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

Article abstract of DOI:10.1073/pnas.1720140115

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 structureguided 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 Ki 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 watermediated 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.

Full text of DOI:10.1073/pnas.1720140115

Structure-based discovery of glycomimetic FmlH
ligands as inhibitors of bacterial adhesion
during urinary tract infection
Vasilios Kalasa,b,c, Michael E. Hibbinga,b, Amarendar Reddy Maddiralac, Ryan Chuganic, Jerome S. Pinknera,b
Laurel K. Mydock-McGranec, Matt S. Conovera,b, James W. Janetkaa,c,1, and Scott J. Hultgrena,b,1
,
aCenter for Women’s Infectious Disease Research, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Molecular Microbiology,
Washington University School of Medicine, St. Louis, MO 63110; and cDepartment of Biochemistry and Molecular Biophysics, Washington University School
of Medicine, St. Louis, MO 63110
Edited by Roy Curtiss III, University of Florida, Gainesville, FL, and approved February 13, 2018 (received for review November 20, 2017)
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 spec-
ificities 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 Ki of ∼90 nM,
representing a major advancement in potency relative to the char-
acteristically 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 signifi-
cantly reduced bladder and kidney bacterial burdens, and coadmin-
istration 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 specif-
ically binds healthy human kidney tissue in a 29β-NAc–inhibitable
manner, suggesting a key role for F9 pili in human kidney coloniza-
tion. Thus, these glycoside antagonists of FmlH represent a rational
antivirulence strategy for UPEC-mediated UTI treatment.
tive antibiotic-sparing approaches to combat bacterial pathogens.
Recently, promising efforts have been made to target the virulence
mechanisms that cause bacterial infection. These studies have
provided much-needed therapeutic alternatives, which simulta-
neously reduce the burden of antibiotic resistance and minimize
disruption of gastrointestinal microbial communities that are
beneficial to human health (16).
Uropathogenic Escherichia coli (UPEC) is the main etiological
agent of UTIs, accounting for greater than 80% of community-
acquired UTIs (17, 18). Comparative genomic studies have revealed
that UPEC strains are remarkably diverse, such that only 60% of the
genome is shared among all strains (19). As a consequence, UTI risk
Significance
The emergence of multidrug-resistant bacteria, including uro-
pathogenic Escherichia coli (UPEC), makes the development of
targeted antivirulence therapeutics a critical focus of research.
During urinary tract infections (UTIs), UPEC uses chaperone–
usher pathway pili tipped with an array of adhesins that recog-
nize distinct receptors with sterochemical specificity to facilitate
persistence in various tissues and habitats. We used an inter-
disciplinary approach driven by structural biology and syn-
thetic glycoside chemistry to design and optimize glycomimetic
inhibitors of the UPEC adhesin FmlH. These inhibitors compet-
itively blocked FmlH in vitro, in in vivo mouse UTI models, and
in ex vivo healthy human kidney tissue. This work demon-
strates the utility of structure-driven drug design in the effort
to develop antivirulence therapeutic compounds.
urinary tract infection glycomimetics structure-based drug design
|
|
|
host–pathogen interactions antibiotic-sparing therapeutic
|
Author contributions: V.K., M.E.H., A.R.M., J.S.P., J.W.J., and S.J.H. designed research; V.K.
and J.W.J. designed compounds; V.K., M.E.H., A.R.M., R.C., J.S.P., and L.K.M.-M. per-
formed research; A.R.M., R.C., and L.K.M.-M. synthesized compounds; V.K., M.S.C., and
J.S.P. purified proteins; V.K. and J.S.P. performed virtual screening, X-ray crystallography,
ELISA, bio-layer interferometry, and immunofluorescence experiments; M.E.H. performed
animal experiments; V.K., A.R.M., R.C., J.S.P., L.K.M.-M., and M.S.C. contributed new
reagents/analytic tools; V.K., M.E.H., A.R.M., J.S.P., J.W.J., and S.J.H. analyzed data;
V.K., M.E.H., J.S.P., J.W.J., and S.J.H. interpreted all data; and V.K., M.E.H., A.R.M.,
J.W.J., and S.J.H. wrote the paper.
rinary tract infections (UTIs) are one of the most prevalent
infections, afflicting 15 million women per year in the United
U
States alone, with annual healthcare costs exceeding $2 to $3 billion
(1–3). Nearly 50% of women will experience at least one UTI in
their lifetime. Despite appropriate and often successful clearance of
bacteriuria by antibiotic treatment, 20% to 30% of women will
experience a recurrence within 6 mo of the initial acute UTI (1, 4).
Kidney infection, or pyelonephritis, represents a severe manifes-
tation of UTI, with ∼250,000 cases and 100,000 hospitalizations
per year in the United States (5). Acute pyelonephritis requires
hospital admission and i.v. antibiotics to thwart the long-term
sequelae of kidney failure and renal scarring, and, together with
bacteremia, results in a mortality rate of 10% to 20% (6–8). With
the global dissemination and increase of antibiotic resistance,
treatment of UTI is becoming a serious clinical challenge (9). Anti-
biotic susceptibility tests indicate that many uropathogens are resistant
to traditional first-line antibiotics like trimethoprim-sulfamethoxazole
(TMP-SMZ) and even to last-line antibiotics such as ciprofloxacin
and colistin (10–15). The diminishing efficacy of antibiotic thera-
pies toward UTIs and other infectious diseases demands alterna-
Conflict of interest statement: J.W.J. and S.J.H. are inventors on US patent US8937167 B2,
which covers the use of mannoside-based FimH ligand antagonists for the treatment of
disease. J.W.J., M.E.H., and S.J.H. have ownership interests in Fimbrion Therapeutics and
may benefit if the company is successful in marketing mannosides.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDerivatives License 4.0 (CC BY-NC-ND).
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.wwpdb.org (PDB ID codes 6AOW, 6AOX, 6AOY, 6ARM, 6ARN,
6ARO, and 6AS8).
1To whom correspondence may be addressed. Email: janetkaj@wustl.edu or hultgren@
wustl.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1720140115/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
PNAS Latest Articles
|
1 of 10

and outcome are determined by complex interactions between host antagonists of FmlH through an X-ray structure-guided me-
susceptibility and diverse bacterial urovirulence potentials, which dicinal chemistry approach. This strategy entailed (i) screening
can be driven by differences in the expression and regulation of a select library of galactosides through multiplex ELISA arrays
conserved functions. The ability of UPEC to colonize various
habitats, such as the gut, kidney, and bladder, depends in large
for initial lead compund identification; (ii) an iterative process of
cocrystal structure determination, virtual screening, structure-
part on the repertoire of adhesins encoded in their genome. The based ligand design, and in vitro biochemical characterization;
most common mechanism for adhesion utilized by UPEC is medi- and (iii) evaluation of the top lead compound in a mouse model of
ated through the chaperone–usher pathway (CUP), which generates UTI (Fig. 1A). Toward these goals, we first investigated whether
extracellular fibers termed pili that can confer bacterial adhesion
to host and environmental surfaces, facilitate invasion into host
Gal, GalNAc, and TF could be adapted to function as soluble,
competitive inhibitors of FmlH. To that end, an ELISA-based
tissues, and promote interaction with other bacteria to form biofilms competition assay was developed to detect binding of FmlHLD to
(20). Phylogenetic analysis of Escherichia genomes and plasmids surface-immobilized desialylated bovine submaxillary mucin (ds-
predicts at least 38 distinct CUP pilus types, with single organisms BSM) in the presence or absence of soluble compounds (Fig. 1A).
capable of maintaining as many as 16 distinct CUP operons (21). As expected, Gal, GalNAc, and TF were each capable of inhibiting
Many of these CUP pilus operons contain two-domain, tip-localized FmlHLD at a concentration of 1 mM, with GalNAc exerting greater
adhesins, each of which likely recognize specific ligands or receptors
to mediate colonization of a host and/or environmental niche.
