Biology subjects

Rowland, C. E.

Publications and source records attributed to Rowland, C. E..

3 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↗

bioRxiv2025

CDH1 loss remodels gene expression and lineage identity in human mammary epithelial cells

Invasive lobular carcinoma (ILC) is a common subtype of breast cancer, molecularly defined by genetic loss of CDH1, and subsequent loss of cell adhesion protein E-cadherin, in [~]95% of ILC. Though CDH1 loss occurs early in ILC oncogenesis, it is unclear how this facilitates transformation. We modeled early CDH1 loss using "normal" human mammary epithelial cells (HMEC), i.e. finite lifespan cells reflecting early hyperplasia, and targeted E-cadherin signaling using antibodies versus causing genetic CDH1 loss using siRNA or CRISPR/Cas9-knockout. Transcriptome analysis across four HMEC models showed that the mode of E-cadherin targeting is critical for the subsequent phenotype. Antibody-mediated inhibition of cell-cell contacts induced gene signatures of epithelial-mesenchymal transition (EMT), consistent with the role of E-cadherin suppression during the EMT process. Conversely, genetic CDH1 loss - as in ILC oncogenesis - repressed EMT signatures, and instead remodeled gene expression toward a luminal epithelial phenotype. RNA-seq, single cell transcriptomics, flow cytometry, microscopy, and ATACseq analyses support that CDH1 loss induces lineage remodeling to a luminal state, which is mirrored in transcriptomic analysis of clinical ILC precursor lesions. By isolating luminal versus basal cells prior to CDH1 knockout, we found that CDH1 loss led to remodeling of lineage identity in both populations, converging on a new lineage homeostasis with a luminal progenitor-like phenotype. Consistent with the shift to a luminal progenitor phenotype, CDH1 loss enhanced proliferative capacity over the finite lifespan of the HMECs, highlighting a feature of early CDH1 loss that may contribute to clonal advantage during tumor initiation. Moreover, CDH1 loss enhanced anoikis resistance, a defining feature of ILC cells. Our findings support that genetic loss of CDH1 in mammary epithelial cells induces transcriptional and phenotypic changes consistent with lineage identity remodeling toward a luminal progenitor-like state, which may underpin the mechanism by which early CDH1 loss mediates ILC oncogenesis.

cancer 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↗