Fragment-assisted hit investigation involving integrated HTS and fragment screening: Application to the identification of phosphodiesterase 10A (PDE10A) inhibitors.

Fragment-based drug design (FBDD) relies on direct elaboration of fragment hits and typically requires high resolution structural information to guide optimization. In fragment-assisted drug discovery (FADD), fragments provide information to guide selection and design but do not serve as starting points for elaboration. We describe FADD and high-throughput screening (HTS) campaign strategies conducted in parallel against PDE10A where fragment hit co-crystallography was not available. The fragment screen led to prioritized fragment hits (IC50's ∼500µM), which were used to generate a hypothetical core scaffold. Application of this scaffold as a filter to HTS output afforded a 4µM hit, which, after preparation of a small number of analogs, was elaborated into a 16nM lead. This approach highlights the strength of FADD, as fragment methods were applied despite the absence of co-crystallographical information to efficiently identify a lead compound for further optimization.

Human stem cell-derived cardiomyocytes in cellular impedance assays: bringing cardiotoxicity screening to the front line.

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.

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.

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.

Development of a plate-based optical biosensor fragment screening methodology to identify phosphodiesterase 10A inhibitors.

We describe the development of a novel fragment screening methodology employing a plate-based optical biosensor system that can operate in a 384-well format. The method is based on the "inhibition in solution assay" (ISA) approach using an immobilized target definition compound (TDC) that has been specifically designed for this purpose by making use of available structural information. We demonstrate that this method is robust and is sufficiently sensitive to detect fragment hits as weak as KD 500 µM when confirmed in a conventional surface plasmon resonance approach. The application of the plate-based screen, the identification of fragment inhibitors of PDE10A, and their structural characterization are all discussed in a forthcoming paper.

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.

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.

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 G(i)-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 ([(35)S]GTPgammaS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPgammaS data. CellKey detection of positive allosteric modulators appeared superior to GTPgammaS 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.