Channa Jayawickreme

Discovery of 3-Aryl-4-isoxazolecarboxamides as TGR5 Receptor Agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.

Constitutive receptor systems for drug discovery

This paper discusses the use of constitutively active G-protein-coupled receptor systems for drug discovery. Specifically, the ternary complex model is used to define the two major theoretical advantages of constitutive receptor screening-namely, the ability to detect antagonists as well as agonists directly and the fact that constitutive systems are more sensitive to agonists. In experimental studies, transient transfection of Chinese hamster ovary cyclic AMP response element (CRE) luciferase reporter cells with cDNA for human parathyroid hormone receptor, glucagon receptor, and glucagon-like peptide (GLP-1) receptor showed cDNA concentration-dependent constitutive activity with parathyroid hormone (PTH-1) and glucagon. In contrast, no constitutive activity was observed for GLP-1 receptor, yet responses to GLP-1 indicated that receptor expression had taken place. In another functional system, Xenopus laevi melanophores transfected with cDNA for human calcitonin receptor showed constitutive activity. Nine ligands for the calcitonin receptor either increased or decreased constitutive activity in this assay. The sensitivity of the system to human calcitonin increased with increasing constitutive activity. These data indicate that, for those receptors which naturally produce constitutive activity, screening in this mode could be advantageous over other methods.

Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120).

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

Synthesis and structure–activity relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5 agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent agonists of the human TGR5 G-protein-coupled receptor is described. Many analogues were readily accessible via solution-phase synthesis which resulted in the rapid identification of key structure-activity relationships (SAR), and the discovery of potent exemplars (up to pEC50=9). Details of the SAR and optimization of this series are presented herein.

Gene expression systems in the development of high-throughput screens

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Exploration of phenylpropanoic acids as agonists of the free fatty acid receptor 4 (FFA4): Identification of an orally efficacious FFA4 agonist

The long chain free fatty acid receptor 4 (FFA4/GPR120) has recently been recognized as lipid sensor playing important roles in nutrient sensing and inflammation and thus holds potential as a therapeutic target for type 2 diabetes and metabolic syndrome. To explore the effects of stimulating this receptor in animal models of metabolic disease, we initiated work to identify agonists with appropriate pharmacokinetic properties to support progression into in vivo studies. Extensive SAR studies of a series of phenylpropanoic acids led to the identification of compound 29, a FFA4 agonist which lowers plasma glucose in two preclinical models of type 2 diabetes.

Identification of potent, nonabsorbable agonists of the calcium-sensing receptor for GI-specific administration

Modulation of gastrointestinal nutrient sensing pathways provides a promising a new approach for the treatment of metabolic diseases including diabetes and obesity. The calcium-sensing receptor has been identified as a key receptor involved in mineral and amino acid nutrient sensing and thus is an attractive target for modulation in the intestine. Herein we describe the optimization of gastrointestinally restricted calcium-sensing receptor agonists starting from a 3-aminopyrrolidine-containing template leading to the identification of GI-restricted agonist 19 (GSK3004774).

Functional Screening in the Melanophore Bioassay

The melanophore bioassay is a robust, sensitive, and versatile procedure for screening G protein–coupled receptors in a variety of formats. Because melanophores contain a wide variety of G proteins, they can be employed as a sensitive, real‐time response system for studying transfected receptors and for defining equilibria for drug effects. This assay can be run in 96‐well microtiter plates or in open‐lawn 1536 format, and can yield conventional agonist‐antagonist as well as constitutive assays.

Identification of nonabsorbable inhibitors of the scavenger receptor-BI (SR-BI) for tissue-specific administration

The identification of a low-permeability scavenger receptor BI (SR-BI) inhibitor starting from the ITX-5061 template is described. Structure-activity and structure-permeability relationships were assessed for analogs leading to the identification of compound 8 as a potent and nonabsorbable SR-BI inhibitor.