For example, the type 1 pilus adhesin FimH binds mannosylated
glycoproteins on the surface of the bladder epithelium, which is
crucial for the establishment of cystitis (22, 23). The structural
basis of mannose (Man) recognition by the N-terminal–receptor
inhibitory potency than TF or Gal. However, neither Man nor
glucose (Glc) had any detectable effect on the ability of FmlHLD
to bind ds-BSM (Fig. 1B). Lactose (Lac), or Gal(β1-4)Glc, was
also incapable of inhibiting FmlHLD, demonstrating the high selec-
tivity in which FmlHLD engages Gal-containing glycans (Fig. 1B). O-
nitrophenyl β-galactoside (ONPG) and isopropyl β-thiogalactoside
binding domain, or lectin domain (LD), of FimH has been lever- (IPTG) were also tested for inhibition in this exploratory phase
aged to rationally develop high-affinity aryl mannosides (24–32). In of our search for FmlH inhibitors. While IPTG exerted minor in-
mouse models of UTI, we have previously demonstrated that orally hibitory activity at 100 μM, ONPG was found to block FmlHLD from
bioavailable mannosides that tightly bind FimH can prevent acute interacting with ds-BSM more effectively than Gal, GalNAc, or
UTI, treat chronic UTI, and potentiate the efficacy of existing an- TF (Fig. 1B). The strong inhibitory potency of ONPG suggested
tibiotic treatments like TMP-SMZ, even against antibiotic-resistant that β-galactosides could potentially be rationally designed with
E. coli strains (28). Thus, use of mannosides that target the adhesin
FimH represents the first successful application of an antivirulence
strategy in the treatment of UTI.
A homolog of the type 1 pilus, the F9 pilus, is one of the most
common CUP pili in the E. coli pan genome and an important
urovirulence factor employed by UPEC for the maintenance of
UTI (21, 33). Our recent work has demonstrated that UPEC up-
higher affinity by specifically targeting residues within and sur-
rounding the sugar binding pocket of FmlH.
Therefore, X-ray crystallography was implemented to eluci-
date the 3D structures of both apo and ligand-bound FmlHLD
(SI Appendix, Table S1). First, a crystal structure of apo FmlHLD
was solved at 1.6 Å resolution by molecular replacement using
FimHLD [Protein Data Bank (PDB) ID 3MCY] as the search model.
regulates the expression of F9 pili in response to bladder inflam- Within this structure, two copies of FmlHLD are found in the asym-
mation and epithelial remodeling induced upon UPEC infection
(34). These pili display the FimH-like adhesin FmlH, which is
capable of binding terminal galactose (Gal), N-acetylgalactosamine
(GalNAc), or Thomsen-Friedenreich antigen (TF) [Gal(β1-3)Gal-
metric unit, each of which adopts a canonical β-sandwich fold,
with three distinct binding loops (loop 1: residues 10 to 15; loop
2: residues 44 to 53; and loop 3: residues 132 to 142) that form a
wide, shallow, solvent-exposed binding pocket (Fig. 1 C and D).
NAc(α)]. FmlH was shown to bind TF within naïve or infected kid- Within the binding pocket of both copies resides a sulfate ion,
neys and to Thomsen nouvelle antigen (Tn) (GalNAc) within the which interacts with residues implicated in Gal binding (Fig. 1D).
inflamed bladder epithelium during chronic, unresolved UTI. De-
letion of FmlH in the urosepsis isolate CFT073 resulted in a
competitive defect in the ability of this strain to maintain murine
UTI in C3H/HeN female mice. Furthermore, vaccination with the
LD of FmlH (FmlHLD) as the challenge antigen significantly
protected mice from developing UTI. Thus, we have shown that
FmlH serves a key role in the UPEC pathogenesis cascade and
represents a promising target for antivirulence therapies for UTI
in both the bladder and kidney habitats.
Cocrystal structures of FmlHLD bound to TF and of FmlHLD bound
to ONPG were also solved to 2.1 Å and 1.8 Å, respectively. Struc-
tural overlay of the apo and ligated crystal structures yields root-
mean-square deviation (RMSD) values that fall within 0.6 Å,
suggesting that FmlHLD generally adopts the same active or func-
tional conformational state in the absence or presence of ligand
(Fig. 1C). This functional conformational state most likely cor-
responds to a high-affinity conformation of FmlH, as the FmlHLD
structures exhibit a higher degree of structural homology to the
Herein, we describe the discovery and structure-based optimiza- high-affinity conformation of FimH (RMSD values of 0.8 to 0.9 Å)
tion of high-affinity aryl galactoside and N-acetylgalactosaminoside than to the low-affinity conformation of FimH (RMSD values of
FmlH ligands that potently inhibit the function of FmlH. Treatment
with these FmlH antagonists significantly reduced bacterial burdens
in the kidneys and bladders of infected mice, thereby demonstrating
promising translational value in the treatment of UTI in humans.
The results of these studies, together with our previous work on FimH
1.7 to 1.9 Å) (34–38).
The cocrystal structure of FmlHLD-TF reveals two copies of
FmlHLD-TF in the unit cell, in which each TF adopts a distinct
ligand conformation (Fig. 1D). In both copies, the terminal Gal
in TF occupies the cleft of the binding pocket through direct
mannosides, further support the mechanistic and therapeutic value of polar interactions with residues F1, D53, K132, and N140. In
antivirulence strategies that leverage structure-function relationships
of diverse bacterial adhesins for the rational design of high-affinity
glycosides for the treatment of UTI and other bacterial infections.
contrast, the orientation of the GalNAc in TF differs signifi-
cantly between the two copies of FmlH. In chain A, the GalNAc
sugar points toward loop 3, with the carbonyl group of GalNAc
forming a hydrogen bond (H-bond) with the guanidinium group
of R142. In chain B, however, the GalNAc packs against and
forms a H-bond with the hydroxyl group of Y46. Accordingly, the
differences in the orientation of bound ligand across the two
copies are accompanied by slight differences in orientation of the
side chains of the interacting residues Y46 and R142. The mul-
tiple binding modes observed for a single ligand suggests that the
Results
O-nitrophenyl β-Galactoside Identified as Early Lead Inhibitor of the
F9 Pilus Adhesin FmlH. We revealed in a previous communication
that FmlH binds surface glycan receptors containing terminal
Gal, GalNAc, or TF residues (34). Given the role of FmlH in UTI
pathogenesis, we aimed to develop high-affinity galactoside
2 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
Kalas et al.

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 FmlHLD by blue rectangles,
and galactosides shown as colored circles. Cocrystal structures of FmlHLD 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 FmlHLD from an apo FmlHLD crystal structure (PDB ID 6AOW), a FmlHLD-TF cocrystal structure (PDB ID 6AOX), and a
FmlHLD-ONPG cocrystal structure (PDB ID 6AOY). (D) Crystal structures of sulfate ions or ligands bound in the FmlHLD 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).
Virtual Screen Identifies and Informs the Design of FmlH-Targeting
Galactosides. An exhaustive virtual screen was performed using
AutoDock Vina to computationally dock ∼1,800 known galac-
wide, shallow nature of the Gal binding pocket in FmlH would
enable galactosides to possibly bind FmlH with diverse interac-
tions and conformations.
tosides in the binding pocket of FmlHLD (from an FmlHLD
-
The FmlHLD-ONPG cocrystal structure also shows two copies
of FmlHLD in the unit cell, in which a sulfate ion occupies the
binding pocket of chain A while ONPG occupies the binding
pocket of chain B (Fig. 1D). As expected, the Gal component of
ONPG resides in the cleft of the binding pocket, while the solvent-
exposed nitrophenyl group mediates a polar or salt-bridge inter-
action with R142 through an intricate network of H-bonds with
water molecules. Furthermore, the positioning of the Gal compo-
nent of ONPG aligns with that of the Gal residue of TF (Fig. 1C).
