Michael P. Graziano

Specific activation of Gs by synthetic peptides corresponding to an intracellular loop of the β-adrenergic receptor

Peptides corresponding to the amino acid sequence of the hamster β2‐adrenergic receptor (β2AR) were synthesized and their ability to activate purified G‐proteins determined. Two peptides, comprising the N‐ and C‐terminal 15 amino acids of the putative third intracellular loop region of the β2AR were found to activate the G‐protein Gs but not to activate a preparation of Gi/Go. Other peptides corresponding to the internal portions of this loop and the C‐terminal tail region failed to activate either G‐protein. The presence of phospholipid vesicles was required for this activation. The observation that peptides with sequences corresponding to the ends of the third intracellular loop of the βAR can specifically activate Gs confirms the results of previous mutagenesis studies on the receptor and demonstrates that the secondary structure conferred by the amino acid sequences in these regions is sufficient for the activation of G‐proteins.

Characterisation of a chimeric hD3/D2 dopamine receptor expressed in CHO cells

The D2 dopamine receptor is known to be functionally coupled when expressed in CHO cells, whereas the effector systems for the D3, dopamine receptor remain unclear. A chimeric, human D3/D2 receptor (hD3/D2) was constructed containing the third intracellular loop region of the D2 receptor. CHO cells stably expressing the D2, D3, or hD3/D2 receptors were created and the pharmacology of the receptors was examined. The chimeric hD3/D2 receptor retained D3‐like affinities for dopaminergic ligands. However, in contrast to the D2 receptor neither the D3 receptor nor the hD3/D2 receptor could functionally couple to the adenylate cyclase or arachidonic acid release mechanisms.

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 μM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

Alterations in Receptor Activation and Divalent Cation Activation of Agonist Binding by Deletion of Intracellular Domains of the Glucagon Receptor

Deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein with high affinity for glucagon but with attenuated agonist activation of adenylyl cyclase. The Δ252-259 mutant has 4-fold higher affinity for glucagon than does the wild type receptor. The nonhydrolyzable GTP analog, guanosine 5′-(β,γ-imido)triphosphate (Gpp(NH)p), inhibits binding of 125I-glucagon to the wild type receptor but not to the Δ252-259 mutant. Divalent cations such as MgCl2 and CaCl2 stimulate the binding of 125I-glucagon to the wild type receptor by increasing glucagon affinity. The rate of dissociation of 125I-glucagon is decreased 4-fold by MgCl2 and increased 6-fold by Gpp(NH)p. However, divalent cations do not affect the binding of 125I-glucagon to the Δ252-259 mutant. The rate of dissociation of 125I-glucagon from the Δ252-259 mutant protein is equivalent to the rate of dissociation from the wild type receptor in the presence of MgCl2. These data suggest that at least three conformations of the glucagon receptor can exist in the membrane based on their differing affinities for 125I-glucagon. Deletion of residues 252-259 appears to lock the protein in the conformation promoted by divalent cations and prevents the protein from normal coupling to Gs.

Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider 2 cells.

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

Fluorescein-Trp25-Exendin-4, a Biologically Active Fluorescent Probe for the Human GLP-1 Receptor

Exendin-4, a reptilian GLP-1 analogue, has been fluorescently labeled by covalently linking a fluorescein moiety onto the Trp residue yielding fluorescein-Trp25-exendin-4 (FLEX). FLEX is equipotent to GLP-1(7-36)-amide and exendin-4 as an inhibitor of [125I] GLP-1 binding to the human GLP-1 receptor stably expressed in CHO cells, and maintains full biological potency and efficacy as measured by the stimulation of cAMP accumulation in these cells. FLEX binding to CHO/hGLP-1R membranes results in an increase in fluorescence anisotropy. The binding is specific and saturable (Kd = 2.0 +/- 0.4 nM), and GLP-1(7-36)-amide and exendin-4 are equipotent inhibitors of FLEX binding to the human GLP-1 receptor. Thus, FLEX is a potent, biologically active ligand that is useful for the study of the binding and functional characteristics of the human GLP-1 receptor.

Zucker Diabetic Fatty rats exhibit hypercoagulability and accelerated thrombus formation in the Arterio-Venous shunt model of thrombosis

INTRODUCTIONnDiabetes is a significant risk factor for thrombosis. The present study aimed at assessing coagulability, platelet reactivity, and thrombogenicity of the diabetic female Zucker Diabetic Fatty (ZDF) rat model and its relevance in studying antithrombotic mechanisms.nnnMATERIALS AND METHODSnThe basal coagulant state in ZDF rats was evaluated by clotting times, thromboelastography, and thrombin generation assay. A 14-day treatment with dapagliflozin in ZDF rats was pursued to investigate if glycemic control can improve coagulability. Thrombus formation in the Arterio-Venous (A-V) shunt model and the FeCl3-induced arterial thrombosis model was studied, with the antithrombotic effect of apixaban in the former model further investigated.nnnRESULTSnZDF rats exhibited significantly shortened clotting times, enhanced thrombin generation, and decreased fibrinolysis at baseline. Effective glycemic control achieved with dapagliflozin did not improve any of these parameters. ZDF rats displayed accelerated thrombus formation and were amenable to apixaban treatment in the A-V shunt model albeit with less sensitivity than normal rats. ZDF rats exhibited less platelet aggregation in response to ADP, collagen and PAR-4, and attenuated thrombotic response in the FeCl3 model.nnnCONCLUSIONSnZDF rats are at a chronic hypercoagulable and hypofibrinolytic state yet with compromised platelet reactivity. They display accelerated and attenuated thrombosis in the A-V shunt and FeCl3 model of thrombosis, respectively. Results shed new light on the pathophysiology of the ZDF rat model and illustrate its potential value in translational research on anticoagulant agents in diabetics. Caution needs to be exerted in utilizing this model in assessing antiplatelet mechanisms in diabetes-associated atherothrombosis.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.