Biology subjects

Walker, S. G.

Publications and source records attributed to Walker, S. G..

4 recordsLinked to original sources

bioRxiv2025

Selectivity of the time-dependent M. tuberculosis LeuRS inhibitor ganfeborole is driven by target vulnerability

Ganfeborole (GSK3036656) inhibits the Mycobacterium tuberculosis leucyl-tRNA-synthetase (mtLeuRS) and is in phase 2a clinical trials for the treatment of tuberculosis. Here we show that ganfeborole is a time-dependent inhibitor of mtLeuRS (IC50 1 nM) and generates a post-antibiotic effect of 77 h at 50xMIC (MIC 0.058 M) with M. tuberculosis H37Rv (Mtb), indicating that mtLeuRS is a highly vulnerable drug target and supporting the excellent in vivo efficacy of the drug. Ganfeborole is also a potent time-dependent inhibitor of Escherichia coli LeuRS (ecLeuRS, IC50 2 nM), however no antibacterial activity is observed towards E. coli up to 1 mM ganfeborole despite the observation that less potent ganfeborole analogs have antibacterial activity. To rationalize this observation, we propose that ganfeborole forms a complex with AMP that binds to the ecLeuRS editing site but does not impact aminoacylation. In support, addition of 12.5 M norvaline generates a ganfeborole MIC of 0.4 M since ecLeuRS is unable to hydrolyze norvaline-tRNALeu. Additionally, mutations that reduce the affinity and residence time of ganfeborole-AMP on ecLeuRS result in antibacterial activity. We propose that the activity of ganfeborole towards Mtb is because mtLeuRS is a highly vulnerable target so that only low levels of enzyme need to be inhibited by the ganfeborole-tRNALeu complex in contrast to ecLeuRS, which we previously demonstrated is a low vulnerability target.

biochemistry↗

bioRxiv2025

Identifying Modulators of the Post-Antibiotic Effect

The post-antibiotic effect (PAE) is the delay in bacterial regrowth following antibiotic removal. It has important implications for dosing regimens since drugs that have extended activity following their elimination can be dosed less frequently, widening the therapeutic window. While the PAE has been associated with target vulnerability and the rate of target turnover, little is known about the genetic components that modulate the PAE. Here, we developed a high-throughput assay to screen the Escherichia coli Keio collection of [~]4000 deletion strains, identifying genes that enhance the PAE for CHIR-090, an inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). This screen revealed approximately 400 gene knockouts that enhanced the PAE of CHIR-090. The list of PAE enhancers was enriched for genes involved in transmembrane transport and outer membrane synthesis. Notably, deletion of the rfaE gene, which is involved in lipopolysaccharide (LPS) biosynthesis, increased the PAE of the LpxC inhibitors CHIR-090 and LPC-058 by 2 h and 3 h, respectively. Consistent with this phenotype, co-treatment of wild-type E. coli with an RfaE inhibitor increased the PAE of CHIR-090 or LPC-058 by 1 h. To probe the mechanism of this interaction, we measured the rate of LpxC turnover and found that knocking out rfaE reduced its half-life by 2-fold, suggesting that disrupting RfaE increases the stability of LpxC, increasing target vulnerability and enhancing the PAE of LpxC inhibitors. SIGNIFICANCEAntibiotics are generally dosed at very high levels leading to unwanted side effects and non-compliance, which in turn results in the emergence of drug-resistant bacterial infections. The goal of this work was to develop strategies that will enable antibiotics to be dosed at lower levels, thereby improving safety and compliance. In the present work, we have screened 4,000 strains of Escherichia coli to identify compounds that result in an increase in the post-antibiotic effect (PAE), which is the delay in bacterial regrowth following antibiotic exposure and removal. Drugs that cause a PAE are dosed less frequently, and the method we describe will provide a new approach to developing safer drugs.

microbiology↗

bioRxiv2022

A screen for modulation of nucleocapsid protein condensation identifies small molecules with anti-coronavirus activity

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase separated condensates that are hypothesized to play critical roles in viral replication, transcription and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43 and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

molecular biology↗

bioRxiv2019

RNA polymerase mutations cause cephalosporin resistance in clinical Neisseria gonorrhoeae isolates

Increasing Neisseria gonorrhoeae resistance to ceftriaxone, the last antibiotic recommended for empiric gonorrhea treatment, poses an urgent public health threat. However, the genetic basis of reduced susceptibility to ceftriaxone is not completely understood: while most ceftriaxone resistance in clinical isolates is caused by target site mutations in penA, others lack these mutations. Here, we show that penA-independent ceftriaxone resistance has evolved multiple times through distinct mutations in rpoB and rpoD. We identify five mutations in these genes that each increase resistance to ceftriaxone, including one mutation that arose independently in two lineages, and show that clinical isolates from multiple lineages are a single nucleotide change from ceftriaxone resistance. These RNA polymerase mutations result in large-scale transcriptional changes without altering susceptibility to other antibiotics, reducing growth rate, or deranging cell morphology. These results underscore the unexpected diversity of pathways to resistance and the importance of continued surveillance for novel resistance mutations.

microbiology↗