Moreover, the conformation of the FmlH binding pocket observed
in this FmlHLD-ONPG cocrystal structure resembles the binding
pocket conformation in the FmlHLD-TF cocrystal structure, reflect-
ing a high-affinity binding orientation that can be targeted for
drug discovery. These results and observations strongly suggested
that the FmlHLD-ONPG cocrystal structure represents an appro-
priate structural candidate for use in virtual screening to aid in the
design of galactoside compounds specific for FmlH.
ONPG cocrystal structure; PDB ID 6AOY), generating a ranked
list of top binding poses and associated docking scores for each
galactoside (SI Appendix, Fig. S1A). Top hits from the virtual
screen were filtered according to group efficiency values and then
visually inspected to aid and inform structure-guided drug design.
In all cases, the Gal component of top-scoring galactosides bound
to the cleft of the binding pocket, as expected. In addition, most of
the high-scoring hits also interacted with specific hot-spot residues
near the Gal binding pocket, which we sought to leverage for com-
pound optimization. These hot-spot residues included (i) residue
Y46, which caps the top of the binding pocket and can contribute
hydrophobic interactions; (ii) residue K132, which lies at the bottom
of the sugar binding pocket and can engage polar groups linked to
the Gal sugar; and (iii) residue R142, which points toward an empty,
solvent-exposed cleft near the binding pocket and can contribute
electrostatic interactions (SI Appendix, Fig. S1B). These visual
Kalas et al.
PNAS Latest Articles
|
3 of 10

insights were then considered in our rational design strategy for
FmlH-targeting galactoside antagonists.
Design and Synthesis of FmlH-Targeting Galactoside Antagonists. To
increase FmlH binding affinity and explore structure-activity
relationships (SARs), we constructed a large library of galacto-
side analogs (Fig. 2). Based on the docking results, we predicted
that β-Gal isomers would be preferred over α-Gal and that ortho
positioning of functional groups on a phenyl scaffold would best
facilitate interactions with specific sites within the binding pocket,
namely hot-spot residues Y46 and R142. Accordingly, we synthe-
sized and evaluated small sets of phenyl galactosides with ortho-
substituted functional groups (2 to 6; Fig. 2A). We also either
purchased or synthesized several other phenyl galactosides, which
contained meta or para substituents on the aglycone ring (7 to 11;
Fig. 2A), and other aryl and heterocyclic galactosides (12 to 22;
Fig. 2 B and C). This allowed us to derive meaningful SARs for
informing further design and optimization of improved galacto-
sides. In addition, we tested natural-product galactosides iso-
lated from cranberries and other natural sources (23 to 27; Fig.
2D). The promising activity of the simple galactoside ONPG (4β)
in the initial screen, coupled with the hot-spot residues identified
in virtual screening, prompted us to expand our FmlH-ligand design
strategy with a compound series containing biphenyl aglycones (28
to 32; Fig. 2E), such as 29β-NAc, the N-acetyl-β-galactosaminoside
with an m-carboxylic acid on the B-ring designed to directly in-
teract with the hot-spot residue R142 (SI Appendix, Fig. S1 B and
C). To confirm the predicted preference for the β-Gal isomers, we
also synthesized and tested many corresponding α-Gal isomers.
Compounds were synthesized by using one of two general synthetic
glycosylation methods involving either a reaction between Gal
pentaacetate and phenols promoted by boron trifluoride or a
Koenigs–Knorr-type reaction of galactosyl halide with aryl alcohols
(SI Appendix, Fig. S2).
Biochemical Characterization of FmlH Antagonists. Selected top-hit
glycosides and a few low-scoring analogs from the virtual screen,
as well as synthetic galactosides, were tested in the ELISA-based
competition assay for their ability to inhibit binding of FmlHLD
to ds-BSM. Direct comparison of inhibitory potency among
galactosides led to delineation of basic SARs (Fig. 3 A–C and
SI Appendix, Table S2). When tested at 100 μM, the phenyl
β-galactoside 1β (beta isomer of 1; Fig. 2A) exhibited significantly
higher binding inhibition (77%) than Gal (8.1%), indicating that the
phenyl group significantly enhances binding to FmlHLD (Fig. 3A).
Various ortho substituents on the phenyl ring additionally conferred
substantial improvements in inhibitory potency, as observed with 2β
(87%), 3β (95%), 4β (ONPG; 93%), 5β (97%), and 6β (90%). In
contrast, the meta-methoxy groups in compound 7β (76%) did not
enhance binding strength compared with 1β. Further, para-
substituted functional groups displayed variable inhibitory poten-
cies relative to 1β, with enhancements observed in 8β (86%) and
9β (86%), with no significant effect observed in 11β (78%) or
11β-thio (72%), and with a reduction observed in 10β (65%).
Thus, we deduced that the ortho-substituted phenyl β-galactosides
generally outperformed other simple phenyl galactosides.
Complex heterocyclic galactosides, such as coumarins 12β (85%)
and 14β (89%), which differ only by a methyl group, displayed
significant inhibitory potencies against FmlHLD, while the related
galactoside 13β (50%) displayed reduced inhibitory activity, likely
because of its fluoro substituents (Fig. 3A and SI Appendix, Table
S2). Resorufin galactoside 15β (80%) also showed similar potency
compared with the phenyl β-galactoside 1β. These combined results
suggest that the substituents of 12β are responsible for augmenting
affinity relative to 1β. In contrast, indoles 16β (22%) and 17β
(41%) performed poorly as inhibitors of FmlHLD. Naphthyl galac-
tosides 18β (46%) and 19β (79%), in addition to isoquinoline 21β
Fig. 2. Grouped organization of galactosides evaluated for FmlHLD in-
hibition. The major groups include the phenyl (A), heterocyclic (B), napthyl/
quinoline/phenylethyl (C), natural product (D), and biphenyl (E) series.
quinoline 20β (95%) displayed significantly higher inhibition
than 1β and 18β. This advocates that the electron pair-donating
nitrogen atom in 20β is making a specific interaction with FmlH.
This observation is consistent with the pattern of SARs, in-
dicating that the ortho position is key to enhancing inhibitory
(15%), showed no improvement in activity relative to 1β. However, potency against FmlHLD
.
4 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
Kalas et al.

Fig. 3. In vitro screening and affinity determination of galactosides against FmlHLD. (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 in-
hibition, 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 FmlHLD (blue rectangles). (Right) Equilibrium analysis of soluble FmlHLD 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 FmlHLD in the presence of varying concentrations of galactoside (yellow circles). (Right) Equilibrium constants
of soluble galactoside-mediated inhibition of FmlHLD in binding immobilized Ser-TF in accord with R2 > 0.85.
We also evaluated naturally occurring galactosides derived
from cranberries and other natural sources in this screen (Fig. 3A
and SI Appendix, Table S2). These compounds included antho-
cyanidin (pelargonidin, 23β; cyanidin, 24β; peonidin, 25β) and
flavonol (quercetin, 26β; myricetin, 27β) β-galactosides. Gener-
ally, these compounds exhibited moderate to weak inhibition of
FmlHLD binding, with little enhancement in inhibition relative to
Gal (8.1%). The only significant binders were 24β (29%) and
26β (14%). Comparison of the anthocyanidin family indicates
that the 3′ or meta-substituted hydroxyl group on the B-ring of
24β is critical for its specific interaction with FmlH. Absence of
this meta substituent in 23β (0.7%) or methylation of the hy-
droxyl group in 25β (3.6%) abrogates potency, suggesting that
the hydroxyl group of 24β might participate in a H-bond to a
specific residue in the FmlHLD binding pocket. Additional in-
hibitory screens performed with cranberry-derived compounds
Kalas et al.
