Gram-negative bacteria utilize extracellular fibers called chaperone-usher pathway (CUP) pili to mediate adhesion to host and environmental surfaces, facilitate invasion into host tissues, and promote interaction with other bacteria to form biofilms. Uropathogenic E. coli (UPEC) use a CUP adhesion protein (lectin) called FimH on the type 1 pilus to bind to mannosylated glycoproteins on bladder epithelial cells to mediate the onset and progression of urinary tract infections (UTIs). This binding event initiates bacterial invasion and formation of intracellular bacterial communities (IBCs) in the eukaryotic cell. Using rational drug design, mannoside antagonists of FimH have been developed as orally bioavailable therapeutics for the treatment and prevention of UTIs. Acute infection can either self-resolve or develop into chronic cystitis, which is characterized by: i) persistent, high titer bacteriuria and bacterial bladder burdens at sacrifice >4 weeks post- infection; ii) chronic inflammation and urothelial necrosis; iii) lymphonodular hyperplasia in the bladder submucosa and; iv) urothelial hyperplasia with a lack of uroplakin expression, which is a marker for terminal differentiation in superficial facet cells. Similar histological findings have been observed in humans suffering persistent bacteriuria and recurrent UTI. FmlH, the tip-associated adhesin of Fml/F9/Yde pili (previously denoted FmlD) functions in UPEC pathogenesis by providing a fitness advantage during chronic cystitis. FmlH specifically binds to Gal(?1-3)GalNac epitopes which appear as part of a remodeled glycan/galactose profile of the mouse bladder during chronic cystitis. FmlH is also upregulated in urines directly isolated from patients with UTI compared to expression during in vitro growth in media or normal urine, suggesting a host-specific induction. In this proposal, innovative strategies will be taken to rationally develop ?Gal and ?GalNAc ligands as antagonists of FmlH, alone or in combination with FimH inhibitors, as new preclinical therapeutics for treatment of acute and chronic cystitis. Using an X-ray structure of FmlH and virtual screening, new FmlH ligands, including O-nitrophenyl-?-galactoside (ONPG) were identified. Subsequently, the X-ray structures of Gal?-1-3-GalNAc (TF) and ONPG bound to FmlH were used to design improved FmlH ligands, with binding affinities several times higher than ONPG, as determined by an ELISA binding assay. This structure-based design strategy will be used to optimize new ligands with higher affinity and good drug-like properties. This project's aims will be accomplished by integrating: i) X-ray structure-based drug design with medicinal chemistry (Aim 1); ii) biochemical screening, functional cell and tissue binding assays Aim 2) and; rigorous pharmacokinetic (PK) evaluation and pharmacodynamic (PD) efficacy testing of the most promising FmlH ligands, in murine animal models of chronic cystitis, urosepsis/kidney infection, and GIT colonization (Aim 3). The overall goal is to develop preclinical candidate FmlH antagonists as standalone anti-virulence therapeutics and/or as combination therapy with existing FimH antagonists for the treatment and prevention of UTIs.
We will integrate reiterative X-ray structure-based drug design and synthetic medicinal chemistry with testing in a panel of biochemical and cellular assays followed by evaluation for pharmacokinetics and efficacy in animal models of urinary tract infections, to develop Gal- and GalNAc-based antagonists of the UPEC adhesin FmlH, which is used by uropathogenic E. coli (UPEC) to bind to the inflamed bladder tissue during chronic cystitis. Mannosides, inhibitors of FimH, an adhesin critical in acute and chronic infections, are efficacious in reducing UPEC bladder colonization. Thus, antagonists against FmlH will be tested either alone or together with high affinity mannosides in combination or as dual activity compounds. The goal is to identify new bioavailable small molecules as preclinical therapeutics for acute and chronic cystitis (bladder infection).
|Maddirala, Amarendar; Klein, Roger D; Pinkner, Jerome et al. (2018) Biphenyl Gal and GalNAc FmlH Lectin Antagonists of Uropathogenic E. coli (UPEC): Optimization through iterative rational drug design. J Med Chem :|
|Kalas, Vasilios; Hibbing, Michael E; Maddirala, Amarendar Reddy et al. (2018) Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection. Proc Natl Acad Sci U S A 115:E2819-E2828|
|Conover, Matt S; Ruer, Ségolène; Taganna, Joemar et al. (2016) Inflammation-Induced Adhesin-Receptor Interaction Provides a Fitness Advantage to Uropathogenic E. coli during Chronic Infection. Cell Host Microbe 20:482-492|