Biology subjects

Lanyon-Hogg, T.

Publications and source records attributed to Lanyon-Hogg, T..

4 recordsLinked to original sources

bioRxiv2025

Chemical proteomics identifies signal peptidase IB (SpsB) as a target of the SOS response inhibitor OXF-077 and a regulator of quinolone resistance emergence in Staphylococcus aureus

Antimicrobial resistance (AMR) is an existential threat to health globally, and novel compounds that act through new targets are urgently required to combat increasing levels of resistance. One emerging strategy is the development of antibiotic adjuvants that can slow the evolution of resistance by inhibiting the mutagenic SOS response in bacteria, in order to prolong the clinical lifetime of antibiotics. OXF-077 is a potent SOS response inhibitor that suppresses the emergence of ciprofloxacin resistance in Staphylococcus aureus; however, the cellular target of OXF-077 is unknown. We report here the use of affinity-based protein profiling to identify signal peptidase IB (SpsB) as a target of OXF-077. Genetic and chemical studies demonstrated that SpsB is required for upregulation of the SOS response gene recA, increased frequency of resistance emergence to ciprofloxacin, and activation of the mutagenic SOS response in S. aureus. SpsB is therefore postulated to regulate the quinolone-activated SOS response in S. aureus, and can be targeted by small-molecule inhibitors such as OXF-077 to slow the evolution of resistance. Collectively, this work delivers SpsB as an attractive new drug target for the development of antibiotic adjuvants to combat the urgent threat of AMR. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/689737v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@f547e1org.highwire.dtl.DTLVardef@1422a32org.highwire.dtl.DTLVardef@e06979org.highwire.dtl.DTLVardef@12cc0d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Live-Cell Chemoproteomic Profiling Identifies the Uncharacterised Protein YbaA as a Direct Target of Ciprofloxacin in Escherichia coli

Fluoroquinolone antibiotics, such as ciprofloxacin, are important broad-spectrum agents for a range of bacterial infections; however, fluoroquinolone usage is increasingly challenged by the emergence of resistance. Ciprofloxacin resistance mechanisms include mutations in the antibiotic target DNA gyrase, downregulation of porins required for bacterial cell penetration, and upregulation of efflux pumps to expel the antibiotic. However, new pathways driving bacterial tolerance to fluoroquinolones are still being discovered, suggesting that additional ciprofloxacin-binding proteins may exist in bacteria. In this study, we report the use of affinity-based protein profiling (AfBPP) with photo-crosslinking chemical probes to identify protein binding partners of ciprofloxacin in live E. coli cells. AfBPP identified novel ciprofloxacin binding proteins including YjdN and YbaA, whose molecular functions are as yet unannotated. Target engagement was validated using genetic knockout and biophysical binding assays, and key interactions identified in the ciprofloxacin binding site of YbaA. Collectively, this study demonstrates that additional and previously unreported biological interactions can exist for well-established antibiotics, and provides methodology to identify and interrogate these interactions in detail.

microbiology↗

bioRxiv2025

Small molecule inhibitors of the NorA multidrug efflux pump potentiate antibiotic activity by binding the outward-open conformation

Antibiotic resistance is among the greatest threats of the modern era. Multidrug efflux pumps expel antibiotics from bacterial cells and present a particular challenge by conferring resistance to a broad range of antibiotic classes; however, there is currently a lack of potent and selective inhibitors. Here, we report the discovery of IMP-2380, a drug-like chemical probe for the multidrug efflux pump NorA that delivers low-nanomolar potentiation of ciprofloxacin activity in vitro and activity in an in vivo S. aureus infection model. A phenotypic high-throughput screen for inhibitors of the ciprofloxacin-activated SOS DNA repair pathway in methicillin-resistant Staphylococcus aureus (MRSA) identified hit compounds targeting NorA, and subsequent optimization established IMP-2380 as the most potent NorA inhibitor discovered to date. The structure of NorA bound to IMP-2380 was solved by cryo-electron microscopy at 2.52 [A] resolution, revealing that the small molecule locks the pump in the outward-open conformation. This closes the inner face and prevents antibiotics binding from the cytosol, providing an explanation for the exceptional potency of IMP-2380 and structure-activity relationship across the series. IMP-2380 represents an in vivo active NorA inhibitor, functioning via a structurally defined outward-open binding mode, and will enable future exploration of NorA as a druggable target to combat antibiotic resistance.

biochemistry↗

bioRxiv2020

Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

biochemistry↗