Typically, high throughput screening (HTS) campaigns are conducted with assay systems comprising protein binding assay or functional assays with cell lines recombinantly expressing the target of interest. The results obtained from such assays have a high risk to contain false positive and negative hits whereas the drug interaction with other potential cardiac targets cannot be displayed.
In order to avoid costly late stage failures of drug candidates, HTS assays for cardiac toxicity and drug efficacy, which are predictive for multiple cardiac targets, are thus desired early in the drug development process.
Directed differentiation and a transgenic approach allow for a specific selection of pure, human induced pluripotent stem cell-derived cardiomyocytes (hiPSCM). The hiPSCM reveal spontaneous contraction and typical physiological properties compared to their primary counterparts allowing for a multi-parametric, high content analysis (HCA). Furthermore, hiPSCM can be generated in the quality, stability and amounts required in HTS. Transients of cytosolic free calcium ([Ca2+]i) appear during excitation of hiPSCM as a result of calcium-induced calcium release from the sarcoplasmatic reticulum.
We have used the FLIPR Calcium 5 Assays Kit together with the Hamamatsu FDSS μCell and the FLIPR to monitor and analyze the effect of more than 30 standard compounds on the parameter of [Ca2+]i transients from hiPSCM in a 384 well format. Each compound was tested in a full dose response with at least 3 replicates for each concentration. The compounds tested comprise known modulators of cardiac sodium, calcium, potassium (including several hERG blockers), and hyperpolarization-activated ion channels as well as GPCRs, connexins , and ion exchangers and pumps. Typical modulations of [Ca2+]i transients in terms of frequency, duration, peak amplitude, slope (rise and decay), and rhythmicity were analyzed and correlated to the known mode of action of the compounds. The results from our study reveal that modulation of [Ca2+]i transients is predictive for a broad range of cardiac targets. Based on these results, the described assay system is a cost effective, predictive in vitro system to assess cardiac liabilities and drug efficacies in a HTS assay system with HCA at an early state of the drug development process.
