Biology subjects

Dowson, C. G.

Publications and source records attributed to Dowson, C. G..

5 recordsLinked to original sources

bioRxiv2026

Discovery, characterisation and optimisation of bicyclic peptide inhibitors that disarm Staphylococcus aureus a-hemolysin

-Hemolysin (Ahly) is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation; antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable. Here we demonstrate that phage-derived bicyclic peptides can serve as compact, chemically tractable Ahly neutralisers. Using TATB-scaffolded M13 phage libraries we identified WNP-motif containing bicyclic binders, with a lead hit of Peptide 14 (KD = 1792nM) and progressed the lead by iterative affinity maturation to Peptide 20 (KD = 609 nM) and by incorporation of strategically chosen non-canonical amino acids to yield Peptide 88 (KD = 96 nM). A 2.2 [A] co-crystal structure with AhlyH35A locates the binding footprint on the rim domain and explains the critical role of the WNP motif in target engagement. Functional assays show that the Peptide 88 blocks Ahly mediated hemolysis, inhibits Ahly driven ADAM10 activation, and elucidate its inhibitory mechanism of preventing Ahly binding to human A549 epithelial cells. Peptide 88 protects A549 cells from recombinant toxin and attenuates cytotoxicity in S. aureus co-culture experiments, whilst showing no toxicity to A549 cells. Bicyclic peptides thus represent a new and promising anti-virulence modality: small, synthetically accessible molecules that mimic antibody recognition, with therapeutic potential against S. aureus infections.

molecular biology↗

bioRxiv2024

Discovery and chemical optimisation of a Potent, Bi-cyclic (Bicycle(R)) Antimicrobial Inhibitor of Escherichia coli PBP3

Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of {beta}-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of novel, bicyclic peptide (Bicycle(R)) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent a novel non-{beta}-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors.

biochemistry↗

bioRxiv2022

Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram negative bacteria

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.

evolutionary biology↗

bioRxiv2019

Structure-guided enhancement of selectivity of chemical probe inhibitors targeting bacterial seryl-tRNA synthetase

Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than two orders of magnitude compared to their human homologue, demonstrating a route to selective chemical inhibition of these bacterial targets.

biochemistry↗

bioRxiv2018

Rapid covalent-probe discovery by electrophile fragment screening

Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.

biochemistry↗