Global analysis reveals families of chemical motifs enriched for HERG inhibitors.

Promiscuous inhibition of the human ether-à-go-go-related gene (hERG) potassium channel by drugs poses a major risk for life threatening arrhythmia and costly drug withdrawals. Current knowledge of this phenomenon is derived from a limited number of known drugs and tool compounds. However, in a diverse, naïve chemical library, it remains unclear which and to what degree chemical motifs or scaffolds might be enriched for hERG inhibition. Here we report electrophysiology measurements of hERG inhibition and computational analyses of >300,000 diverse small molecules. We identify chemical 'communities' with high hERG liability, containing both canonical scaffolds and structurally distinctive molecules. These data enable the development of more effective classifiers to computationally assess hERG risk. The resultant predictive models now accurately classify naïve compound libraries for tendency of hERG inhibition. Together these results provide a more complete reference map of characteristic chemical motifs for hERG liability and advance a systematic approach to rank chemical collections for cardiotoxicity risk.

Deorphanizing the human transmembrane genome: A landscape of uncharacterized membrane proteins.

The sequencing of the human genome has fueled the last decade of work to functionally characterize genome content. An important subset of genes encodes membrane proteins, which are the targets of many drugs. They reside in lipid bilayers, restricting their endogenous activity to a relatively specialized biochemical environment. Without a reference phenotype, the application of systematic screens to profile candidate membrane proteins is not immediately possible. Bioinformatics has begun to show its effectiveness in focusing the functional characterization of orphan proteins of a particular functional class, such as channels or receptors. Here we discuss integration of experimental and bioinformatics approaches for characterizing the orphan membrane proteome. By analyzing the human genome, a landscape reference for the human transmembrane genome is provided.

Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay.

Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100µM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.

Integrated analysis of drug-induced gene expression profiles predicts novel hERG inhibitors.

Growing evidence suggests that drugs interact with diverse molecular targets mediating both therapeutic and toxic effects. Prediction of these complex interactions from chemical structures alone remains challenging, as compounds with different structures may possess similar toxicity profiles. In contrast, predictions based on systems-level measurements of drug effect may reveal pharmacologic similarities not evident from structure or known therapeutic indications. Here we utilized drug-induced transcriptional responses in the Connectivity Map (CMap) to discover such similarities among diverse antagonists of the human ether-à-go-go related (hERG) potassium channel, a common target of promiscuous inhibition by small molecules. Analysis of transcriptional profiles generated in three independent cell lines revealed clusters enriched for hERG inhibitors annotated using a database of experimental measurements (hERGcentral) and clinical indications. As a validation, we experimentally identified novel hERG inhibitors among the unannotated drugs in these enriched clusters, suggesting transcriptional responses may serve as predictive surrogates of cardiotoxicity complementing existing functional assays.

Inside job: ligand-receptor pharmacology beneath the plasma membrane.

Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon.

hERG channel function: beyond long QT.

To date, research on the human ether-a-go-go related gene (hERG) has focused on this potassium channel's role in cardiac repolarization and Long QT Syndrome (LQTS). However, growing evidence implicates hERG in a diversity of physiologic and pathological processes. Here we discuss these other functions of hERG, particularly their impact on diseases beyond cardiac arrhythmia.

Modulation of hERG potassium channel gating normalizes action potential duration prolonged by dysfunctional KCNQ1 potassium channel.

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, ∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.

Profiling diverse compounds by flux- and electrophysiology-based primary screens for inhibition of human Ether-à-go-go related gene potassium channels.

Compound effects on cloned human Ether-à-go-go related gene (hERG) potassium channels have been used to assess the potential cardiac safety liabilities of drug development candidate compounds. In addition to radioactive ligand displacement tests, two other common approaches are surrogate ion-based flux assays and electrophysiological recordings. The former has much higher throughput, whereas the latter measures directly the effects on ionic currents. Careful characterization in earlier reports has been performed to compare the relative effectiveness of these approaches for known hERG blockers, which often yielded good overall correlation. However, cases were reported showing significant and reproducible differences in potency and/or sensitivity by the two methods. This raises a question concerning the rationale and criteria on which an assay should be selected for evaluating unknown compounds. To provide a general basis for considering assays to profile large compound libraries for hERG activity, we have conducted parallel flux and electrophysiological analyses of 2,000 diverse compounds, representative of the 300,000 compound collection of NIH Molecular Library Small Molecular Repository (MLSMR). Our results indicate that at the conventional testing concentration 1.0 µM, the overlap between the two assays ranges from 32% to 50% depending on the hit selection criteria. There was a noticeable rate of false negatives by the thallium-based assay relative to electrophysiological recording, which may be greatly reduced under modified comparative conditions. As these statistical results identify a preferred method for cardiac safety profiling of unknown compounds, they suggest an efficient method combining flux and electrophysiological assays to rapidly profile hERG liabilities of large collection of naive compounds.