Joseph W. Gunnet

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System:

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate spots within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

In vitro permeability of eight β-blockers through Caco-2 monolayers utilizing liquid chromatography/electrospray ionization mass spectrometry

It is demonstrated that the apparent permeability (P(app)) coefficients of beta-adrenoceptor antagonist drugs can easily be determined for Caco-2 cell culture intestinal models utilizing liquid chromatography/mass spectrometry (LC/MS). The LC/MS method with electrospray ionization in the single ion monitoring mode showed an increased sensitivity of 1000-fold compared with LC/UV detection and enhanced selectivity with respect to both LC/UV and radioactivity assays. The P(app) coefficients of beta-adrenoceptor antagonists determined by LC/MS have the same ranking order as those determined by LC/UV and radioactivity assays. However, the P(app) coefficients determined in this study showed significant discrepancies from those determined in other laboratories. There are several experimental factors that directly affect the absolute value of the P(app) coefficients, including pH gradients, additional diffusion barriers (i.e. unstirred water layer and type of filter support), analyte concentration, detection method and possibly cell culture variations. These parameters should be controlled when generating Caco-2 P(app) coefficients for different compounds.

Pharmacological characterization of RWJ-676070, a dual vasopressin V1A/V2 receptor antagonist

The dysregulation of arginine vasopressin (AVP) release and activation of vasopressin V(1A) and V(2) receptors may play a role in disease. The in vitro and in vivo pharmacology of RWJ-676070, a potent, balanced antagonist of both the V(1A) and V(2) receptors is described. RWJ-676070 binding and intracellular functional antagonist activity was characterized using cells expressing V(1A), V(1B) or V(2) receptors. Its inhibition of V(1A) receptor-mediated contraction of vascular rings and platelet aggregation was determined. V(2) receptor-medated aquaresis was determined in rats, dogs and monkeys. V(1A) receptor-mediated inhibitory activity was assessed in vivo in a vasopressin-induced hypertension model and in normotensive rats and in two hypertensive rat models. RWJ-676070 inhibited AVP binding to human V(1A) and V(2) receptors (Ki=1 and 14 nM, respectively). RWJ-676070 inhibited V(1A) receptor-induced intracellular calcium mobilization and V(2) receptor-induced cAMP accumulation with Ki values of 14 nM and 13 nM, respectively. The compound was slightly less potent against rat V(1A) receptors. RWJ-676070 inhibited V(1A) receptor-mediated vasoconstriction in rat and dog vascular rings and AVP-induced human platelet aggregation. Dose dependent aquaresis was demonstrated in rats, dogs and monkeys following oral administration. RWJ-676070 inhibited AVP-induced hypertension in rats but had no effect on arterial pressure in normotensive and spontaneously hypertensive rats but did decrease arterial pressure in Dahl, salt-sensitive hypertensive rats. RWJ-676070 is a new, potent antagonist of V(1A) and V(2) receptors that may be useful for treatment of diseases benefiting from balanced inhibition of both V(1A) and V(2) receptors.

Human TE671 cells express functional motilin receptors

In our search for a cell line expressing endogenous human motilin receptor, we have discovered that theTE671 cell line, a neuron-derived medulloblastoma human line, expresses functional motilin receptors. The cDNA of the receptor was isolated from the cells and its sequence was confirmed to be identical to the previously reported cDNA sequence isolated from human thyroid. The function of the receptor protein was evaluated both for its ability to inhibit the binding of 125I-motilin to a crude membrane preparation of TE671 cells and for activation of the phospholipase C signal transduction pathway by calcium mobilization assay. The precise numbers of motilin receptor RNA molecule in TE671 cell and 24 human tissues were quantitatively determined by real-time PCR. TE671 cell line should be a useful tool for the study of motilin receptor-involved signal transduction in humans.

Discovery of an Isothiazole-Based Phenylpropanoic Acid GPR120 Agonist as a Development Candidate for Type 2 Diabetes

We have discovered a novel series of isothiazole-based phenylpropanoic acids as GPR120 agonists. Extensive structure-activity relationship studies led to the discovery of a potent GPR120 agonist 4x, which displayed good EC50 values in both calcium and β-arrestin assays. It also presented good pharmaceutical properties and a favorable PK profile. Moreover, it demonstrated in vivo antidiabetic activity in C57BL/6 DIO mice. Studies in WT and knockout DIO mice showed that it improved glucose handling during an OGTT via GPR120. Overall, 4x possessed promising antidiabetic effect and good safety profile to be a development candidate.