An impedance-based approach using human iPSC-derived cardiomyocytes significantly improves in vitro prediction of in vivo cardiotox liabilities.

Current in vitro approaches to cardiac safety testing typically focus on mechanistic ion channel testing to predict in vivo proarrhythmic potential. Outside of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, structural and functional cardiotoxicity related to chronic dosing effects are of great concern as these effects can impact compound attrition. Development and implementation of an in vitro cardiotoxicity screening platform that effectively identifies these liabilities early in the discovery process should reduce costly attrition and decrease preclinical development time. Impedence platforms have the potential to accurately identify structural and functional cardiotoxicity and have sufficient throughput to be included in a multi-parametric optimization approach. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) have demonstrated utility in cardiac safety and toxicity screening. The work described here leverages these advantages to assess the predictive value of data generated by two impedance platforms. The response of hIPSC-CMs to compounds with known or predicted cardiac functional or structural toxicity was determined. The compounds elicited cardiac activities and/or effects on "macro" impedance often associated with overt structural or cellular toxicity, detachment, or hypertrophy. These assays correctly predicted in vivo cardiotox findings for 81% of the compounds tested and did not identify false positives. In addition, internal or literature Cmax values from in vivo studies correlated within 4 fold of the in vitro observations. The work presented here demonstrates the predictive power of impedance platforms with hIPSC-CMs and provides a means toward accelerating lead candidate selection by assessing preclinical cardiac safety earlier in the drug discovery process.

Cell-free assay of G-protein-coupled receptors using fluorescence polarization.

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM).

Agonist versus antagonist induce distinct thermodynamic modes of co-factor binding to the glucocorticoid receptor.

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.