A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat.

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.

The synthesis and antiviral activity of 4-fluoro-1-beta-D-ribofuranosyl-1H-pyrazole-3-carboxamide.

A novel fluoropyrazole ribonucleoside has been shown to have significant anti-influenza activity in vitro. The compound is compared and contrasted with the structurally-related compound ribavirin in attempts to identify factors having significant bearing on the mode of action of both compounds.

N-(2-Benzoylphenyl)-L-tyrosine PPARgamma agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

Novel 1,3-disubstituted-5,10-dihydro-5,10-dioxo-1H-benzo[g] isochromene-3 carboxamides as potent antitumor agents.

Novel antitumor 5,10-dihydro-5,10-dioxo-1H- benzo[g]isochromene-3-carboxamides were discovered.

GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2.

The principal inhibitory neurotransmitter GABA (gamma-aminobutyric acid) exerts its effects through two ligand-gated channels, GABA(A) and GABA(C) receptors, and a third receptor, GABA(B) , which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABA(B)R1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABA(B) receptors in the brain. Here we describe a new member of the GABA(B) polypeptide family, GABA(B)R2, that shows sequence homology to GABA(B)R1. Neither GABA(B)R1 nor GABA(B)R2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABA(B)R1 and GABA(B)R2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABA(B)R1 and GABA(B)R2, belonging to the same subfamily is essential for signal transduction by GABA(B) receptors.

Strand-invasion of duplex DNA by peptide nucleic acid oligomers.

Polyamide oligomers, termed peptide nucleic acids (PNAs), bind with high affinity to both DNA and RNA and offer both antisense and antigene approaches for regulating gene expression. When a PNA binds to a complementary sequence in a double-stranded DNA, one strand of the duplex is displaced, and a stable D-loop is formed. Unlike oligodeoxynucleotides for which binding polarity is determined by the deoxyribose sugar, the unrestrained polyamide backbone of the PNA could permit binding to a DNA target in an orientation-independent manner. We now provide evidence that PNAs can, in fact, bind to their complementary sequence in DNA independent of the DNA-strand polarity--that is, a PNA binds to DNA in both "parallel" and "antiparallel" fashion. With a mixed-sequence 15-mer PNA, kinetic studies of PNA.DNA interactions revealed that D-loop formation was rapid and the complex was stable for several hours. However, when measured either by gel-mobility-shift analysis or RNA polymerase II-elongation termination, D-loop formation was salt dependent, but PNA-strand dissociation was not salt dependent. We observed that D-loop-containing DNA fragments had anomalous gel mobilities that varied as a function of the position of the D-loop relative to the DNA termini. On the basis of permutation analysis, the decreased mobility of the PNA.DNA complex was attributed to a bend in the DNA at or near the D-loop.

Synthesis and anti-HIV-1 activity of a series of imidazo[1,5-b]pyridazines.

A series of substituted imidazo[1,5-b]pyridazines have been prepared and tested for inhibitory activity against the reverse transcriptase of HIV-1 (RT) and their ability to inhibit the growth of infected MT-4 cells. Crystal data are reported on two compounds, 15c and 33. From the structure-activity relationships developed within this and other series, it is proposed that key features of the interaction with RT include hydrogen-bond acceptor and aromatic pi-orbital bonding with the imidazopyridazine nucleus and a benzoyl function separated from the heterocycle by a suitable spacer group. Exceptional activity against the reverse transcriptase of HIV-1 (IC50 = 0.65 nM) was obtained with a 2-imidazolyl-substituted derivative, 7-[2-(1H-imidazol-1- yl)-5-methylimidazo-[1,5-b]pyridazin-7-yl]-1-phenyl-1-heptanone (33) which is attributed to additional binding of the imidazole sp2 nitrogen atom. A number of the compounds in this series also inhibit the replication of HIV-1 in vitro in MT-4 and C8166 cells at levels observed with the nucleoside AZT.

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.

Effects of the acetylcholinesterase inhibitor pinacolyl methylphosphonofluoridate (soman) on selected endocrine, glucose, and catecholamine levels in fasted and fed rats.

