Treatment of Clostridium difficile Infection with a Small-Molecule Inhibitor of Toxin UDP-Glucose Hydrolysis Activity.

Clostridium difficile infection (CDI) is the leading cause of hospital-acquired infectious diarrhea, with significant morbidity, mortality, and associated health care costs. The major risk factor for CDI is antimicrobial therapy, which disrupts the normal gut microbiota and allows C. difficile to flourish. Treatment of CDI with antimicrobials is generally effective in the short term, but recurrent infections are frequent and problematic, indicating that improved treatment options are necessary. Symptoms of disease are largely due to two homologous toxins, TcdA and TcdB, which are glucosyltransferases that inhibit host Rho GTPases. As the normal gut microbiota is an important component of resistance to CDI, our goal was to develop an effective nonantimicrobial therapy. Here, we report a highly potent small-molecule inhibitor (VB-82252) of TcdA and TcdB. This compound inhibits the UDP-glucose hydrolysis activity of TcdB and protects cells from intoxication after challenge with either toxin. Oral dosing of the inhibitor prevented inflammation in a murine intrarectal toxin challenge model. In a murine model of recurrent CDI, the inhibitor reduced weight loss and gut inflammation during acute disease and did not cause the recurrent disease that was observed with vancomycin treatment. Lastly, the inhibitor demonstrated efficacy similar to that of vancomycin in a hamster disease model. Overall, these results demonstrate that small-molecule inhibition of C. difficile toxin UDP-glucose hydrolysis activity is a promising nonantimicrobial approach to the treatment of CDI.

Synthesis and SAR studies of novel benzodiazepinedione-based inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

Synthesis and structure-activity relationships (SAR) of a novel series of benzodiazepinedione-based inhibitors of Clostridium difficile toxin B (TcdB) are described. Compounds demonstrating low nanomolar affinity for TcdB, and which possess improved stability in mouse plasma vs. earlier compounds from this series, have been identified. Optimized compound 11d demonstrates a good pharmacokinetic (PK) profile in mouse and hamster and is efficacious in a hamster survival model of Clostridium difficile infection.

Identification and initial optimization of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB).

The discovery, synthesis and preliminary structure-activity relationship (SAR) of a novel class of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB) is described. A high throughput screening (HTS) campaign resulted in the identification of moderately active screening hits 1-5 the most potent of which was compound 1 (IC50 = 0.77 µM). In silico docking of an early analog offered suggestions for structural modification which resulted in the design and synthesis of highly potent analogs 13j(IC50 = 1 nM) and 13 l(IC50 = 7 nM) which were chosen as leads for further optimization.

Novel antifungal drug discovery based on targeting pathways regulating the fungus-conserved Upc2 transcription factor.

Infections by Candida albicans and related fungal pathogens pose a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat infections, target the sterol biosynthetic pathway. Adaptation to azole therapy develops as drug-stressed cells compensate by upregulating several genes in the pathway, a process mediated in part by the Upc2 transcription factor. We have implemented a cell-based high-throughput screen to identify small-molecule inhibitors of Upc2-dependent induction of sterol gene expression in response to azole drug treatment. The assay is designed to identify not only Upc2 DNA binding inhibitors but also compounds impeding the activation of gene expression by Upc2. An AlphaScreen assay was developed to determine whether the compounds identified interact directly with Upc2 and inhibit DNA binding. Three compounds identified by the cell-based assay inhibited Upc2 protein level and UPC2-LacZ gene expression in response to a block in sterol biosynthesis. The compounds were growth inhibitory and attenuated antifungal-induced sterol gene expression in vivo. They did so by reducing the level of Upc2 protein and Upc2 DNA binding in the presence of drug. The mechanism by which the compounds restrict Upc2 DNA binding is not through a direct interaction, as demonstrated by a lack of DNA binding inhibitory activity using the AlphaScreen assay. Rather, they likely inhibit a novel pathway activating Upc2 in response to a block in sterol biosynthesis. We suggest that the compounds identified represent potential precursors for the synthesis of novel antifungal drugs.

Synthesis of (3,4-dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists.

The discovery and synthesis of a series of (dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists from a small-molecule combinatorial library using a high-throughput calcium mobilization functional assay (HEK293-human OX2-R cell line) is described. Active compounds show a good correlation between high-throughput single concentration screening data and measured IC(50)s. Specific examples exhibit IC(50) values of approximately 20 nM using human orexin A as the peptide agonist for the orexin-2 receptor.

Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3. EXPERIMENTAL APPROACH: As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined. KEY RESULTS: The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues. CONCLUSION AND IMPLICATIONS: We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.

Identification of CXCR3 receptor agonists in combinatorial small-molecule libraries.

In a high-throughput screen of four million compounds from combinatorial libraries for small-molecule modulators of the chemokine receptor CXCR3, two classes of receptor agonists, based on tetrahydroisoquinoline and piperidinyl diazepanone templates, were identified. Several of these compounds stimulated calcium flux in HEK293 cells expressing the recombinant human CXCR3 receptor with efficacies and kinetics similar to those of native ligand CXCL11/I-TAC and stimulated chemotaxis of activated human T-cells. The agonist small molecules also inhibited binding of another CXCR3 ligand, CXCL10/IP-10, to the receptor. The response to small-molecule agonists was inhibited by a CXCR3-specific small-molecule antagonist previously identified within the same combinatorial compound collection but structurally unrelated to the agonists. Remarkably, while other, non-amino acid substituents were present in the majority of the library compounds screened, the agonists from both classes contained a positively charged amino acid component, with preference for Arg>Lys, as well as a hydrophobic component.

Identification and initial evaluation of 4-N-aryl-[1,4]diazepane ureas as potent CXCR3 antagonists.

The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor CXCR3 are described. Specific examples exhibit IC(50) values of approximately 60 nM in a calcium mobilization functional assay, and dose-dependently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell alpha chemoattractant/I-TAC) as measured by T-cell chemotaxis, with a potency of approximately 100 nM.