PNAS Latest Articles
|
5 of 10

Fig. 4. Structural basis of galactoside inhibition of FmlHLD. (A) Crystal structures of sulfate ions or galactosides bound in the FmlHLD 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 FmlHLD crystal structure (PDB ID 6AOW), a FmlHLD-4β cocrystal structure (PDB ID 6ARM), a FmlHLD-5β cocrystal
structure (PDB ID 6ARN), and a FmlHLD-20β cocrystal structure (PDB ID 6ARO). (B) Cocrystal structure of 29β-NAc bound to FmlHLD (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.
and fractions at 1 mM confirmed the specificity and necessity of
the Gal sugar for inhibiting the binding pocket of FmlH (SI
Appendix, Fig. S3 A and B).
of 15.0
0.8 μM (Fig. 3D). Next, immobilized Ser-TF was in-
cubated in solutions comprising a fixed concentration of FmlHLD
but varying concentrations of galactosides to determine their in-
hibitory or dissociation constant (Ki or Kd) values (Fig. 3E). The
BLI-based affinity determinations correlated well with the relative
binding strengths measured in the ELISA-based competition assay
(Fig. 3 A–C and SI Appendix, Table S2). The two lead compounds,
29β-NAc and 29β, bound tightly to FmlHLD, with respective Ki
values of ∼90 nM and 2.1 μM, which represent a ∼7,800-fold and
∼330-fold enhancement in binding affinity relative to Gal. Another
promising compound, 4β-NAc, bound FmlHLD with a Ki value of
2.3 μM. In summary, a combinatorial approach based on virtual
Interestingly, the tested GalNAc-derived compounds pos-
sessed significantly higher inhibitory potency compared with
their matched-pair Gal-derived counterparts, as exemplified with
4β-NAc (87%) relative to 4β (31%) when tested for inhibition at
10 μM (Fig. 3B and SI Appendix, Table S2). These results taught
us that the N-acetyl group, together with other functional groups,
contributes to binding by targeting distinct components of the
binding pocket of FmlH. In contrast, the galactosides with
α-linkages (28α-30α) or disaccharides with aglycone moieties (33
to 35) were generally poor inhibitors of FmlH, except for 11α- /screening and structure-guided ligand design led to the discovery of
NAc (82%) (Fig. 3A and SI Appendix, Table S2).
small–molecular weight monomeric glycosides derived from Gal and
GalNAc that function as effective antagonists of FmlH. Optimiza-
tion of early hits to high-affinity o-biphenyl Gal and GalNAc an-
tagonists was realized via ortho substitution on phenyl aglycones to
facilitate interactions that significantly enhanced binding to FmlH.
Consistent with the above-mentioned SARs, the ortho biphenyl
galactoside 28β (91%) was more potent than the meta 31β (57%)
or para 32β (30%) analogs (Fig. 3A and SI Appendix, Table S2).
Next, we installed a carboxylate group at the meta position on the
biphenyl B-ring (29β), intended to target the pocket formed by
N140 and R142, and found that 29β exhibited greater inhibition
(99%) compared with 28β when tested at 100 μM. This pro-
nounced difference in activity was further highlighted when these
compounds were tested for inhibition at 10 μM and 1 μM (Fig. 3
B and C and SI Appendix, Table S2). Importantly, 30β (87%), the
methyl ester of 29β, tested at 100 μM resulted in a reduction in
binding, suggesting that the negative charge of the carboxylic acid
likely mediates a critical electrostatic interaction with R142 of
FmlHLD. Lastly, we synthesized the GalNAc version of 29β to
increase its binding affinity and found that 29β-NAc (93%) had
significant improvement in activity over 29β (75%) when tested at
10 μM. Final evaluation of the highest performing galactosides in
the ELISA-based competition assay at concentrations of 10 μM
and 1 μM allowed for a clearer ranking of compounds, where 29β-
NAc clearly stood out as the most potent (Fig. 3 B and C and SI
Appendix, Table S2).
Structural Basis of Galactoside Inhibition of FmlH. To elucidate the
molecular basis for galactoside inhibition of FmlH, cocrystal
structures of FmlHLD bound to 4β, 5β, 20β, and 29β-NAc were
determined (Fig. 4 A and B). These galactosides share a common
aglycone motif consisting of a phenyl ring with an ortho-substituted
functional group. As predicted from computational studies, the
sugar portion of all these galactosides resides within the cleft of the
binding pocket. The phenyl groups directly attached to the sugar
portion of all four compounds lie along the same 3D plane. In this
nearly identical conformation, the phenyl ring is oriented perpen-
dicularly to the side chain of residue Y46, revealing edge-to-face
π-stacking, which likely contributes to the affinity enhancement ob-
served for all β-galactosides. For 4β, 5β, and 20β, the ortho sub-
stituents point toward R142 but are too distant (>7 Å) for direct
interaction and, instead, form H-bonds with water molecules that,
in turn, interact with residues K132 and R142 (Fig. 4A). Thus, we
deduced that the marked affinity enhancement observed for 4β,
5β, and 20β is due to a combination of (i) indirect interactions
between the ortho substituent and residues K132 and R142 formed
by an intricate network of water-mediated H-bonds and (ii) edge-
to-face π-stacking between the phenyl ring and residue Y46.
In contrast to simple phenyl galactosides, the biphenyl scaffold
of 29β-NAc presents the carboxylic acid to engage in a direct
Determination of FmlH–Galactoside Binding Affinities. Bio-layer in-
terferometry (BLI) was pursued to quantitate the binding affinity
of the most promising FmlH antagonists. First, biotinylated serine-
linked TF (Ser-TF) immobilized on streptavidin pins was incu-
bated with varied titrations of FmlHLD in solution, and steady-state
analysis of binding responses revealed a dissociation constant (Kd)
6 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
Kalas et al.

charge–charge interaction with the guanidinium side chain of
R142 (Fig. 4B). The lower potency of the methyl ester de-
rivative 30β is further evidence that the charge–charge inter-
action likely drives the observed affinity enhancement (Fig.
4C). The improved affinity of 29β-NAc relative to 29β is also
due to additional interactions mediated by the N-acetyl group
in H-bonding to a water molecule captured by the biphenyl
aglycone and the side chain of residue K132 (Fig. 4 B and C).
Altogether, analysis of all X-ray crystal structures of ligand-
bound FmlH offers two general mechanisms for the significant
enhancement in binding affinity of galactosides relative to
Gal: edge-to-face π-stacking with Y46 and polar or electro-
static charge–charge interactions with K132 and R142.
FmlH Antagonist Effectively Treats Murine UTI in Vivo and Prevents
Binding to Human Kidney Tissue. We previously reported that
FmlH binds to naïve kidney and inflamed bladder tissue and
plays a critical role in chronic UTI, as abrogation of its function
through genetic deletion or vaccination results in significant at-
tenuation in the ability of UPEC to cause chronic UTI (34).
Thus, we hypothesized that galactosides that inhibit the function
of FmlH would have efficacy in the treatment and/or prevention
of UTI. To assess therapeutic potential, the lead compound 29β-
NAc was evaluated for its ability to reduce bacterial burdens in
the urinary tracts of C3H/HeN mice during chronic UTI. We
previously defined chronic cystitis in C3H/HeN mice as urine
titers of >104 CFU/mL lasting at least 2 to 4 wk, as well as bladder
inflammation and edema at euthanasia (39). Further, C3H/HeN
mice are genetically predisposed to vesicoureteral reflux (retro-
grade flow of urine from the bladder to the kidneys), which can
lead to bacterial colonization of the kidneys, renal abscess for-
mation, scarring, and atrophy (40). Accordingly, we observed high
levels of kidney colonization by CFT073 in control (vehicle-
treated) animals. When delivered intravesically, 29β-NAc sig-
nificantly reduced bacterial burdens in both the bladder and the
kidneys of these mice (Fig. 5 A and B). For comparison, man-
noside 4Z269, which inhibits the type 1 pilus adhesin FimH, also
significantly reduces titers of CFT073 from the bladders and
kidneys of infected mice relative to vehicle control (Fig. 5 A and
B). When administered together, 29β-NAc and 4Z269 eradicated
bacteria from the kidney in nearly all mice while also reducing
bacterial titers in the bladder, suggesting that FimH mannosides
and FmlH galactosides may function synergistically to target distinct
bacterial adhesins or communities within the kidney habitat (Fig.
