Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection

Abstract

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29β-NAc, is a biphenyl N-acetyl-β-galactosaminoside with a K_i of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29β-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29β-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29β-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29β-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Keywords: antibiotic-sparing therapeutic; glycomimetics; host–pathogen interactions; structure-based drug design; urinary tract infection.

Fig. 1.

Biochemical and structural characterization of early galactoside antagonists of FmlH. (A) Strategy for structure-guided drug design and evaluation of FmlH-targeting galactosides. A select library of galactosides were initially assessed in an ELISA-based competition assay for inhibition of FmlH binding to sialidase-treated BSM, with BSM indicated by gray circles, TF residues indicated by the yellow square-circle conjugates, biotinylated FmlH_LD by blue rectangles, and galactosides shown as colored circles. Cocrystal structures of FmlH_LD bound to a lead compound facilitated virtual screening and structure-guided drug design for biochemical evaluation of an expanded galactoside library. The top lead compound would then be tested as a treatment in a mouse model of UTI. (B) ELISA-based competition assay performed in triplicate in the absence or presence of 1 mM or 0.1 mM compounds with at least two biological replicates. Data are reported as the mean percent inhibition, with the box indicating the 25th to 75th percentiles and the whiskers indicating the 2.5th and 97.5th percentiles. (C) Structural alignment of FmlH_LD from an apo FmlH_LD crystal structure (PDB ID 6AOW), a FmlH_LD-TF cocrystal structure (PDB ID 6AOX), and a FmlH_LD-ONPG cocrystal structure (PDB ID 6AOY). (D) Crystal structures of sulfate ions or ligands bound in the FmlH_LD binding pocket, with H-bonding (black dashed lines) indicated between sulfate ions (yellow sticks), ligands (green sticks), water molecules (red spheres), or side chains (pink sticks).

Fig. 2.

Grouped organization of galactosides evaluated for FmlH_LD inhibition. The major groups include the phenyl (A), heterocyclic (B), napthyl/quinoline/phenylethyl (C), natural product (D), and biphenyl (E) series.

Fig. 3.

In vitro screening and affinity determination of galactosides against FmlH_LD. (A–C) ELISA-based competition assay performed in triplicate in the absence or presence of (A) 100 μM, (B) 10 μM, and (C) 1 μM compounds with at least two biological replicates. Data are reported as the mean percent inhibition, with the box indicating the 25th to 75th percentiles and the whiskers indicating the 2.5th and 97.5th percentiles. a, α; b, β. (D, Left) Schematic of conventional BLI experiment, in which pins coated with streptavidin (orange stars) are loaded with biotinylated Ser-TF (gray ovals and yellow square-circle conjugates) and dipped into solutions of varying concentrations of FmlH_LD (blue rectangles). (Right) Equilibrium analysis of soluble FmlH_LD binding to immobilized Ser-TF according to a 1:1 binding model. (E, Left) Schematic of competitive BLI experiment, in which streptavidin-coated pins are dipped into a solution composed of a fixed concentration of FmlH_LD in the presence of varying concentrations of galactoside (yellow circles). (Right) Equilibrium constants of soluble galactoside-mediated inhibition of FmlH_LD in binding immobilized Ser-TF in accord with R² > 0.85.

Fig. 4.

Structural basis of galactoside inhibition of FmlH_LD. (A) Crystal structures of sulfate ions or galactosides bound in the FmlH_LD binding pocket, with H-bonding (black dashed lines) indicated between sulfate ions (yellow sticks), ligands (green sticks), water molecules (red spheres), or side chains (pink sticks). Crystal structures shown here include an apo FmlH_LD crystal structure (PDB ID 6AOW), a FmlH_LD-4β cocrystal structure (PDB ID 6ARM), a FmlH_LD-5β cocrystal structure (PDB ID 6ARN), and a FmlH_LD-20β cocrystal structure (PDB ID 6ARO). (B) Cocrystal structure of 29β-NAc bound to FmlH_LD (PDB ID 6AS8). (C) SARs for 29β-NAc and related compounds, with their corresponding IC₅₀ values derived from the ELISA-based competition assay. IC₅₀ values are reported for six replicates as the mean with SEM.

Fig. 5.

Evaluation of galactosides for treatment of UTI and relevance in humans. (A and B) Bacterial titers in bladders (A) or kidneys (B) from C3H/HeN mice experiencing chronic cystitis transurethrally inoculated with 10% DMSO (three replicates, n = 13), or 50 mg/kg of 4Z269 (three replicates, n = 13), of 29β-NAc (three replicates, n = 14), or of both 4Z269 and 29β-NAc (two replicates, n = 9). Bars indicate median values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant; two-tailed Mann–Whitney U test. (C) Immunofluorescence analysis of FmlH_LD WT, FmlH_LD K132Q, or FmlH_LD WT in the presence of 29β-NAc binding to human bladder or human kidney tissue. Green corresponds to FmlH, red corresponds to Wheat Germ Agglutinin, and blue corresponds to DAPI. Each image is representative of nine total images (three imaged areas of three tissue slices). (Scale bars: 100 μm.)