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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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 American Society for Microbiology.)

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 (IC 50  = 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(IC 50  = 1 nM) and 13 l(IC 50  = 7 nM) which were chosen as leads for further optimization., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

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.

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.

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.

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.

In Saccharomyces cerevisiae, the STE12 protein mediates transcriptional induction of cell type-specific genes in response to pheromones. STE12 binds in vitro to the pheromone response elements (PREs) present in the control region of a-specific genes. STE12 is also required for transcription of alpha-specific genes, but there is no evidence that it binds directly to these genes. Instead, the MAT alpha-encoded protein alpha 1 and the MCM1 product bind to the DNA element that is responsible for alpha-specific and a-factor-inducible expression. To explore the role of STE12 in the pheromone induction of alpha-specific genes, we cloned STE12 and MAT alpha 1 homologs from the related yeast Kluyveromyces lactis. The K. lactis STE12 protein did not cooperate with the S. cerevisiae alpha 1 protein to promote the overall mating process or the induction of transcription of an alpha-specific gene. However, introduction of both K. lactis STE12 along with K. lactis alpha 1 did restore mating, suggesting that an interaction between STE12 and alpha 1 is important for alpha-specific gene activation. We also show that bacterially expressed STE12 and alpha 1 are able to form a complex in vitro. Thus, we demonstrate a coupling in alpha cells between a protein functioning in cell identity, alpha 1, with a protein responsive to the pheromone-induced signal STE12.

The pheromone response pathway of the yeast Saccharomyces cerevisiae is necessary for the basal level of transcription of cell-type-specific genes, as well as the induced level observed after pheromone treatment. The STE12 protein binds to the DNA sequence designated the pheromone response element and is a target of the pheromone-induced signal. We generated 6-nucleotide linker insertion mutants, internal-deletion mutants, and carboxy-terminal truncation mutants of STE12 and assayed them for their ability to restore mating and transcriptional activity to a ste12 delta strain. Two of these mutant proteins retain the capacity to mediate basal transcription but show little or no induced transcription upon pheromone treatment. Cells producing these proteins cannot mate, formally demonstrating that the ability to respond to pheromone by increasing gene expression is essential for the mating process. Since distinct domains of STE12 appear to be required for basal versus induced transcription, we suggest that the pheromone-induced signal is likely to target residues of the protein different from those targeted by the basal signal because of the constitutive activity of the response pathway. Our analysis of mutant STE12 proteins also indicates that only the DNA-binding domain is sensitive to the small changes caused by the linker insertions. In addition, we show that, while the carboxy-terminal sequences necessary for STE12 to form a complex with the transcription factor MCM1 are not essential for mating, these sequences are required for optimal transcriptional activity.

Multicopy plasmids containing the hisDG region of Klebsiella pneumoniae were mutagenized with transposon Tn5. The resulting plasmids were examined for their ability to complement hisD and hisG mutations in Escherichia coli. The physical location of Tn5 on each of the hisD::Tn5 and hisG::Tn5 plasmids was determined by restriction endonuclease analysis. By combining the two types of data, a precise correlated physical and genetic map of the K. pneumoniae hisDG region was constructed. Based on this analysis, the minimum sizes of the hisD and the hisG genes were calculated to be 1100 bp and 900 bp, respectively. The hisO(P) region was also identified. The insertional specificity of transposon Tn5 was shown to be very low. One unanticipated result was obtained: Tn5 insertions in the plasmid-borne hisG gene were not polar on hisD.

We report here the isolation and primary structure of two genes encoding rat U3 small nuclear RNA. One of the genes encodes U3B RNA; the other encodes an RNA which is almost identical to U3A RNA. Both genes are expressed after microinjection into the nuclei of Xenopus laevis oocytes and can direct the accumulation of mature U3 RNA as well as longer transcripts which may be the U3 precursors. We have also isolated and sequenced four other regions of the rat genome homologous to U3 RNA. One of these almost certainly represents a second U3B gene; the other three are pseudogenes which appear to have been generated by the reverse flow of genetic information from U3 RNA back into the genome. Using genomic blotting techniques, we show that the rat U3 genes are present in only a few copies per haploid genome and are probably not closely linked to one another.

From previous work it was known that U3 RNA is hydrogen bonded to nucleolar 28 S to 35 S RNA and can be covalently crosslinked to RNA of greater than 28 S by irradiation in vivo with long-wave ultraviolet light in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT psoralen). Here we use a novel sandwich blot technique to identify these large nucleolar RNA species as rRNA precursors and to map the site(s) of crosslinking in vivo. The crosslink occurs between one or more residues near the 5' end of U3 RNA and a 380 nucleotide region of the rat rRNA external transcribed spacer (ETS1). We have sequenced this region of the rat ETS and we show that it includes an RNA-processing site analogous to those previously mapped to approximately 3.5 kb upstream from the 5' end of mouse and human 18 S rRNAs.