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Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9-mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.

Modification of a 2-iminobenzimidazole series derived from an HTS hit resulted in compounds with improved in-vitro species selectivity. Incorporation of an 8-quinoline amide and conformational rigidification of an aliphatic tether furnished potent compounds suitable for further lead optimization.

1 Pregnane X receptor (PXR) transactivation and binding assays have been developed into high-throughput assays, which are robust and reproducible (Z' > 0.5). For most compounds, there was a good correlation between the result so f the transactivation and binding assays. EC50 values of compounds in the transactivation assay correlated reasonably well with their IC50 values in the binding assay. However, there were discrepancies with some compounds showing high binding affinity in the binding assay translated into low transactivation. The most likely cause for these discrepancies was an agonist- dependent relationship between binding affinity and transactivation response. In general, compounds that bound to human PXR and transactivated PXR tended to be large hydrophobic molecules. (Journal of Biomolecular Screening 2004:533-540)

A scalable and enantioselective synthesis of a potent S1P1 agonist containing two stereogenic centers on a cyclopentane ring is described. Control of the absolute chirality of an amino alcohol precursor, generated via a robust phase-transfer catalyzed alkylation protocol, allows for substrate directed hydrogenation to install the second stereogenic center providing access to gram-quantities of compound 2.

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

The discovery and optimization of a novel series of prolinol-derived GHSR agonists is described. This series emerged from a 11,520-member solid-phase library targeting the GPCR protein superfamily, and the rapid optimization of low micromolar hits into single-digit nanomolar leads can be attributed to the solid-phase synthesis of matrix libraries, which revealed multiple non-additive structure-activity relationships. In addition, the separation of potent diastereomers highlighted the influence of the alpha-methyl stereochemistry of the phenoxyacetamide sidechain on GHSR activity.

High-throughput screening identified a low molecular weight antagonist of CXCR3 displaying micromolar activity in a membrane filtration-binding assay. Systematic modification of the benzimidazole core and tethered acetophenone moiety established tractable SAR of analogs with improved physicochemical properties and sub-micromolar activity across both human and murine receptors.

Herein we report the first disclosure of biphenyl azoles that are nanomolar binders of adipocyte fatty acid binding protein (aFABP or aP2) with up to thousand-fold selectivity against muscle fatty acid binding protein and epidermal fatty acid binding protein. In addition a new radio-ligand to determine binding against the three fatty acid binding proteins was also synthesized.

Several novel series of tetrahydroisoquinoline 1-carboxamides were prepared and shown to be potent growth hormone (GH) secretagogues. Among them, carbamate 12a-E2 displays excellent in vivo activity by increasing plasma GH 10-fold in an anesthetized IV rat model.

Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.