BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.     

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)     

Resistance to a protein farnesyltransferase inhibitor in Plasmodium falciparum

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. Inhibition of protein farnesyltransferase (PFT) is lethal to the pathogenic protozoa Plasmodium falciparum. Parasites were isolated that were resistant to BMS-388891, a tetrahydroquinoline (THQ) PFT inhibitor. Resistance was associated with a 12-fold decrease in drug susceptibility. Genotypic analysis revealed a single point mutation in the beta subunit in resistant parasites. The resultant tyrosine 837 to cysteine alteration in the beta subunit corresponded to the binding site for the THQ and peptide substrate. Biochemical analysis of Y837C-PFT demonstrated a 13-fold increase in BMS-388891 concentration necessary for inhibiting 50% of the enzyme activity. These data are consistent with PFT as the target of BMS-388891 in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents for effective therapy.     

Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization of a labile ester-containing drug

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D(5)-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid-liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487>314 and 473>300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within +/-5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at -30 degrees C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.     

Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.

The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.     

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR₅) PG-901 (10^?10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR₁ agonist BMS-470539 (10^?5 M) or with the mixed MCR_3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10^?10M) but not by the MCR_3/4 agonist MTII (0.30 nmol) or the MCR₁ agonist BMS-470539 (10^?5 M). The MCR₅-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR₅ agonist in the ARPE-19 cells. Altogether, these data suggest that MCR₅ is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.     

S1319: a novel beta2-andrenoceptor agonist from a marine sponge Dysidea sp.

In the course of screening of potential leads for beta2-receptor agonists, we found a novel beta2-adrenoceptor selective agonist, S1319, from a marine sponge Dysidea sp. The active compound was isolated and structurally characterized as 4-hydroxy-7-[1-(1-hydroxy-2-methylamino)ethyl]-1,3-benzothiazole-2(3H)-o ne, a new member of the beta2-adrenoceptor agonist. This is the first example of a sponge-derived beta2-adrenoceptor agonist.     

Phosphonooxymethyl prodrugs of the broad spectrum antifungal azole, ravuconazole: synthesis and biological properties

Synthesis of phosphonooxymethyl derivatives of ravuconazole, 2 (BMS-379224) and 3 (BMS-315801) and their biological evaluation as potential water-soluble prodrugs of ravuconazole are described. The phosphonooxymethyl ether analogue 2 (BMS-379224) and N-phosphonooxymethyl triazolium salt 3 (BMS-315801) were both prepared from ravuconazole (1) and bis-tert-butyl chloromethylphosphate, but under two different conditions. Both derivatives were highly soluble in water and converted to the parent in alkaline phosphatase, and also in vivo (rat). However, BMS-315801 was found to be less stable than BMS-379224 in water at neutral pH. BMS-379224 (2) has proved to be one of the most promising prodrugs of ravuconazole that we tested, and it is currently in clinical evaluation as an intravenous formulation of the broad spectrum antifungal azole, ravuconazole.     

Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052

BMS-790052, targeting nonstructural protein 5A (NS5A), is the most potent hepatitis C virus (HCV) inhibitor described to date. It is highly effective against genotype 1 replicons and also displays robust genotype 1 anti-HCV activity in the clinic (M. Gao et al., Nature 465:96-100, 2010). BMS-790052 inhibits genotype 2a JFH1 replicon cells and cell culture infectious virus with 50% effective concentrations (EC(50)s) of 46.8 and 16.1 pM, respectively. Resistance selection studies with the JFH1 replicon and virus systems identified drug-induced mutations within the N-terminal region of NS5A. F28S, L31M, C92R, and Y93H were the major resistance mutations identified; the impact of these mutations on inhibitor sensitivity between the replicon and virus was very similar. The C92R and Y93H mutations negatively impacted fitness of the JFH1 virus. Second-site replacements at NS5A residue 30 (K30E/Q) restored efficient replication of the C92R viral variant, thus demonstrating a genetic interaction between NS5A residues 30 and 92. By using a trans-complementation assay with JFH1 replicons encoding inhibitor-sensitive and inhibitor-resistant NS5A proteins, we provide genetic evidence that NS5A performs the following two distinct functions in HCV RNA replication: a cis-acting function that likely occurs as part of the HCV replication complex and a trans-acting function that may occur outside the replication complex. The cis-acting function is likely performed by basally phosphorylated NS5A, while the trans-acting function likely requires hyperphosphorylation. Our data indicate that BMS-790052 blocks the cis-acting function of NS5A. Since BMS-790052 also impairs JFH1 NS5A hyperphosphorylation, it likely also blocks the trans-acting function.     

Synthesis and nicotinic receptor activity of a hydroxylated tropane

(+/-)-3alpha-hydroxy homoepibatidine 4 has been synthesized from the alkaloid scopolamine 5 and its properties as a nicotinic agonist assessed. While still binding strongly, the compound showed reduced agonist potency for the alpha(4)beta(2) nAChR compared with the parent compound epibatidine 1. Compound 4 also displayed generally similar binding and selectivity profiles at alpha(4)beta(2), alpha(2)beta(4), alpha(3)beta(4), and alpha(4)beta(4) nAChR subtypes to those for nicotine.     

