Biomedical subjects

Sujatha M Gopalakrishnan

Publications and source records attributed to Sujatha M Gopalakrishnan.

5 recordsLinked to original sources

PubMed2005

An offline-addition format for identifying GPCR modulators by screening 384-well mixed compounds in the FLIPR.

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca(2+) with an EC(50) value of 0.30 +/- 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors.

Cell Line, Tumor↗

PubMed2003

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors.

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening microARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the G alpha(q) protein can readily be assessed by fluorescence-based Ca(2+) release measurements, many GPCRs that are coupled to G alpha(s) or G alpha(i/o) proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D(4.4) receptor, which normally couples through the G alpha(i/o) protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca(2+) release by stably coexpressing this receptor with a chimeric G(alpha qo5) protein in HEK-293 cells. In microARCS format, the cells expressing D(4.4) receptor and G alpha(qo5) protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable microARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.

Apomorphine↗

PubMed2002

Structure-activity relationships of non-imidazole H(3) receptor ligands. Part 1.

SAR studies for novel non-imidazole containing H(3) receptor antagonists with high potency and selectivity for rat H(3) receptors are described. A high throughput screening lead, A-923, was further elaborated in a systematic manner to clarify a pharmacophore for this class of aryloxyalkyl piperazine based compounds.

Amines↗

PubMed2002

Functional characterization of adenosine receptors and coupling to ATP-sensitive K+ channels in Guinea pig urinary bladder smooth muscle.

Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (K(ATP)) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the K(ATP) channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5'-N-ethyl-carboxamido adenosine (NECA; -log EC50 = 7.97) approximately 2-p-(2-carboxethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) approximately 2-chloro-N6-cyclopentyladenosine (CCPA; 5.51) approximately N6-cyclopentyladenosine approximately N6-(R)-phenylisopropyladenosine > 2-chloro- N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A(2A)-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K+-stimulated guinea pig bladder strips, NECA (-log EC50 = 6.41) approximately CGS-21680 (6.38) > 2-chloro adenosine (5.90) >> CCPA approximately 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A(2A) receptor-mediated activation of K(ATP) channels via adenylate cyclase and elevation of cAMP.

ATP-Binding Cassette Transporters↗

PubMed2002

Application of Micro Arrayed Compound Screening (microARCS) to identify inhibitors of caspase-3.

Micro Arrayed Compound Screening (microARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8,640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the microARCS assay format were compared to those from a conventional 96-well plate-screening format.

Apoptosis↗