Bicyclic cyanothiazolidines as novel dipeptidyl peptidase 4 inhibitors.

The synthesis and biochemical evaluation of novel cyanothiazolidine inhibitors of dipeptidyl peptidase 4 (DPP4) is described. Their main structural feature is a constrained bicyclic core that prevents the intramolecular formation of inactive cyclic species. The inhibitors show good to moderate biochemical potency against DPP4 and display distinct selectivity profiles towards DPP7, DPP8 and DPP9 depending on their substitution.

Synthesis and antiviral activity of HCV NS3/4A peptidomimetic boronic acid inhibitors.

A new series of NS3/4A protease boronic acid inhibitors is described. The compounds show good biochemical potency and cellular activity. The peptidomimetic inhibitors were evaluated against proteases from different HCV genotypes and clinically relevant NS3/4A mutants. Compound 28 displayed subnanomolar to single digit nanomolar potencies in the enzymatic assays and an EC50 of 25 nM in the replicon cell-based assay.

Pharmacokinetic and pharmacodynamic assessments of the dipeptidyl peptidase-4 inhibitor PHX1149: double-blind, placebo-controlled, single- and multiple-dose studies in healthy subjects.

BACKGROUND: PHX1149 is a dipeptidyl peptidase-4 (DPP4) inhibitor that is currently in clinical development for the treatment of type 2 diabetes mellitus. PHX1149 is a small (molecular weight = 241.16 Da), highly water-soluble (>2 g/mL), orally active molecule with a selectivity index of 15- to 319-fold relative to those of other members of the DPP family. The biochemical median inhibitory concentration of DPP4 is 2.5 nmol/L. OBJECTIVE: The aim of these 2 double-blind, randomized, placebo-controlled studies was to examine the pharmacokinetic (PK) parameters and pharmacodynamic (PD) properties and tolerability of single and multiple ascending doses of PHX1149 in healthy human subjects. METHODS: Healthy men and women aged 18 to 60 years were recruited to participate in a single- or a multiple-dose study in which sequential dose escalation paradigm was used. In the single-dose study, subjects were given a single oral dose of PHX1149 50 to 500 mg or placebo; in the multiple-dose study, subjects were given PHX1149 at doses from 50 to 400 mg or placebo QD for 13 days. There was no intrasubject dose escalation. Blood samples were collected from each subject at a series of time points ranging from 1 hour before to 24 hours after dosing on day 1 in the single- dose study and on days 1, 7, and 13 in the multiple-dose study. PK and PD analyses were performed in plasma samples to determine Cmax, Tmax, AUC0-t, AUC0-infinity, and DPP4 enzymatic activity. The drug accumulation index was also calculated for each dose of PHX1149 in the multiple-dose study. Adverse events (AEs) were monitored in both studies through physical examinations, including measurement of vital signs, and clinical laboratory testing. In both studies, electrocardiography was performed. RESULTS: The single- and multiple-dose studies enrolled 30 and 28 subjects, respectively, for a total enrollment in the 2 studies of 58 healthy adult subjects. The distribution of male and female subjects was 14 (47%) and 16 (53%), respectively, in the single- dose study and 16 (57%) and 12 (43%) in the multiple- dose study. In the single-dose study, 28 (93%) subjects were white; in the multidose study, all subjects were white. The mean (SD) ages in the 2 studies were 51 (10) and 51 (12) years, respectively; and mean (SD) body weights were 89.0 (10.8) and 81.1 (10.9) kg, respectively. PHX1149 exhibited dose-proportional increases in mean Cmax AUC0-t, and AUC0-infinity across the evaluated dose ranges. Tmax ranged from 2 to 4 hours, and t1/2 ranged from approximately 10 to 13 hours. No drug accumulation was observed. Plasma DPP4 inhibition at 24 hours was >or=50% in the multiple-dose study for doses of >or=100 mg. PHX1149 400 mg achieved approximately 90% 24-hour plasma DPP4 inhibition in the multiple-dose study. All AEs were characterized as mild, with the exception of 1 case of moderate edema, which occurred 17 days after the end of dosing in the multiple-dose study (50-mg dose group) and was considered unrelated to the study drug. Adverse events were experienced by 47% of all subjects studied in the single-dose study and 93% of subjects in the multiple-dose study. The rates of AEs were comparable between the study and placebo groups. CONCLUSIONS: The PK parameters and PD properties of PHX1149 were suitable (eg, tl/2, DPP4 inhibition) for once-daily dosing in this group of 58 healthy subjects. All doses were well tolerated.

Pharmacological characterization of recombinant N-type calcium channel (Cav2.2) mediated calcium mobilization using FLIPR.

The N-type voltage-gated calcium channel (Ca(v)2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca(v)2.2 pharmacology in two stable Ca(v)2.2 cell lines: alpha1(B), alpha2delta, beta(3)-HEK-293 and alpha1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca(2+) mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR(96)). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC(50) values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca(v)2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC(50) values correlated with literature EP-derived values. Novel Ca(v)2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na(+) channel inhibitors and antidepressants. Novel Na(+) channel compounds with high potency at Ca(v)2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca(v)2.2 tricyclic antidepressant identified was desipramine.

Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response.

The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. In an effort to identify novel proteins that might be involved in stabilizing stalled replication forks, Saccharomyces cerevisiae mutant wss1Delta was isolated from a high-throughput screening of approximately 5000 deletion strains for genes involved in the response to continuous, low-intensity UV irradiation. Disruption of WSS1 resulted in synergistic increases in UV sensitivity with null mutants of genes involved in recombination (RAD52) and cell cycle control (RAD9 and RAD24). WSS1 was also found to interact genetically with SGS1, TOP3, SRS2 and CTF4, which are involved in recombination, repair of replication forks and the establishment of sister chromatid cohesion. A yeast two-hybrid screen identified a potential physical interaction between Wss1 and both Psy2 and Tof1. Genetic interactions were also detected between PSY2 and TOF1, as well as between each gene and RAD52 and SRS2, and between WSS1 and TOF1. Tof1 is known to be involved in stabilizing stalled replication forks and our data suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.

A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).

Preliminary characterization of light harvesting in E. coli DNA photolyase.

E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.

Previously uncharacterized genes in the UV- and MMS-induced DNA damage response in yeast.

A competitive growth assay has been used to identify yeast genes involved in the repair of UV- or MMS-induced DNA damage. A collection of 2,827 yeast strains was analyzed in which each strain has a single ORF replaced with a cassette containing two unique sequence tags, allowing for its detection by hybridization to a high-density oligonucleotide array. The hybridization data identify a high percentage of the deletion strains present in the collection that were previously characterized as being sensitive to the DNA-damaging agents. The assay, and subsequent analysis, has been used to identify six genes not formerly known to be involved in the damage response, whose deletion renders the yeast sensitive to UV or MMS treatment. The recently identified genes include three uncharacterized ORFs, as well as genes that encode protein products implicated in ubiquitination, gene silencing, and transport across the mitochondrial membrane. Epistatsis analysis of four of the genes was performed to determine the DNA damage repair pathways in which the protein products function.