The role of beta93 Cys in the inhibition of Hb S fiber formation

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the beta93 Cys residue. In this study, the role of beta93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of beta93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the beta93 and alpha104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the beta93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the beta93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the alpha104 residue in addition to the beta93 residue significantly perturbs the alpha(1)beta(2) interface, while modification of only beta93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the alpha104 Cys residue and not the beta93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.     

Novel (4-piperidin-1-yl)-phenyl sulfonamides as potent and selective human beta(3) agonists

A series of novel (4-piperidin-1-yl)-phenyl sulfonamides was prepared and evaluated for their biological activity on the human beta(3)-adrenergic receptor (AR). Replacement of the 3,4-dihydroxyl group of the catechol moiety with 4-hydroxyl-3-methyl sulfonamide on the left-hand side of the compounds resulted in a number of potent full agonists at the beta(3) receptor. Modification of the right-hand side of the compounds by incorporation of a free carboxylic acid resulted in a few potent human beta(3) agonists with low affinities for beta(1)- and beta(2)-ARs. N-Alkyl substitution on the 4-piperidin-1-yl-phenylamine further increased the beta(3) potency while maintaining the selectivity. For example, sulfonamide 48 is a potent full beta(3) agonist (EC(50)=0.004 microM, IA=1.0) with > 500-fold selectivity over beta(1)- and beta(2)-ARs.     

Atypical beta-adrenergic effects on insulin signaling and action in beta(3)-adrenoceptor-deficient brown adipocytes

Cross talk between adrenergic and insulin signaling systems may represent a fundamental molecular basis of insulin resistance. We have characterized a newly established beta(3)-adrenoceptor-deficient (beta(3)-KO) brown adipocyte cell line and have used it to selectively investigate the potential role of novel-state and typical beta-adrenoceptors (beta-AR) on insulin signaling and action. The novel-state beta(1)-AR agonist CGP-12177 strongly induced uncoupling protein-1 in beta(3)-KO brown adipocytes as opposed to the beta(3)-selective agonist CL-316,243. Furthermore, CGP-12177 potently reduced insulin-induced glucose uptake and glycogen synthesis. Neither the selective beta(1)- and beta(2)-antagonists metoprolol and ICI-118,551 nor the nonselective antagonist propranolol blocked these effects. The classical beta(1)-AR agonist dobutamine and the beta(2)-AR agonist clenbuterol also considerably diminished insulin-induced glucose uptake. In contrast to CGP-12177 treatment, these negative effects were completely abrogated by metoprolol and ICI-118,551. Stimulation with CGP-12177 did not impair insulin receptor kinase activity but decreased insulin receptor substrate-1 binding to phosphatidylinositol (PI) 3-kinase and activation of protein kinase B. Thus the present study characterizes a novel cell system to selectively analyze molecular and functional interactions between novel and classical beta-adrenoceptor types with insulin action. Furthermore, it indicates insulin receptor-independent, but PI 3-kinase-dependent, potent negative effects of the novel beta(1)-adrenoceptor state on diverse biological end points of insulin action.     

Arylpropanolamines: selective beta3 agonists arising from strategies to mitigate phase I metabolic transformations

Utilization of N-substituted-4-hydroxy-3-methylsulfonanilidoethanolamines 1 as selective beta(3) agonists is complicated by their propensity to undergo metabolic oxidative N-dealkylation, generating 0.01-2% of a very potent alpha(1) adrenergic agonist 2. A summary of the SAR for this hepatic microsomal conversion precedes presentation of strategies to maintain the advantages of chemotype 1 while mitigating the consequences of N-dealkylation. This effort led to the identification of 4-hydroxy-3-methylsulfonanilidopropanolamines 15 for which the SAR for the unique stereochemical requirements for binding to the beta adrenergic receptors culminated in the identification of the potent, selective beta(3) agonist 15f.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.     

Understanding the binding mode and function of BMS-488043 against HIV-1 viral entry

A recently discovered small-molecule inhibitor, BMS-488043 (BMS-488), for the invasion of Human immunodeficiency virus Type 1 (HIV-1), shows a high activity against the entry of diversified HIV-1. Docking and molecular dynamic studies have been carried out to understand the binding mode of BMS-488 to gp120 as well as the effect of the small molecule on the conformational change of gp120 induced by CD4 binding. The results indicate that BMS-488 can accommodate in the CD4 binding pocket and interfere the CD4 binding in a noncompetitive mode. The piperazine group of BMS-488 prevents the bridging sheet formation of gp120 induced by the CD4 binding mainly through blocking the rotation of the Trp112 located on the α1 helix of gp120. The bridging sheet formation cannot be blocked for the W112A mutant of gp120 due to the reduced steric hindrance, in agreement with its significant resistance to the BMS inhibitor. The aza-indole ring is likely to interfere the exposure of gp41 by stacking within the β3-β5 and LB loops to disrupt the close packing of Pro212-His66-Phe210. The mode of action of BMS-488 also accommodates many mutagenesis results related to BMS-488 activity.