Matthew F. Peters

Label-free whole-cell assays: expanding the scope of GPCR screening.

A new class of instruments offers an unprecedented combination of label-free detection with exquisite sensitivity to live-cell responses. These instruments can quantify G-protein-coupled receptor (GPCR) signaling through G(s), G(i) and G(q) pathways and in some cases distinguish G-protein coupling, with sensitivity high enough to detect endogenous receptors. Here, we review emerging data evaluating impedance- and optical-based label-free instruments for GPCR drug discovery. In comparison with traditional GPCR assays, we highlight strengths, weaknesses and future opportunities for label-free biosensors. The ability to qualitatively distinguish G-protein coupling has groundbreaking potential for assessing functional selectivity, a concept that is changing the way GPCR pharmacology is defined and screening strategies are designed.

Human induced pluripotent stem cells and their use in drug discovery for toxicity testing

Predicting human safety risks of novel xenobiotics remains a major challenge, partly due to the limited availability of human cells to evaluate tissue-specific toxicity. Recent progress in the production of human induced pluripotent stem cells (hiPSCs) may fill this gap. hiPSCs can be continuously expanded in culture in an undifferentiated state and then differentiated to form most cell types. Thus, it is becoming technically feasible to generate large quantities of human cell types and, in combination with relatively new detection methods, to develop higher-throughput in vitro assays that quantify tissue-specific biological properties. Indeed, the first wave of large scale hiSC-differentiated cell types including patient-derived hiPSCS are now commercially available. However, significant improvements in hiPSC production and differentiation processes are required before cell-based toxicity assays that accurately reflect mature tissue phenotypes can be delivered and implemented in a cost-effective manner. In this review, we discuss the promising alignment of hiPSCs and recently emerging technologies to quantify tissue-specific functions. We emphasize liver, cardiovascular, and CNS safety risks and highlight limitations that must be overcome before routine screening for toxicity pathways in hiSC-derived cells can be established.

Evaluating Cellular Impedance Assays for Detection of GPCR Pleiotropic Signaling and Functional Selectivity

G-protein—coupled receptors can couple to different signal transduction pathways in different cell types (termed cell-specific signaling) and can activate different signaling pathways depending on the receptor conformation(s) stabilized by the activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy and reduce side effects. Despite significant interest, functional selectivity has been difficult to exploit in drug discovery, in part due to the burden of multiple assays. Cellular impedance assays use an emerging technology that can qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay and is thereby suited for studying these pharmacological concepts. Cellular impedance confirmed cell-specific Gs and Gq coupling for the melanocortin-4 receptor and dual Gi and Gs signaling with the cannabinoid-1 (CB1) receptor. The balance of Gi versus Gs signaling depended on the cell line. In CB1-HEKs, Giand Gs-like responses combined to yield a novel impedance profile demonstrating the dynamic nature of these traces. Cellspecific signaling was observed with endogenous D1 receptor in U-2 cells and SK-N-MC cells, yet the pharmacological profile of partial and full agonists was similar in both cell lines. We conclude that the dynamic impedance profile encodes valuable relative signaling information and is sufficiently robust to help evaluate cell-specific signaling and functional selectivity. (Journal of Biomolecular Screening 2009:246-255)

Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs.

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range of whole-cell responses in a label-free manner. A new CDS-based instrument, CellKey, has been developed that is optimized for G-protein coupled receptor (GPCR) detection and has automated liquid handling in microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition to having sufficient throughput, new assay technologies must pass rigorous standards for assay development, signal window, dynamic range, and reproducibility to effectively support drug discovery SAR studies. Here, the authors evaluated CellKey with 3 different Gi-coupled GPCRs for suitability in supporting SAR studies. Optimized assay conditions compatible with the precision, reproducibility, and throughput required for routine screening were quickly achieved for each target. Across a 1000-fold range in compound potencies, CellKey results correlated with agonist and antagonist data obtained using classical methods ([ 35S]GTPγS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPγS data. CellKey detection of positive allosteric modulators appeared superior to GTPγS methodology. Agonist and antagonist activity could be accurately quantified under conditions of low receptor expression. CellKey is a new technology platform that uses label-free detection in a homogeneous assay that is unaffected by color quenching and is easily integrated into existing microtiter-based compound testing and data analysis procedures for drug discovery. (Journal of Biomolecular Screening 2007:312-319)

