Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

Prospective Assessment of Virtual Screening Heuristics Derived Using a Novel Fusion Score.

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5.

The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7, resulting in either loss-of-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5 as confirmed in electrophysiology.

High-Throughput Screen of GluK1 Receptor Identifies Selective Inhibitors with a Variety of Kinetic Profiles Using Fluorescence and Electrophysiology Assays.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC50 values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors.

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization.

Identification of novel and selective Kv2 channel inhibitors.

Identification of selective ion channel inhibitors represents a critical step for understanding the physiological role that these proteins play in native systems. In particular, voltage-gated potassium (K(V)2) channels are widely expressed in tissues such as central nervous system, pancreas, and smooth muscle, but their particular contributions to cell function are not well understood. Although potent and selective peptide inhibitors of K(V)2 channels have been characterized, selective small molecule K(V)2 inhibitors have not been reported. For this purpose, high-throughput automated electrophysiology (IonWorks Quattro; Molecular Devices, Sunnyvale, CA) was used to screen a 200,000-compound mixture (10 compounds per sample) library for inhibitors of K(V)2.1 channels. After deconvolution of 190 active samples, two compounds (A1 and B1) were identified that potently inhibit K(V)2.1 and the other member of the K(V)2 family, K(V)2.2 (IC(50), 0.1-0.2 µM), and that possess good selectivity over K(V)1.2 (IC(50) >10 µM). Modeling studies suggest that these compounds possess a similar three-dimensional conformation. Compounds A1 and B1 are >10-fold selective over Na(V) channels and other K(V) channels and display weak activity (5-9 µM) on Ca(V) channels. The biological activity of compound A1 on native K(V)2 channels was confirmed in electrophysiological recordings of rat insulinoma cells, which are known to express K(V)2 channels. Medicinal chemistry efforts revealed a defined structure-activity relationship and led to the identification of two compounds (RY785 and RY796) without significant Ca(V) channel activity. Taken together, these newly identified channel inhibitors represent important tools for the study of K(V)2 channels in biological systems.

A miniaturized homogenous assay of mitochondrial membrane potential.

Mitochondrial dysfunction is increasingly associated with disease states. These organelles, responsible for adenosine triphosphate production, have been targeted for improved function in such diseases as Parkinson's, Alzheimer's, type 2 diabetes, and sarcopenia. In addition, the importance of determining if a clinical drug candidate adversely effects mitochondria function, which could lead to overt toxicity, has been recognized. Hence, assays that measure mitochondria activity have become essential in early stage drug development. Limitations of current assays that measure mitochondria membrane potential have prohibited the high-throughput performance necessary to screen current chemical space. Here, we describe a homogeneous assay to measure mitochondria membrane potential that can utilize either adherent or suspension cell types. The assay has been miniaturized to 1,536-well plate format, and was used to perform a fully automated robotic high-throughput screen of a small molecule chemical library.

A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-à-go-go related gene potassium channel.

The voltage-gated potassium channel, human Ether-à-go-go related gene (hERG), represents the molecular component of IKr, one of the potassium currents involved in cardiac action potential repolarization. Inhibition of IKr increases the duration of the ventricular action potential, reflected as a prolongation of the QT interval in the electrocardiogram, and increases the risk for potentially fatal ventricular arrhythmias. Because hERG is an appropriate surrogate for IKr, hERG assays that can identify potential safety liabilities of compounds during lead identification and optimization have been implemented. Although the gold standard for hERG evaluation is electrophysiology, this technique, even with the medium capacity, automated instruments that are currently available, does not meet the throughput demands for supporting typical medicinal chemistry efforts in the pharmaceutical environment. Assays that could provide reliable molecular pharmacology data, while operating in high capacity mode, are therefore desirable. In the present study, we describe a high-capacity, 384- and 1,536-well plate, functional thallium flux assay for the hERG channel that fulfills these criteria. This assay was optimized and validated using different structural classes of hERG inhibitors. An excellent correlation was found between the potency of these agents in the thallium flux assay and in electrophysiological recordings of channel activity using the QPatch automated patch platform. Extension of this study to include 991 medicinal chemistry compounds from different internal drug development programs indicated that the thallium flux assay was a good predictor of in vitro hERG activity. These data suggest that the hERG thallium flux assay can play an important role in supporting drug development efforts.

Miniaturization and HTS of a FRET-based membrane potential assay for K(ir) channel inhibitors.

The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.

High-throughput screening of 11beta-hydroxysteroid dehydrogenase type 1 in scintillation proximity assay format.

11beta-Hydroxysteroid dehydrogenase type-1 (11beta-HSD1) is a potential target for the treatment of diabetes, obesity, and hyperlipidemia. This enzyme is mainly responsible for reactivating glucocorticoid hormone inside cells such as adipose cells and liver cells by converting the inactive cortisone to active cortisol. Enzyme assays for 11beta-HSD1 involve either a thin-layer chromatography or high-performance liquid chromatography step to separate cortisol from the substrate cortisone. This additional step is labor intensive and increases the assay time, which limits assay throughput. A homogenous scintillation proximity assay-based method has been recently developed that enables high-throughput screening of 11beta-HSD1 inhibitors. We have applied this novel 11beta-HSD1 assay to screening a large-size compound collection and identified several structural classes of lead compounds that selectively inhibit the activity of 11beta-HSD1.

Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells.

Cell-based functional assays are becoming popular in many HTS laboratories because of recent advances in detection and automation technologies. However, the supply of large amounts of live cells with consistent cellular response for day-to-day screening operations over several days/weeks is a tremendous challenge. The high cost of cell culture, labor-intensive nature of the work, and inherent variability in cellular responses from time to time tend to be prohibitive for extensive applications of cell-based assays in HTS. We therefore tested division-arrested cells that were prepared in a single batch and frozen at -80 degrees C before use in several cell-based assays and in a robotic screening campaign. Chinese hamster ovary cells expressing a Gq-coupled receptor were analyzed for the agonist-induced intracellular Ca2+ response measured on a fluorescent imaging plate reader. In this case, the division-arrested cells showed consistent agonist-induced intracellular Ca2+ concentration response as reflected by signal-to-basal ratio and EC50 even 48 h after cell plating. In comparison, the responses from untreated frozen cells and fresh cells declined significantly approximately 30 h after cell plating. In other cell-based assays tested (cyclic AMP assay, reporter gene beta-lactamase assay, and ion-channel assay), the division-arrested cells performed as well as frozen, or fresh cells. We thus conclude that the use of alternate strategies such as frozen cells or division-arrested cells may alleviate the need for several batches of cell plating each day during HTS while maintaining the desired robotic throughput and assay quality.

Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays.

Label-free detection emerges as a new approach in the development of technologies for cell-based screening assays. Unlike the classic detection methods that use fluorescence, radioisotope, luminescence, or light absorption, label-free detection directly measures the cell function without using a labeled molecule. The advantages of label-free detection include a simple homogeneous assay format, noninvasive measurement, less interference with normal cell function, kinetic measurement, and reduced time for assay development. Here, we have applied the electrical impedance detection method in a real-time cell electronic sensing (RT-CES trade mark ) system for cell-based assays. The cell growth rate measured by this RT-CES system was comparable to actual cell number counted manually. In addition, cell proliferation, cytotoxicity, cytoprotection, cell growth inhibition, and apoptosis data generated by this RT-CES system correlated with those determined by the classic methods. The conclusion is that the RT-CES system is a useful tool for label-free detection of certain cell-based parameters.