5 A and B).
To show relevance to human UTI, we assessed FmlH and
FmlH-targeting galactosides through immunofluorescence anal-
ysis of FmlHLD binding to human kidney and bladder biopsied
tissue determined to be nonmalignant. While FmlHLD does not
appear to bind to healthy human bladder tissue, FmlHLD does
bind to healthy human kidney tissue, particularly in regions re-
sembling the collecting ducts and distal tubules of the kidney
(Fig. 5C and SI Appendix, Figs. S4 and S5). As a negative control,
the binding null mutant FmlHLD K132Q, which lacks the ability
to bind ds-BSM in vitro (SI Appendix, Fig. S6), was incapable of
binding kidney tissue, suggesting that FmlHLD specifically recog-
nizes receptors naturally present in human kidney tissue (Fig. 5C).
These observations are consistent with the previously reported
binding phenotypes in mice, in which FmlH can bind naïve mouse
kidney tissue, but not naïve mouse bladder tissue, and can bind to
receptors in inflamed bladder tissue (34). Moreover, incubation of
29β-NAc with FmlHLD prevented binding to human kidney tissue,
suggesting that these results may translate to humans. Importantly,
these collective data provide substantial evidence that aryl glycoside–
based FmlH antagonists derived from β-Gal or β-GalNAc can
serve as an effective therapy for persistent UTIs, including pyelo-
nephritis, for which there is an enormous unmet medical need.
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 FmlHLD WT, FmlHLD K132Q, or FmlHLD
WT in the presence of 29β-NAc binding to human bladder or human kidney
tissue. Green corresponds to FmlH, red corresponds to Wheat Germ Agglu-
tinin, and blue corresponds to DAPI. Each image is representative of nine
total images (three imaged areas of three tissue slices). (Scale bars: 100 μm.)
Discussion
UPEC is the causative agent of most UTIs, a common and very
costly disease in women, children, and the elderly that is becoming
increasingly resistant to antibiotic treatment. By leveraging our
expertise in UPEC pathogenesis and structure-based drug de-
sign, we developed small-molecule Gal-based FmlH antagonists
that show in vivo efficacy in the treatment of chronic UTI in
mouse models. Virtual screening combined with rational design
led to the identification of several naturally occurring cranberry
and synthetic galactosides, the most potent of which binds FmlH
with nanomolar affinity. X-ray crystallography revealed that potent
galactosides achieve significant enhancements in binding affinity
through interactions on opposite sides of the wide Gal binding
pocket of FmlH. Appropriately substituted aryl groups, like those
found in 4β/4β-NAc, 5β, and 20β, are seen to mediate edge-to-face
π-stacking interactions with Y46 of FmlHLD. Further, the optimized
biphenyl aglycone of compound 29β-NAc contains an ideally po-
sitioned carboxyl group to mediate electrostatic interactions with
R142 in addition to π-stacking interactions with Y46. Evaluation
of the lead candidate 29β-NAc in a mouse model of chronic UTI
demonstrated significant reductions of bacterial burdens in the
mouse kidney and bladder. Combination dosing with mannoside
and galactoside resulted in near complete clearance of bacteria
from the kidney and significant elimination of bacteria from the
bladder. Furthermore, FmlH was shown to bind specifically to
Kalas et al.
PNAS Latest Articles
|
7 of 10

human kidney tissue, which could be inhibited by 29β-NAc.
antagonists for the development of small-molecule glycoside-based
Additionally, FmlH has been shown to be up-regulated in urine
drugs aimed at treating infections mediated by E. coli or other mi-
samples directly isolated from human patients with UTI compared crobes (44).
with expression during in vitro growth in media or normal urine
(41), suggesting an important role for FmlH in human UTI. Thus,
The rapid increase and spread of antibiotic resistance, in-
cluding multidrug-resistant forms of bacteria, has rendered many
FmlH-targeting galactosides represent a rational antivirulence antibacterial therapies ineffective and threatens to undermine
modality for the effective treatment of UPEC-mediated UTI.
Our rational strategy to discover receptor-mimicking galacto-
sides targeting FmlH was similar to the strategy we followed for
the development of FimH mannosides. However, the design of
the galactoside and N-acetylgalactosaminoside antagonists of
FmlH was met with distinct challenges. The most striking dif-
ference between FmlH and FimH is the binding affinity for their
respective ligands: FimH binds soluble Man with a moderate
binding affinity of ∼5 to 10 μM, and FmlH binds soluble Gal with
a weak binding affinity of ∼700 μM (34, 42, 43). The weak binding
affinity of FmlH, which is quite common for most carbohydrate–
lectin interactions, rendered the development of high-affinity ga-
lactosides much more challenging. This disparity in affinity is a
direct consequence of the substantial variance in the shape of the
binding pocket. FimH binds Man with high affinity because of the
deep, narrow pocket formed by loops 1, 2, and 3, in which loops
2 and 3 mediate specific polar interactions directly to Man and a
water molecule and loop 1 serves as an affinity clamp to stymie
dissociation of Man (35). In contrast, loop 1 in FmlH is more
distant from loops 2 and 3 than it is in FimH and does not con-
tribute to binding, which results in a widened, solvent-exposed
pocket for weak Gal binding (Fig. 1D). In addition, the differ-
ences in binding pocket architecture dictate the sterically allowed
linkage types. FimH has space at the tip of the LD between its
parallel tyrosine gate (residues Y48 and Y137) to accept α-linked
moieties, of which biaryl groups confer drastic enhancements in af-
finity through strong parallel face-to-face π-stacking interactions. In
contrast, FmlH is capped at the very tip of the pocket with Y46,
which biases specificity toward β-linked moieties, of which biaryl
groups confer moderate enhancements in affinity through signifi-
cant edge-to-face π-stacking interactions. Having accounted for
these variations, our structure-guided medicinal chemistry approach,
coupled with our in vivo work, has clearly demonstrated the future
translational impact of galactosides as treatments for UTI.
It is noteworthy that our collective search for high-affinity
antagonists of FmlH and FimH has led to discovery of biphenyl
moieties as the preferred aglycone groups for high-affinity galac-
tosides and mannosides, respectively. Pocket geometry dictates the
type of biphenyl scaffold that is optimal. Thus, the best FimH-
targeting mannosides contain para biphenyls in the alpha stereo-
chemistry, while the best FmlH-targeting galactosides contain ortho
biphenyls in the beta orientation. However, in both cases, H-
bonding donors or acceptors on the B-ring result in significant
enhancement in binding affinity through specific interactions
outside the sugar binding pocket. Intriguingly, the inhibitory
potency conferred by the meta carboxyl on the B-ring of 29β-NAc
is also appreciated in the significant inhibitory role of the meta-
substituted group on the B-ring in cranberry compounds 24β and
26β, which suggests a common pharmacophore between our
optimal synthetic compound and natural-product compounds in
targeting FmlH. This study provides evidence that specific gly-
cosidic compounds in cranberry can specifically bind and inhibit
a bacterial adhesin. Furthermore, our work exposes a trend in-
dicating that π-stacking of aromatic aglycones with binding pocket
residues in the adhesin is essential in mimicking glycoprotein re-
ceptors and for developing tight-binding ligands in each lectin.