Comparing Label-Free Biosensors for Pharmacological Screening With Cell-Based Functional Assays

The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes of label-free instruments, using either an electrical impedance-based or an optical-based biosensor, are now available for investigating the effects of ligands on cellular targets. Studies of GPCR function have been especially prominent with these instruments due to the importance of this target class in drug discovery. Although both classes of biosensors share similar high sensitivity to changes in cell shape and structure, it is unknown whether these biosensors yield similar results when comparing the same GPCR response. Furthermore, since cell morphology changes induced by GPCRs differ depending on which G-protein is activated, there is potential for these instruments to have differential sensitivities to G-protein signaling. Here 1 impedance (CellKey)- and 2 optical-based instruments (BIND and Epic) are compared using Gi-coupled (ACh M2), Gq-coupled (ACh M1), and Gs-coupled (CRF1) receptors. All 3 instruments were robust in agonist and antagonist modes yielding comparable potencies and assay variance. Both the impedance and optical biosensors showed similar high sensitivity for detecting an endogenous D1/D5 receptor response and a melanocortin-4 receptor inverse agonist (agouti-related protein). The impedance-based biosensor was uniquely able to qualitatively distinguish G-protein coupling and reveal dual signaling by CRF1. Finally, responses with a ligand-gated ion channel, TRPV1, were similarly detectable in each instrument. Thus, despite some differences, both impedance- and optical-based platforms offer robust live-cell, label-free assays well suited to drug discovery and typically yield similar pharmacological profiles for GPCR ligands.

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

Abstract Cardiovascular (CV) toxicity is a leading cause of drug attrition and withdrawal. Introducing in vitro assays with higher throughput should permit earlier CV hazard identification and enable medicinal chemists to design-out liabilities. Heretofore, development of in vitro CV assays has been limited by the challenge of replicating integrated cardiovascular physiology while achieving the throughput and consistency required for screening. These challenges appear to be met with a combination of human stem cell-derived cardiomyocytes (CM) which beat spontaneously and monitoring the response with technology that can assess drug-induced changes in voltage dependent contraction such as cellular impedance which has been validated with excellent predictivity for drug-induced arrhythmia and contractility. Here, we review advances in cardiomyocyte impedance with emphasis on stem cell-derived cardiomyocyte models for toxicity screening. Key perspectives include: the electrical principles of impedance technology, impedance detection of cardiomyocyte beating, beat parameter selection/analysis, validation in toxicity and drug discovery, and future directions. As a conclusion, an in vitro screening cascade is proffered using the downstream, inclusive detection of CM impedance assays as a primary screen followed by complementary CM assays chosen to enable mechanism-appropriate follow-up. The combined approach will enhance testing for CV liabilities prior to traditional in vivo models.

An impedance-based cellular assay using human iPSC-derived cardiomyocytes to quantify modulators of cardiac contractility.

Cardiovascular toxicity, a prominent reason for late-stage failures in drug development, has resulted in a demand for in vitro assays that can predict this liability in early drug discovery. Current in vitro cardiovascular safety testing primarily focuses on ion channel modulation and low throughput cardiomyocyte (CM) contractility measurements. We evaluated both human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and rat neonatal CMs (rat CMs) on the xCELLigence Cardio system which uses impedance technology to quantify CM beating properties in a 96-well format. Forty-nine compounds were tested in concentration-response mode to determine potency for modulation of CM beating, a surrogate biomarker for contractility. These compounds had previously been tested in vivo and in a low throughput in vitro optical-based contractility assay that measures sarcomere shortening in electrically paced dog CMs. In comparison with in vivo contractility effects, hiPSC-CM impedance had assay sensitivity, specificity, and accuracy values of 90%, 74%, and 82%, respectively. These values compared favorably to values reported for the dog CM optical assay (83%, 84%, and 82%) and were slightly better than impedance using rat CMs (77%, 74%, and 74%). The potency values from the hiPSC-CM and rat CM assays spanned four orders of magnitude and correlated with values from the dog CM optical assay (r(2 )= 0.76 and 0.70, respectively). The Cardio system assay has >5× higher throughput than the optical assay. Thus, hiPSC-CM impedance testing can help detect the human cardiotoxic potential of novel therapeutics early in drug discovery, and if a hazard is identified, has sufficient throughput to support the design-make-test-analyze cycle to mitigate this liability.

