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

Significance The emergence of multidrug-resistant bacteria, including uropathogenic 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 recognize distinct receptors with sterochemical specificity to facilitate persistence in various tissues and habitats. We used an interdisciplinary approach driven by structural biology and synthetic glycoside chemistry to design and optimize glycomimetic inhibitors of the UPEC adhesin FmlH. These inhibitors competitively blocked FmlH in vitro, in in vivo mouse UTI models, and in ex vivo healthy human kidney tissue. This work demonstrates the utility of structure-driven drug design in the effort to develop antivirulence therapeutic compounds. </jats:p

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue

Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling

Mannose-derived FimH antagonists: a promising anti-virulence therapeutic strategy for urinary tract infections and Crohn’s disease

<p><b>Introduction</b>: Type 1 pili are utilized by Gram-negative bacteria to adhere to host tissue and thus are a key virulence factor in urinary tract infections (UTIs) and Crohn’s disease (CD). This adhesion is mediated through specific binding of the terminal adhesin, FimH, to mannosylated host glycoproteins. FimH is essential for UTI pathogenesis and thus is a promising therapeutic target.</p> <p><b>Areas Covered</b>: Herein, we review the structural frameworks of FimH antagonists disclosed in the patent literature. X-ray crystallographic binding studies of D-mannose and early FimH antagonists have uncovered key molecular interactions. Exploiting this knowledge, mannosides with extraordinarily high binding affinities have been designed. Structure-activity relationships (SAR) and structure-property relationship (SPR) studies have resulted in the rapid development of orally bioavailable FimH antagonists with promising therapeutic potential for UTI and CD.</p> <p><b>Expert opinion</b>: It is our opinion that biaryl or ‘two-ring’ mannosides, which represent the largest and most thoroughly tested class of FimH antagonists, also hold the most promise as a novel treatment for UTIs. These antagonists have also been shown to have efficacy in treating CD. Judging from the strong preclinical data, we predict that one or more FimH antagonists will be entering the clinic within the next 1–2 years.</p

Synthesis of a Smoothened Cholesterol: 18,19-Di-nor-cholesterol

Herein, we report the first synthesis of a demethylated form of cholesterol (18,19-di-nor-cholesterol), in which the C18 and C19 methyl groups of the β-face were eliminated. Recent molecular simulations modeling 18,19-di-nor-cholesterol have suggested that cholesterol’s opposing rough β-face and smooth α-face play necessary roles in cholesterol’s membrane condensing abilities and, additionally, that specific facial preferences are displayed as cholesterol interacts with different neighboring lipids and transmembrane proteins. Inspired by these poorly characterized biochemical interactions, an extensive 18-step synthesis was completed as part of a collaborative effort, wherein synthesizing a “smoothened” cholesterol analogue would provide a direct way to experimentally measure the significance of the β-face methyl groups. Starting from known perhydrochrysenone <b>A</b>, the synthesis of 18,19-di-nor-cholesterol was accomplished with an excellent overall yield of 3.5%. The use of the highly stereoselective Dieckmann condensation and the employment of Evans’ chiral auxiliary were both key to ensuring the success of this synthesis