D-peptide Inhibitors of Uropathogenic E. coli Adhesion Proteins to Treat Urinary Tract Infections

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PROJECT SUMMARY Adhesion proteins on the pilus-covered surface of Uropathogenic E. coli (UPEC) are required for attachment to host cells to facilitate colonization and disease progression in urinary tract infections (UTIs). The primary adhesion proteins associated with UPEC virulence are FimH, which binds D-mannose on uroepithelial cells, and FmlH, which binds N-acetylgalactosamine on kidney and inflamed bladder cells. This proposal aims to develop UTI treatments that prevent the attachment and subsequent internalization of UPEC in uroepithelial cells, eliminating bacteria from the urinary tract. Treatments that block the highly conserved binding pockets of FimH/FmlH prevent UPEC attachment to host cells and clear bacteria from the urinary tract6,9,11. However, designing inhibitors that reach the urinary tract to reduce the risk for recurrent infections (e.g., through a long half-life and without disrupting commensal bacteria) remains challenging. Our goal is to use mirror-image phage display to identify D-peptide inhibitors of UPEC adhesion to eliminate bacteria from the urinary tract. We hypothesize that D-peptide inhibitors that prevent attachment to host cells could serve as novel antibiotic-sparing UTI treatments. D-peptides are ideal for this application because they are cleared via kidney filtration (accumulate in urine), have a long half-life, and can be formulated as a long-acting injection. To screen for D-peptide inhibitors, we will first chemically synthesize the mirror-image D-target proteins (FimH/FmlH) using solid-phase peptide synthesis (SPPS) with D-amino acids and native chemical ligation (NCL). These D-proteins will be validated by comparison to recombinant L-proteins for secondary structure and binding activity. Using mirror-image phage display, we will screen diverse phage libraries for L-peptide binders to the synthesized D-target proteins. Next-generation sequencing (NGS) will be used to identify high-affinity hits that will then be synthesized in D- to inhibit the natural L-target. We will determine the affinity of our inhibitors via direct binding and competition studies. The crystal structures of our D-peptides in complex with FimH or FmlH will be obtained using X-ray crystallography and will inform inhibitor affinity optimization. In vitro assays will be performed using a panel of 40 UPEC strains to validate D- peptide inhibition of host cell attachment, hemagglutination, and biofilm formation. We will determine the pharmacokinetic (PK) profile of the most promising D-peptides in mice. Finally, we will evaluate the efficacy of treatments in mouse acute and chronic UTI models. This project will contribute valuable information about UPEC pili binding interactions, advance our chemical protein synthesis knowledge, and generate D-peptide inhibitor leads for precision UTI treatment.