, J. E..

3 recordsLinked to original sources

bioRxiv2025

A multiplexed, target-based phenotypic screening platform using CRISPR interference in Mycobacterium abscessus

The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 809 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step towards accelerating early-stage drug discovery.

microbiology↗

bioRxiv2025

Reference-based chemical-genetic interaction profiling to elucidate small molecule mechanism of action in Mycobacterium tuberculosis

In an era of increasing resistance, new and effective strategies are needed for antibiotic discovery. Whole-cell active screens yield candidate compounds lacking mechanism-of-action (MOA) information and thus do not provide biological insight for prioritization. We previously reported PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of Mycobacterium tuberculosis mutants, each depleted of a different essential protein target. PROSPECT facilitates efficient hit prioritization by simultaneously identifying whole-cell active compounds with high sensitivity and providing early insights into their MOA. Here, we report a reference-based approach to infer MOA from often complex PROSPECT data. For this aim, we curated a reference set of 437 compounds with published, annotated MOA and known or suspected antitubercular activity, and applied PROSPECT to it. We then developed Perturbagen CLass (PCL) analysis, a computational method that predicts MOA by comparing chemical-genetic interaction profiles of unknown compounds to those of this reference set. In leave-one-out cross-validation, PCL analysis correctly predicted MOA with 70% sensitivity and 75% precision. When applied to 75 antitubercular leads with known MOA previously reported by GlaxoSmithKline (GSK), PCL analysis similarly achieved 69% sensitivity and 87% precision. We also analyzed 98 GSK compounds lacking MOA information, predicting 60 of them to act via a reference MOA, and followed up with functional validation of 29 compounds predicted to target respiration-related MOAs. Finally, we applied PROSPECT and PCL analysis to [~]5,000 compounds from larger unbiased libraries that had not been preselected for antitubercular activity. PCL analysis identified a novel scaffold lacking wild-type activity but predicted to inhibit respiration via QcrB, and we confirmed this prediction while chemically optimizing this scaffold to achieve wild-type activity. PCL analysis of PROSPECT data thus enables rapid MOA assignment and hit prioritization, advancing the discovery of new, potent antitubercular compounds.

microbiology↗

bioRxiv2024

Structure and inhibition mechanisms of Mycobacterium tuberculosis essential transporterefflux protein A

A broad chemical genetics screen in Mycobacterium tuberculosis (Mtb) to identify inhibitors of established or previously untapped targets for therapeutic development yielded compounds (BRD-8000.3 and BRD-9327) that inhibit the essential efflux pump EfpA. To understand the mechanisms of inhibition by these compounds, we determined the structures of EfpA with inhibitors bound at 2.7 -3.4 [A] resolution. Our structures reveal different mechanisms of inhibition for the two inhibitors. BRD-8000.3 binds in a tunnel making contact with the lipid bilayer and extending toward the central cavity to displace the fatty acid chain of a lipid molecule bound in the apo structure, suggesting its blocking of an access route for a natural lipidic substrate, in contrast to its uncompetitive mechanism for the small molecule substrate ethidium bromide which likely enters through an alternative tunnel. Meanwhile, BRD-9327 binds in the outer vestibule without complete blockade of the substrate path to the outside, suggesting its possible inhibition of the dynamical motion necessary for "alternate access" to the two different sides of the membrane, as is characteristic of major facilitator superfamily (MFS) transporters. Both inhibitors may have a role in inhibiting the "alternate access" mechanism that could account for the uncompetitive nature of their efflux of some substrates. Our results explain the basis of the synergy of these inhibitors and their potential for combination in a multi drug strategy for anti-tuberculosis therapy. They also potentially point to a possible function for this essential efflux pump as a lipid transporter. The structures provide a foundation for rational modification of these inhibitors to increase potency.

biochemistry↗