Mimicking carbohydrates with small molecules is a long-sought-
after goal in medicinal chemistry and chemical biology (44–46),
and we believe that these results add significantly to this under-
standing and goal. This information can now be utilized not only in
the future optimization of lead compound 29β-NAc as a treatment
for UTIs, but also in the rational design of numerous other lectin
the biomedical strides made to promote human health and
longevity (9). Selection pressures imposed by antibiotics on
bacterial pathogens have promoted their proliferation, especially
through overuse of antibiotics within the farming industry and
inappropriate use or misuse among patients (47–50). Recent
reports indicate that patients are now succumbing to bacterial
strains which possess broad-spectrum resistance to all last-resort
antibiotics, which many fear signals that antibiotic resistance will
pave the way for the “next pandemic” (15). Antivirulence strat-
egies that aim to reduce the pathogenicity of bacterial pathogens
promise to provide the same therapeutic efficacy as antibiotics
without introducing selective pressures that would promote wide-
spread dissemination of resistance (16). Multiple antivirulence ef-
forts will be required to combat the multiple mechanisms by which
diverse bacterial pathogens colonize the host, which can include, for
example, the targeting of CUP pilus adhesins or the biogenesis
machinery responsible for the assembly of CUP pili (51). As
highlighted in this work, UPEC employs an armament of diverse
CUP pili to colonize and persist within changing local environ-
ments encountered during UTI pathogenesis, which suggests that
targeting more than one CUP adhesin may indeed be a more ef-
fective strategy for combating UTIs. Herein, we have highlighted
the overwhelming value of applying a deep mechanistic under-
standing of structure-function-virulence relationships of bacterial
adhesins to the rational design of high-affinity carbohydrate gly-
comimetics for the treatment of UTI. This demonstration serves as
a general model for the rational approach necessary to target viru-
lence factors and disrupt their role in bacterial infections.
Materials and Methods
Ethics Statement. All animal experiments were conducted according to the
National Institutes of Health (NIH) guidelines for housing and care of labo-
ratory animals and performed in accordance with institutional regulations
after pertinent review and approval by the Institutional Animal Care and Use
Committee at Washington University School of Medicine (protocol 20150226).
Deidentified human tissue was obtained from the Tissue Procurement Core at
Washington University School of Medicine.
Protein Expression and Purification. FmlH residues 1 to 160 from UPEC strain
UTI89 with a C-terminal six-histidine tag (i.e., FmlHLD) were cloned into the
IPTG-inducible plasmid pTrc99A. This construct was then transformed into
and expressed in E. coli strain C600. Periplasms were isolated as previously
described and dialyzed four times against PBS plus 250 mM NaCl (34). FmlHLD
was purified from this periplasmic fraction by cobalt affinity chromatogra-
phy through elution with 150 mM imidazole. FmlHLD was buffer exchanged
into 10 mM Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH
7.5) and 50 mM NaCl, concentrated to 6 mg/mL, and stored stably at 4 °C for
use in biochemical and biophysical assays.
In Silico Docking and Virtual Screening. Structure-based virtual screening
through in silico docking was performed with AutoDock Vina (52). Existing
Gal-based derivatives were identified through the ZINC12 database (53).
Their 3D structures were extracted from the downloaded mol2 file as pdb
coordinates and converted to pdbqt format using Open Babel (54). The
crystal structure of apo FmlH was converted to its topology file using
AutoDock Tools. The grid box was centered at the Gal binding pocket of
FmlH, and its dimensions (26 × 26 × 26 Å3) were chosen to accommodate
bulky compounds and multiple potential binding modes at or near the
binding pocket. The exhaustiveness of the search was set to a value of 15.
The top binding modes and scores within this grid space were generated by
AutoDock Vina. Custom in-house scripts in Bash and MATLAB were used to
link these binding scores with compound properties such as molecular
weight. Top binding modes were visualized in PyMOL.
Virtual screening of this library, which comprised galactosides ranging
from 150 to 900 Da in molecular mass, yielded a mean docking score of
8 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
Kalas et al.

6.3 kcal/mol (1 kcal = 4.18 kJ), with a standard deviation of 0.73 kcal/mol and
a range of 4 to 9 kcal/mol (SI Appendix, Fig. S1A). To prioritize hits, we
abstained from directly comparing raw binding scores, as large, lipophilic
molecules tend to have artificially high predicted binding interactions due to
their contribution to hydrophobic interactions as calculated by the empirical
scoring function of AutoDock Vina (52). Instead, the results of the virtual
screen were evaluated per group efficiency (GE), which, in this context, mea-
sures the contribution of the aglycone group within each galactoside (indi-
cated as X in the following equation) to the docking score (DS) with respect to
and 2% PEG8000) and equilibrated against 1 mL of mother liquor in the
reservoir. These crystals were transferred into cryoprotectant (1 M LiSO4,
10% PEG8000, and 25% glycerol). Diffraction data for TF, 4β (in space group C
1 2 1), and 29β-NAc structures were collected at 100 K at an in-house facility
equipped with a rotating anode Rigaku MicroMax 007 generator, a Rayonix
Marmux X-ray source, and a Mar345 image plate detector. Diffraction data for
apo, 4β, 5β, and 20β structures were collected at 100 K at the ALS Beamline
4.2.2. Data were indexed and integrated in iMosflm (55), XDS (56), or
HKL2000 and scaled by Scala (57). The phase problem was solved by molecular
replacement using Phaser-MR in PHENIX (58) with FimHLD from PDB ID 3MCY.
Several rounds of refinements were performed in PHENIX to improve the
final models.
the number of heavy atoms (HA) present in the aglycone group [GE = (DSX
−
DSGal)/(HAX − HAGal)]. Top hits were defined as galactosides with a GE value
greater than 1.25 times the SD (σ = 0.0016 kcal/mol per HA) above the library
mean (μ = 0.0011 kcal/mol per HA), which constituted the top ∼10% of highest
scoring galactosides (SI Appendix, Fig. S1A).
Mouse Infections. Seven- to 8-wk-old female C3H/HeN mice were obtained
from Envigo. Mice were anesthetized and inoculated via transurethral cathe-
terization with 50 μL of CFT073 bacterial suspension (∼1 × 108 to 2 × 108 CFU in
total) in PBS. Mice experiencing high titers of bacteriuria (>104 CFU/mL) and
edematous and inflamed bladders when killed after 2 wk, or chronic cystitis
(39), were then transurethrally inoculated either with 50 mg/kg compound or
vehicle control (10% DMSO). Mice were killed 6 h posttreatment, and bacteria
colonizing the bladder or kidney were plated for quantification.
Synthesis of Galactosides and N-acetyl Aminogalactosides. Galactosides and
N-acetyl aminogalactosides were synthesized by standard glycosylation
chemistry, including boron trifluoride-mediated glycosidation and the Koenigs–
Knorr reaction, respectively (SI Appendix, Fig. S2). In method A, boron trifloride-
promoted glycosylation of phenols with Gal pentaacetate yielded corresponding
acetylated aryl galactosides, which were treated with sodium methoxide in
methanol to provide the corresponding aryl galactosides (1β to 3β, 5β to 9β, 18β
to 19β, and 28β to 32β; 2α to 3α, 18α to 19α, and 28α to 32α). In method B, final
GalNAc and Gal analogs (20β-NAc, 21β, 28β-NAc, and 29β-NAc) were synthesized
from galactosyl halide and aryl alcohols via a Koenigs–Knorr-type reaction, which
yielded aryl galactosides that were then deacetylated by treatment with
methylamine in ethanol.
Immunofluorescence. Frozen, deidentified human bladder and kidney sections
were obtained from the Tissue Procurement Core and stored stably at −80 °C.
These tissue section slides were removed from the freezer and allowed to
thaw at room temperature for 10 to 20 min. After applying a hydrophobic
barrier pen around the tissue, slides were rehydrated in 200 μL buffer (5%
BSA and 0.2% Triton X-100 in PBS) for 10 min. Buffer was gently aspirated
and slides were blocked for 1 h at room temperature with 200 μL of buffer.
