Hepatitis C virus (HCV) replication is dependent on the formation of specialized membrane structures; however, the host factor requirements for the formation of these HCV complexes remain unclear. ...Herein, we demonstrate that inhibition of stearoyl-CoA desaturase 1 (SCD-1) halts the biosynthesis of unsaturated fatty acids, such as oleic acid, and negatively modulates HCV replication. Unsaturated fatty acids play key roles in membrane curvature and fluidity. Mechanistically, we demonstrate that SCD-1 inhibition disrupts the integrity of membranous HCV replication complexes and renders HCV RNA susceptible to nuclease-mediated degradation. Our work establishes a novel function for unsaturated fatty acids in HCV replication.
The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist (3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopentabindol-3-yl-acetic acid (13) ...is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP K i = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (C max = 1100 μM for 6 and 3900 μM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.
To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying ...bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that ...cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prostaglandin G/H synthase (PGHS), a key enzyme leading to the formation of prostaglandins, is the target of nonsteroidal antiinflammatory drugs. Two forms of the enzyme have been identified, PGHS-1 ...and PGHS-2. Epidemiological evidence has suggested that aspirin and other nonsteroidal antiinflammatory drugs may reduce the risk of colorectal cancer. We examined by immunoblot analyses the expression of human PGHS-1 and PGHS-2 protein in 25 matched colon cancer and nontumor tissues, 4 premalignant polyps, 5 control colon tissues from noncancer patients, and 3 matched normal and cancerous breast tissue samples. PGHS-1 was detected in all normal and tumor tissue. In contrast, PGHS-2 was not detected in 23 of 25 normal colon tissues but was detected in 19 of 25 colon tumors. PGHS-2 protein was not observed in four human premalignant polyp samples, control colon from noncancer patients, or matched normal or cancerous breast tissues. These results suggest that the beneficial effects of nonsteroidal antiinflammatory drugs in colon cancer may be mediated by inhibition of PGHS-2.
The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory ...effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.
Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and ...duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.
Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp "safety-catch" regulatory tripeptide contained within a flexible loop near the ...large-subunit/small-subunit junction. Removal of this "safety catch" results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.
Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to ...successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.
Abstract
Reactivation of androgen receptor (AR) signaling by active splice variants (AR-Vs) is one of key drivers of castration resistant prostate cancer (CRPC). AR-v7 is the most prevalent AR-V and ...its expression has been clinically associated with poor overall survival, resistance to the AR inhibitors enzalutamide and abiraterone, as well as taxane resistance. Given that treatment with AR inhibitors and taxanes are the only effective therapeutic modalities in CRPC, development of specific AR-v7 inhibitors is urgently needed. Mechanistically, AR-v7 re-activates AR signaling by being constitutively active in the nucleus. While taxane chemotherapy inhibits the nuclear import of AR which is significantly associated with clinical outcomes in CRPC, it has no effect on AR-v7 nuclear localization and activity. Mechanistically, AR-v7 lacks the microtubule-binding domain and—unlike AR—does not utilize the canonical importin-α/β pathway, or RanGTP for nuclear import. Using wheat germ agglutinin to block active protein nuclear uptake resulted in AR-v7 cytoplasmic sequestration, indicating a requirement for an alternative transport receptor. Further, mutation of AR-v7 dimerization domain (D-box) led to its cytoplasmic sequestration, indicating that the D-box is also required for nuclear import. As inhibition of AR nuclear import is a clinically validated therapeutic strategy, we developed a novel drug discovery platform to identify compounds that specifically inhibit AR-v7 nuclear import. Using cells stably expressing inducible AR-v7 in conjunction with an enzyme complementation assay we tested 166,000 compounds by high throughput screening (HTS). The robust HTS performance (Z>0.8) together with subsequent counter screens including confirmation and compound titration, cell toxicity, a tertiary imaging based screen, led to identification of lead compounds that inhibit AR-v7 nuclear import. The lead compounds share structural features, across two main chemotypes, which are amenable to structure-activity relationship studies to identify the most desirable compound for in vivo studies. Using newly synthesized compounds from each of the two chemotypes, we showed specific dose-dependent inhibition of AR-v7 nuclear import. Currently, we are testing these compounds, on inhibition of AR-v7 transcriptional activity across several cell models including enzalutamide-resistant cells as well as inhibition of tumor growth in AR-v7 xenograft models. In parallel we are testing the lead compound for potential direct binding to the AR-v7 dimerization domain or to candidate alternative nuclear transport receptors. Further development of our lead small molecules will yield novel chemotypes, with desirable pharmacological properties that target the unique AR-v7 nuclear import pathway and can be clinically combined with existing AR therapies.
Note: This abstract was not presented at the meeting.
Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Eiman Mukhtar, Michael Miller, Leigh Baxt, Stacia Kargman, Andrew Stamford, Peter Meinke, Paraskevi Giannakakou. Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1017.
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