Identification of short-acting κ-opioid receptor antagonists with anxiolytic-like activity.

The κ-opioid receptor plays a central role in mediating the response to stressful life events. Inhibiting κ-opioid receptor signaling is proposed as a mechanism for treating stress-related conditions such as depression and anxiety. Preclinical testing consistently confirms that disruption of κ-opioid signaling is efficacious in animal models of mood disorders. However, concerns about the feasibility of developing antagonists into drugs stem from an unusual pharmacodynamic property of prototypic κ-opioid receptor-selective antagonists; they inhibit receptor signaling for weeks to months after a single dose. Several fundamental questions include - is it possible to identify short-acting antagonists; is long-lasting inhibition necessary for efficacy; and is it safe to develop long-acting antagonists in the clinic. Here, we test representative compounds (AZ-ECPC, AZ-MTAB, and LY-DMPF) from three new chemical series of κ-opioid receptor ligands for long-lasting inhibition. Each compound dose-dependently reversed κ-opioid agonist-induced diuresis. However, unlike the prototypic antagonist, nBNI, which fully inhibited evoked diuresis for at least four weeks, the new compounds showed no inhibition after one week. The two compounds with greater potency and selectivity were tested in prenatally-stressed rats on the elevated plus maze, an exploration-based model of anxiety. Spontaneous exploration of open arms in the elevated plus maze was suppressed by prenatal stress and restored with both compounds. These findings indicate that persistent inhibition is not an inherent property of κ-opioid-selective antagonists and that post-stress dosing with transient inhibitors can be effective in a mood disorder model. This further supports κ-opioid receptor as a promising target for developing novel psychiatric medications.

Evaluation of Cellular Impedance Measures of Cardiomyocyte Cultures for Drug Screening Applications

Cardiovascular toxicity is a leading contributor to drug withdrawal and late-stage attrition. Earlier and broader screening is a validated approach to build-in cardiovascular safety as demonstrated with human Ether-à-go-go-related gene (hERG) screening to reduce drug-induced arrhythmia. There is an urgent need for novel in vitro assays to address other mechanistic aspects of cardiovascular function, including contractility, heart rate, toxicity, hypertrophy, and non-hERG arrhythmia. Recent advances in label-free cellular impedance technology now enable tracking of spontaneous, synchronized beating of cultured cardiomyocytes. Analysis of beating allows integrated detection that is downstream of electrical and mechanical aspects of contraction. Here, we evaluate impedance-based cardiomyocyte responses against criteria required for drug screening. The throughput and sensitivity allowed for rapid assay development. Critical variables for rat neonatal cardiomyocyte assays included cell density and serum levels. Once optimized, consistent, stable beating for at least 3 days was straight-forward to achieve. In tests of compounds spanning a breadth of target classes, the potency values showed excellent precision, wide dynamic range, and consistency across multiple experiments. Cardiomyocyte impedance assays can extract multiple beat-related parameters. In these experiments, rate, amplitude, and rise slope were examined and each yielded acceptable precision. Potency values calculated by beat rate and amplitude were highly correlated for most compounds although selected compounds displayed unique profiles indicative of different mechanisms. Tests with known cardiovascular active drugs revealed concordance with clinical findings. Thus, impedance assays combine novel features including sensitivity to contractile activity, versatile data analysis, and robust/translatable data in a format with sufficient throughput to become a valuable addition to the cardiovascular in vitro screening arsenal.

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

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.