Thereafter, buffer was gently aspirated and slides were incubated with
200 μL of sample overnight at 4 °C. Samples diluted in buffer included 50 μg/mL
FmlHLD wild-type (WT), 50 μg/mL FmlHLD K132Q, and 50 μg/mL FmlHLD WT in-
cubated with 100 μM 29β-NAc. Samples were gently aspirated and slides were
washed three times in buffer for 5 min each. Next, slides were incubated with our
mouse anti-FmlH polyclonal antibody (1:500 dilution in buffer) for 1 h at room
temperature. Slides were washed again three times in buffer and then incubated
in the dark with donkey anti-mouse IgG Alexa Fluor 594 and Wheat Germ
Agglutinin Alexa Fluor 633 (each 1:500 dilution in buffer) for 1 h at room
temperature. Slides were washed once with buffer and then incubated in the
dark with DAPI (1:1,000 dilution in buffer) for 5 min at room temperature. After
washing twice with buffer, coverslips were mounted using 80 μL of mounting
media. Slides were loaded onto a Zeiss LSM 880 Confocal Laser Scanning Mi-
croscope (Carl Zeiss, Inc.) equipped with a diode 405 to 430 laser, a HeNe 543
laser, and a HeNe 633 laser. Images were acquired with a 20×, 0.8 numerical
aperture Zeiss Plan Apochromat objective using ZEN 2 imaging software.
ELISA. Immulon 4HBX 96-well plates were coated overnight with 1 μg of
bovine submaxillary mucin (Sigma). Coated wells were then treated with
100 μL of Arthrobacter ureafaciens sialidase (10 mU/mL) diluted in PBS for
1 h at 37 °C. Thereafter, wells were incubated with 200 μL of blocking buffer
(PBS plus 1% BSA) for 2 h at 23 °C, followed by incubation with 100 μL of
biotinylated FmlHLD diluted in blocking buffer to 20 μg/mL in the presence or
absence of galactoside compounds for 1 h at 23 °C. After washing three
times with PBS plus 0.05% TWEEN-20, 100 μL of streptavidin-HRP conjugate
(BD Biosciences; 1:2,000 dilution in blocking buffer) was added to each well
for 1 h at 23 °C. After a final round of washing, plates were developed with
100 μL of tetramethylbenzidine (BD Biosciences) substrate and quenched
within 1 to 2 min with 50 μL of 1 M H2SO4, and absorbance was measured at
450 nm. This assay was used to determine percent inhibition values and
inhibitory constant (IC50) values where indicated.
BLI. Streptavidin pins were first dipped in a baseline in PBS (pH 7.4) for 120 s,
followed by loading of 5 to 10 μg/mL biotinylated Ser-TF (Toronto Research
Chemicals) in PBS for 300 s, quenching by 10 μg/mL biocytin in PBS for 240 s,
and another baseline step in PBS for 120 s. Thereafter, pins were dipped in
PBS for 120 s and transferred to protein samples (varying concentration of
FmlHLD or fixed concentration of FmlHLD with varying concentration of ga-
lactoside compounds) for association for 300 to 600 s. Equilibrium binding
response values were used to determine the affinity of interaction between
FmlHLD and immobilized Ser-TF under a 1:1 binding model or between FmlHLD
and galactosides in solution under a competitive one-site binding model.
Statistics. Mouse data are compiled from two (4Z269 plus 29β-NAc) or three
(all other treatments) independent experiments, with four or five mice per
group per experiment. These data were analyzed using the uncorrected
two-tailed Mann–Whitney U test in GraphPad Prisim v.5. ELISA data are
reported as box-and-whisker plots indicating the mean, 2.5th, 25th, 75th,
and 97.5th percentiles of at least two independent experiments, with three
technical replicates per experiment.
Protein Crystallization and Structure Determination. Crystals of apo FmlHLD in
10 mM Hepes (pH 7.5) and 50 mM NaCl were grown by mixing 2 μL of
protein (6 mg/mL) with 2 μL of mother liquor [0.2 M ammonium sulfate,
0.1 M NaCl, 0.1 M Mes [2-(N-morpholino)ethanesulfonic acid] (pH 5.6), and
28% PEG 3350] and equilibrated against 1 mL of mother liquor in the res-
ervoir. Cocrystals of FmlHLD bound to TF or galactosides 4β (in space group P
2 21 21), 5β, and 20β were grown by mixing 2 μL of protein (6 mg/mL) in the
presence of 5 mM compound with 2 μL of mother liquor [0.2 M ammonium
sulfate, 0.1 M NaCl, 0.1 M Mes (pH 5.6), and 32% PEG 3350] and equilibrated
against 1 mL of mother liquor in the reservoir. These crystals were trans-
ferred into cryoprotectant [0.2 M ammonium sulfate, 0.1 M NaCl, 0.1 M Mes
(pH 5.6), 35% PEG 4000, and 10% glycerol] and then flash frozen in liquid
nitrogen. Cocrystals of FmlHLD bound to the galactoside 29β-NAc were
grown by mixing 2 μL of protein complex (10 mg/mL FmlHLD with a 1.2:1
molar ratio of 29β-NAc to FmlHLD) with 2 μL of mother liquor (0.7 M LiSO4
ACKNOWLEDGMENTS. We thank members of the S.J.H. laboratory for help-
ful suggestions; Rick Stegeman at Washington University and Jay Nix at ALS
Beamline 4.2.2 for technical assistance in X-ray data collection; and Wandy
Beatty at Washington University for assistance and expertise in confocal
microscopy. We thank Ocean Spray for their helpful advice and the Alvin J.
Siteman Cancer Center at Washington University School of Medicine, the
Barnes-Jewish Hospital, and the Institute of Clinical and Translational Sciences
(ICTS) at Washington University in St. Louis, for the use of the Tissue Procure-
ment Core, which provided human urinary tract tissue. The Alvin J. Siteman
Cancer Center is supported, in part, by National Cancer Institute Cancer Center
Support Grant P30 CA091842. The ICTS is funded by NIH National Center for
Advancing Translational Sciences Clinical and Translational Science Award Pro-
gram Grant UL1 TR002345. J.W.J. and S.J.H. were supported by NIH National
Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK108840.
V.K. was supported by Medical Scientist Training Program Grant T32GM07200.
1. Foxman B (2003) Epidemiology of urinary tract infections: Incidence, morbidity, and
3. Gupta K, Hooton TM, Roberts PL, Stamm WE (2001) Patient-initiated treatment of
economic costs. Dis Mon 49:53–70.
uncomplicated recurrent urinary tract infections in young women. Ann Intern Med
2. Griebling TL (2005) Urologic diseases in America project: Trends in resource use for
135:9–16.
urinary tract infections in women. J Urol 173:1281–1287.
4. Foxman B (2010) The epidemiology of urinary tract infection. Nat Rev Urol 7:653–660.
Kalas et al.
PNAS Latest Articles
|
9 of 10

5. Ramakrishnan K, Scheid DC (2005) Diagnosis and management of acute pyelone-
phritis in adults. Am Fam Physician 71:933–942.
6. Pertel PE, Haverstock D (2006) Risk factors for a poor outcome after therapy for acute
pyelonephritis. BJU Int 98:141–147.
7. Efstathiou SP, et al. (2003) Acute pyelonephritis in adults: Prediction of mortality and
failure of treatment. Arch Intern Med 163:1206–1212.
8. Roberts FJ, Geere IW, Coldman A (1991) A three-year study of positive blood cultures,
with emphasis on prognosis. Rev Infect Dis 13:34–46.
31. Chalopin T, et al. (2016) Second generation of thiazolylmannosides, FimH antagonists
for E. coli-induced Crohn’s disease. Org Biomol Chem 14:3913–3925.
32. Mydock-McGrane LK, Cusumano ZT, Janetka JW (2016) Mannose-derived FimH an-
tagonists: A promising anti-virulence therapeutic strategy for urinary tract infections
and Crohn’s disease. Expert Opin Ther Pat 26:175–197.
33. Wurpel DJ, et al. (2014) F9 fimbriae of uropathogenic Escherichia coli are expressed at
low temperature and recognise Galβ1-3GlcNAc-containing glycans. PLoS One 9:
e93177.
9. WHO (2014) Antimicrobial resistance: Global report on surveillance 2014 (WHO,
Geneva).
34. Conover MS, et al. (2016) Inflammation-induced adhesin-receptor interaction pro-
vides a fitness advantage to uropathogenic E. coli during chronic infection. Cell Host
Microbe 20:482–492.
35. Kalas V, et al. (2017) Evolutionary fine-tuning of conformational ensembles in FimH
during host-pathogen interactions. Sci Adv 3:e1601944.
