Enhancement of kinase selectivity in a potent class of arylamide FMS inhibitors

Structure–activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC₅₀ 0.7 nM, FMS cell IC₅₀ 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.     

Functional Complementation of a Model Target to Study Vpu Sensitivity

HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the ‘Vpu sensitivity sequence’ in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex.     

Synthesis and GABA receptor potency of 3-thiomethyl-4-(hetero)aryl-5-amino-1-phenylpyrazoles

A convenient synthetic route to novel 4-arylpyrazoles is described. The potential for insecticidal activity through GABA channel blockage by this series of compounds, as well as their selectivity for insect versus mammalian receptors, are explored through in vitro and in vivo assays.     

Vpu Downmodulates Two Distinct Targets,Tetherin and Gibbon Ape Leukemia Virus Envelope,through Shared Features in the Vpu Cytoplasmic Tail

During human immunodeficiency virus-1 (HIV-1) assembly, the host proteins CD4 (the HIV-1 receptor) and tetherin (an interferon stimulated anti-viral protein) both reduce viral fitness. The HIV-1 accessory gene Vpu counteracts both of these proteins, but it is thought to do so through two distinct mechanisms. Modulation of CD4 likely occurs through proteasomal degradation from the endoplasmic reticulum. The exact mechanism of tetherin modulation is less clear, with possible roles for degradation and alteration of protein transport to the plasma membrane. Most investigations of Vpu function have used different assays for CD4 and tetherin. In addition, many of these investigations used exogenously expressed Vpu, which could result in variable expression levels. Thus, few studies have investigated these two Vpu functions in parallel assays, making direct comparisons difficult. Here, we present results from a rapid assay used to simultaneously investigate Vpu-targeting of both tetherin and a viral glycoprotein, gibbon ape leukemia virus envelope (GaLV Env). We previously reported that Vpu modulates GaLV Env and prevents its incorporation into HIV-1 particles through a recognition motif similar to that found in CD4. Using this assay, we performed a comprehensive mutagenic scan of Vpu in its native proviral context to identify features required for both types of activity. We observed considerable overlap in the Vpu sequences required to modulate tetherin and GaLV Env. We found that features in the cytoplasmic tail of Vpu, specifically within the cytoplasmic tail hinge region, were required for modulation of both tetherin and GaLV Env. Interestingly, these same regions features have been determined to be critical for CD4 downmodulation. We also observed a role for the transmembrane domain in the restriction of tetherin, as previously reported, but not of GaLV Env. We propose that Vpu may target both proteins in a mechanistically similar manner, albeit in different cellular locations.     

Discovery of novel benzo[b]thiophene tetrazoles as non-carboxylate GPR40 agonists

GPR40 partial agonism is a promising new mechanism for the treatment of type 2 diabetes mellitus with clinical proof of concept. Most of the GPR40 agonists in the literature have a carboxylic acid functional group, which may pose a risk for idiosyncratic drug toxicity. A novel series of GPR40 agonists containing a tetrazole as a carboxylic acid bioisostere was identified. This series of compounds features a benzo[b]thiophene as the center ring, which is prone to oxidation during phase 1 metabolism. Following SAR optimization targeting GPR40 agonist activity and intrinsic clearance in microsomes (human and rat), potent and metabolically stable compounds were selected for in vivo evaluation. The compounds are efficacious at lowering blood glucose in a SD rat oGTT model.     

Optimum spacing between electrodes in an air-cathode single chamber microbial fuel cell with a low-cost polypropylene separator

Bioprocess and Biosystems Engineering - The performance of a single chamber microbial fuel cell (MFC) with a low-cost polypropylene separator was investigated at various electrode interspaces in a...     

Identification,cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.     

EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae

We cloned a gene, ECL_03329, from the chromosome of Enterobacter cloacae ATCC13047, using a drug-hypersensitive Escherichia coli KAM32 cell as the host. We show here that this gene, designated as emmdR, is responsible for multidrug resistance in E. cloacae. E. coli KAM32 host cells containing the cloned emmdR gene (KAM32/pEMMDR28) showed decreased susceptibilities to benzalkonium chloride, norfloxacin, ciprofloxacin, levofloxacin, ethidium bromide, acriflavine, rhodamine6G, and trimethoprim. emmdR-deficient E. cloacae cells (EcΔemmdR) showed increased susceptibilities to several of the antimicrobial agents tested. EmmdR has twelve predicted transmembrane segments and some shared identity with members of the multidrug and toxic compound extrusion (MATE) family of transporters. Study of the antimicrobial agent efflux activities revealed that EmmdR is an H+-drug antiporter but not a Na+ driven efflux pump. These results indicate that EmmdR is responsible for multidrug resistance and pumps out quinolones from E. cloacae.     

A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.     

Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia

Thermosensitive transient receptor potential melastatin 8 (TRPM8) antagonists are considered to be potential therapeutic agents for the treatment of cold hypersensitivity. The discovery of a new class of TRPM8 antagonists that shows in vivo efficacy in the rat chronic constriction injury (CCI)-induced model of neuropathic pain is described.