Fed and 18-h fasted rats were given acute doses of either saline or the acetylcholinesterase inhibitor pinacolyl methylphosphonofluoridate (soman) 40, 60, or 80 micrograms/kg. After 30 min plasma samples were collected and assayed for glucose, insulin, glucagon, corticosterone, epinephrine and norepinephrine and the hypothalamus was isolated and assayed for acetylcholinesterase activity. Toxic sign scores were determined and they indicated that soman may be more toxic in the fasted rat. Soman-induced increases in corticosterone were observed in both fasted and fed rats; these levels were significantly higher in fasted rats given either soman or saline. Also, soman-induced increases in glucagon were more pronounced in fasted rats. Soman also caused an apparent dose-dependent increase in catecholamines and a decrease in hypothalamic acetylcholinesterase activity in both groups of rats. The expected lower insulin and glucose levels in the fasted rats were present in the saline-dosed animals and remained lower than fed rats after each dose of soman. This lack of soman-induced hyperglycemia may contribute to the toxicity of soman in fasted rats.

Expression of atrial natriuretic factor as a cleavable fusion protein with chloramphenicol acetyltransferase in Escherichia coli.

Recombinant fusion proteins containing human atrial natriuretic factor, ANF(1-28) joined to chloramphenicol acetyltransferase (CAT) via cleavable linker sequences have been produced in Escherichia coli. The linker sequences were designed to allow the release of authentic ANF(1-28) following proteolytic cleavage by enterokinase or thrombin, or chemical cleavage with 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine. Proteins, containing ANF(1-28) fused to the carboxyl-terminal region of CAT (using the ScaI restriction site in the cat gene), were largely soluble in E. coli and were obtained in higher yield than analogues containing ANF(1-28) linked to shorter CAT sequences. The longer derivatives also retained CAT activity allowing subsequent purification by affinity chromatography.

Altered Cro repressors from engineered mutagenesis of a synthetic cro gene.

A portion of the gene coding for the Cro repressor protein of bacteriophage lambda has been chemically synthesized, incorporating base pair changes that generate restriction endonuclease sites without altering the amino acid coding sequence. These restriction endonuclease sites were used to remove small segments of the synthetic cro gene and the segments were replaced with duplexes carrying desired mutations. Altered Cro proteins produced by mutants constructed in this manner were then assayed for binding to lambda operator OR3 in vivo. Mutations directed into the region of the cro gene encoding the alpha-3 helix produced altered Cro proteins with a range of affinities for operator DNA. These changes suggest which amino acids play an important role in Cro-OR3 complex formation.

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.

Synthesis and antiviral properties of some 2'-deoxy-5-(fluoroalkenyl)uridines.

The following 5-substituted 2,4-dimethoxypyrimidines were synthesized: 5-(2,2,2-trichloro-1-hydroxyethyl), 5-(2,2,2-trichloro-1-fluoroethyl),5-(2,2-dichloro-1-fluorovinyl) (5), and 5-(perfluoropropen-1-yl) (a mixture of E and Z isomers, 6 and 7). Demethylation of 5 gave 5-(2,2-dichloro-1-fluorovinyl)uracil, and demethylation of the mixture of 6 and 7 gave some pure (E)-5-(perfluoropropen-1-yl)uracil. Compound 5 was converted into its 2'-deoxyribonucleoside (12) and its alpha-anomer by standard procedures. 2'-Deoxy-3,5-dilithio-3',5'-O-bis(trimethylsilyl)uridine was reacted with the appropriate fluoroalkene to give the following 5-substituted 2'-deoxyuridines in low yield (6-24%): 5-(2-chloro-1,2-difluorovinyl) (a mixture of E and Z isomers, 15 and 16, which were separated on a small scale), 5-(perfluoropropen-1-yl), 5-(perfluorocyclohexen-1-yl), and 5-(perfluorocyclopenten-1-yl). In these reactions, 2'-deoxy-5-(trimethylsilyl)uridine and 2'-deoxyuridine were also formed. The 5-substituted 2'-deoxyuridines were tested for activity against herpes simplex virus type 1. Compound 12 and the mixture of 15 and 16 had an ID50 of 20-26 micrograms/mL in Vero cells. The activity of the mixture resided in one isomer, which by analogY with the corresponding (Z)- and (E)-5-(2-bromovinyl)-2'-deoxyuridines was concluded to be the Z isomer (16).