Cellular Impedance Assays for Predictive Preclinical Drug Screening of Kinase Inhibitor Cardiovascular Toxicity

Abstract

Cardiovascular (CV) toxicity is a leading contributor to drug attrition. Implementing earlier testing has successfully reduced human Ether-à-go-go-Related Gene-related arrhythmias. How- ever, analogous assays targeting functional CV effects remain elusive. Demand to address this gap is particularly acute for kinase inhibitors (KIs) that suffer frequent CV toxicity. The drug class also presents some particularly challenging requirements for assessing functional CV toxicity. Specifically, an assay must sense a downstream response that integrates diverse kinase signaling pathways. In addition, sufficient throughput is essential for handling inherent KI nonselectivity. A new opportunity has emerged with cellular impedance technology, which detects spontaneous beating cardiomyocytes. Impedance assays sense morphology changes downstream of cardiomyocyte contraction. To evaluate cardiomyocyte impedance assays for KI screening, we investigated two distinct KI classes where CV toxicity was discovered late and target risks remain unresolved. Microtubule-associated protein/microtubule affinity regulating kinase (MARK) inhibitors decrease blood pressure in dogs, whereas checkpoint kinase (Chk) inhibitors (AZD7762, SCH900776) exhibit dose-limiting CV toxicities in clinical trials. These in vivo effects manifested in vitro as cardiomyocyte beat cessation. MARK effects were deemed mechanism associated because beat inhibition potencies correlated with kinase inhibition, and gene knockdown and microtubule-targeting agents suppressed beating. MARK inhibitor impedance and kinase potencies aligned with rat blood pressure effects. Chk inhibitor effects were judged off-target because Chk and beat inhibition potencies did not correlate and knockdowns did not alter beating. Taken together, the data demonstrate that cardiomyocyte impedance assays can address three unmet needs-detecting KI functional cardiotoxicity in vitro, determining mechanism of action, and supporting safety structure-activity relationships.

Human Stem Cell-Derived Cardiomyocytes in Cellular Impedance Assays: Bringing Cardiotoxicity Screening to the Front Line

“…For example, functional cardiotoxicity of microtubule-affinity regulating kinase (MARK) inhibitors, which initially presented with reduced blood pressure in dogs (see patent [35]), could be recapitulated in vitro as beat inhibition without cytotoxicity. Several lines of data indicate that MARK SMKI cardiotoxicity is on-target-relative potency for MARK inhibition aligned with potency of beat inhibition, genetic knockdown of MARKs decreased beat amplitude, as did targeting the pathway with microtubule inhibitors ( [19] ; Fig. 6).…”

“…Chk inhibitors that presented in clinical trials with heart rate irregularities could be replicated in vitro as beat inhibition without cytotoxicity. However, neither pharmacology nor genetic inhibition indicated that these effects were Chk-mediated, thereby suggesting off-target contribution(s) and offering an assay to explore safer compounds [19].…”

“…SMKIs show robust effects on beating similar to ion channel ligands albeit, with a delayed optimal response window (*2 h vs. *20 min). Approved SMKI drugs with clinical cardiotoxicity can be distinguished with CM beat assays including impedance [33] and mechanisms can be interrogated [19,34]. Evaluations can be focused on individual candidate kinases using a combined approach of pharmacological inhibition, genetic knockdown, and pathway-specific agents.…”

“…Whereas traditional CV testing has relied on in vivo and ex vivo preclinical models followed by thorough clinical evaluation, adding robust in vitro screens will improve downstream efficiency and potentially obviate the need for extensive and expensive clinical assessments [29]. The early screening strategy needs human-relevant assays that predict clinical [19] outcomes and in doing so has the potential to change the paradigm-from the historical screening-out CV toxicity after drug candidates have been developed or worse dropping any molecules with hERG affinity to building-in safety through true understanding of in vivo outcomes across multiple early chemical series.…”

Zebrafish as Toxicological model for screening and recapitulate human diseases

“…The zebrafish has contributed to obtain measurements as action potential trough voltage mapping, to determine cells coupling [20] , and this fact together with calcium signaling, are important for cardiomyocyte proliferation and differentiation [21] . The last decade's large animals, such as mice, rats and rabbits, have been widely used to study cardiotoxicity after drug administration [22][23][24] , presenting some limitations. For instance, rodents can be insensitive to compounds' cardiotoxicity, particularly when the endpoint measurement is left ventricular contractile function [25] .…”