Biomedical subjects

Adam Yasgar

Publications and source records attributed to Adam Yasgar.

3 recordsLinked to original sources

PubMed2006

Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.

High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.

Combinatorial Chemistry Techniques↗

PubMed2006

Electrical alternans and hemodynamics in the anesthetized guinea pig can discriminate the cardiac safety of antidepressants.

INTRODUCTION: The arrhythmogenic risk of fluoxetine, citalopram, and venlafaxine were evaluated through preclinical assays measuring hERG, blood pressure and electrical alternans over their respective clinical unbound concentration ranges. METHODS: Anesthetized guinea pigs were instrumented with jugular and carotid cannulae for drug infusion and blood pressure monitoring respectively; a thoracotomy was performed for placement of a monophasic action potential probe on the left ventricle and for placement of pacing wires on the left ventricular apex. Drugs were infused as a 5-min loading dose immediately followed by a 10-min maintenance dose to achieve clinically relevant plasma concentrations; blood samples were taken at the end of each maintenance dose. Ventricular pacing was performed twice at baseline and at each dose level as follows: 50 preconditioning-beats at S1=220 (or 240) ms immediately followed by 30 test-beats at S2=200 ms. This S1-S2 protocol was repeated for S2=190 to 140 ms. HERG and calcium current measurements were recorded in HEK-293 cells stably expressing hERG potassium currents and freshly isolated guinea pig cardiac myocytes using the whole-cell configuration of the patch clamp technique. RESULTS: Physiologically relevant inhibition (IC(20)) of hERG occurred at concentrations 22-fold (fluoxetine), 9-fold (citalopram), and 11-fold (venlafaxine) beyond their respective clinically effective concentration (C(eff)). At the highest achievable levels, fluoxetine (20-fold C(eff)) and citalopram (28-fold C(eff)) significantly decreased heart rate and/or blood pressure as well as increasing electrical alternans by 5 and 18 ms respectively. Venlafaxine increased blood pressure at only 1.3-fold C(eff), but did not increase electrical alternans at the highest achievable dose (3.1-fold C(eff)). DISCUSSION: These data suggest that evaluating other dose limiting side effects in relation to a drug's therapeutic range may be crucial for accurate assessment of arrhythmia liability.

Action Potentials↗

PubMed2006

Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format.

Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.

Active Transport, Cell Nucleus↗