36. Le Trong I, et al. (2010) Structural basis for mechanical force regulation of the adhesin
FimH via finger trap-like beta sheet twisting. Cell 141:645–655.
37. Sauer MM, et al. (2016) Catch-bond mechanism of the bacterial adhesin FimH. Nat
Commun 7:10738.
10. Guneysel O, Onur O, Erdede M, Denizbasi A (2009) Trimethoprim/sulfamethoxazole
resistance in urinary tract infections. J Emerg Med 36:338–341.
11. Raz R, et al.; Israeli Urinary Tract Infection Group (2002) Empiric use of trimethoprim-
sulfamethoxazole (TMP-SMX) in the treatment of women with uncomplicated urinary
tract infections, in a geographical area with a high prevalence of TMP-SMX-resistant
uropathogens. Clin Infect Dis 34:1165–1169.
12. Aypak C, Altunsoy A, Düzgün
N (2009) Empiric antibiotic therapy in acute un-
complicated urinary tract infections and fluoroquinolone resistance: A prospective
observational study. Ann Clin Microbiol Antimicrob 8:27.
13. Kallen AJ, Welch HG, Sirovich BE (2006) Current antibiotic therapy for isolated urinary
tract infections in women. Arch Intern Med 166:635–639.
14. Karlowsky JA, Hoban DJ, Decorby MR, Laing NM, Zhanel GG (2006) Fluoroquinolone-
resistant urinary isolates of Escherichia coli from outpatients are frequently multidrug
resistant: Results from the North American Urinary Tract Infection Collaborative
Alliance-Quinolone Resistance study. Antimicrob Agents Chemother 50:2251–2254.
15. McGann P, et al. (2016) Escherichia coli harboring mcr-1 and blaCTX-M on a novel IncF
plasmid: First report of mcr-1 in the United States. Antimicrob Agents Chemother 60:
4420–4421.
38. Mayer K, et al. (2017) Urinary tract infection: Which conformation of the bacterial
lectin FimH is therapeutically relevant? J Med Chem 60:5646–5662.
39. Hannan TJ, Mysorekar IU, Hung CS, Isaacson-Schmid ML, Hultgren SJ (2010) Early
severe inflammatory responses to uropathogenic E. coli predispose to chronic and
recurrent urinary tract infection. PLoS Pathog 6:e1001042.
40. Murawski IJ, et al. (2010) The C3H/HeJ inbred mouse is a model of vesico-ureteric
reflux with a susceptibility locus on chromosome 12. Kidney Int 78:269–278.
41. Subashchandrabose S, et al. (2014) Host-specific induction of Escherichia coli fitness
genes during human urinary tract infection. Proc Natl Acad Sci USA 111:18327–18332.
42. Bouckaert J, et al. (2006) The affinity of the FimH fimbrial adhesin is receptor-driven
and quasi-independent of Escherichia coli pathotypes. Mol Microbiol 61:1556–1568.
43. Hung CS, et al. (2002) Structural basis of tropism of Escherichia coli to the bladder
during urinary tract infection. Mol Microbiol 44:903–915.
16. Rasko DA, Sperandio V (2010) Anti-virulence strategies to combat bacteria-mediated
disease. Nat Rev Drug Discov 9:117–128.
17. Ronald A (2003) The etiology of urinary tract infection: Traditional and emerging
pathogens. Dis Mon 49:71–82.
44. Ernst B, Magnani JL (2009) From carbohydrate leads to glycomimetic drugs. Nat Rev
Drug Discov 8:661–677.
18. Ronald AR, et al. (2001) Urinary tract infection in adults: Research priorities and
strategies. Int J Antimicrob Agents 17:343–348.
19. Schreiber HL, 4th, et al. (2017) Bacterial virulence phenotypes of Escherichia coli and
host susceptibility determine risk for urinary tract infections. Sci Transl Med 9:
eaaf1283.
45. Hsu CH, et al. (2016) The dependence of carbohydrate-aromatic interaction strengths
on the structure of the carbohydrate. J Am Chem Soc 138:7636–7648.
46. Hudson KL, et al. (2015) Carbohydrate-aromatic interactions in proteins. J Am Chem
Soc 137:15152–15160.
47. Ter Kuile BH, Kraupner N, Brul S (2016) The risk of low concentrations of antibiotics in
agriculture for resistance in human health care. FEMS Microbiol Lett 363:fnw210.
20. Waksman G, Hultgren SJ (2009) Structural biology of the chaperone-usher pathway of
pilus biogenesis. Nat Rev Microbiol 7:765–774.
48. Llor C, Bjerrum
L (2014) Antimicrobial resistance: Risk associated with antibiotic
21. Wurpel DJ, Beatson SA, Totsika M, Petty NK, Schembri MA (2013) Chaperone-usher
fimbriae of Escherichia coli. PLoS One 8:e52835.
22. Jones CH, et al. (1995) FimH adhesin of type 1 pili is assembled into a fibrillar tip
structure in the Enterobacteriaceae. Proc Natl Acad Sci USA 92:2081–2085.
23. Mulvey MA, et al. (1998) Induction and evasion of host defenses by type 1-piliated
uropathogenic Escherichia coli. Science 282:1494–1497.
24. Mydock-McGrane LK, Hannan TJ, Janetka JW (2017) Rational design strategies for
FimH antagonists: New drugs on the horizon for urinary tract infection and Crohn’s
disease. Expert Opin Drug Discov 12:711–731.
25. Jarvis C, et al. (2016) Antivirulence isoquinolone mannosides: Optimization of the
biaryl aglycone for FimH lectin binding affinity and efficacy in the treatment of
chronic UTI. ChemMedChem 11:367–373.
overuse and initiatives to reduce the problem. Ther Adv Drug Saf 5:229–241.
49. Kardas P, Devine S, Golembesky A, Roberts C (2005) A systematic review and meta-
analysis of misuse of antibiotic therapies in the community. Int J Antimicrob Agents
26:106–113.
50. Chang Q, Wang W, Regev-Yochay G, Lipsitch M, Hanage WP (2015) Antibiotics in
agriculture and the risk to human health: How worried should we be? Evol Appl 8:
240–247.
51. Pinkner JS, et al. (2006) Rationally designed small compounds inhibit pilus biogenesis
in uropathogenic bacteria. Proc Natl Acad Sci USA 103:17897–17902.
52. Trott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of
docking with a new scoring function, efficient optimization, and multithreading.
J Comput Chem 31:455–461.
26. Mydock-McGrane L, et al. (2016) Antivirulence C-mannosides as antibiotic-sparing,
oral therapeutics for urinary tract infections. J Med Chem 59:9390–9408.
27. Han Z, et al. (2010) Structure-based drug design and optimization of mannoside
bacterial FimH antagonists. J Med Chem 53:4779–4792.
28. Cusumano CK, et al. (2011) Treatment and prevention of urinary tract infection with
orally active FimH inhibitors. Sci Transl Med 3:109ra115.
53. Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG (2012) ZINC: A free tool to
discover chemistry for biology. J Chem Inf Model 52:1757–1768.
54. O’Boyle NM, et al. (2011) Open Babel: An open chemical toolbox. J Cheminform 3:33.
55. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: A new
graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D
Biol Crystallogr 67:271–281.
29. Grabosch C, Hartmann M, Schmidt-Lassen J, Lindhorst TK (2011) Squaric acid mono-
amide mannosides as ligands for the bacterial lectin FimH: Covalent inhibition or not?
ChemBioChem 12:1066–1074.
56. Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66:125–132.
57. Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. Acta
Crystallogr D Biol Crystallogr 67:235–242.
30. Kleeb S, et al. (2015) FimH antagonists: Bioisosteres to improve the in vitro and in vivo
PK/PD profile. J Med Chem 58:2221–2239.
58. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro-
molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221.
10 of 10
|
www.pnas.org/cgi/doi/10.1073/pnas.1720140115
Kalas et al.