A phenotypic screening platform for chronic pain therapeutics using all-optical electrophysiology.

ABSTRACT: Chronic pain associated with osteoarthritis (OA) remains an intractable problem with few effective treatment options. New approaches are needed to model the disease biology and to drive discovery of therapeutics. We present an in vitro model of OA pain, where dorsal root ganglion (DRG) sensory neurons were sensitized by a defined mixture of disease-relevant inflammatory mediators, here called Sensitizing PAin Reagent Composition or SPARC. Osteoarthritis-SPARC components showed synergistic or additive effects when applied in combination and induced pain phenotypes in vivo. To measure the effect of OA-SPARC on neural firing in a scalable format, we used a custom system for high throughput all-optical electrophysiology. This system enabled light-based membrane voltage recordings from hundreds of neurons in parallel with single cell and single action potential resolution and a throughput of up to 500,000 neurons per day. A computational framework was developed to construct a multiparameter OA-SPARC neuronal phenotype and to quantitatively assess phenotype reversal by candidate pharmacology. We screened ∼3000 approved drugs and mechanistically focused compounds, yielding data from over 1.2 million individual neurons with detailed assessment of functional OA-SPARC phenotype rescue and orthogonal "off-target" effects. Analysis of confirmed hits revealed diverse potential analgesic mechanisms including ion channel modulators and other mechanisms including MEK inhibitors and tyrosine kinase modulators. Our results suggest that the Raf-MEK-ERK axis in DRG neurons may integrate the inputs from multiple upstream inflammatory mediators found in osteoarthritis patient joints, and MAPK pathway activation in DRG neurons may contribute to chronic pain in patients with osteoarthritis.

Highly Parallelized, Multicolor Optogenetic Recordings of Cellular Activity for Therapeutic Discovery Applications in Ion Channels and Disease-Associated Excitable Cells.

Optogenetic assays provide a flexible, scalable, and information rich approach to probe compound effects for ion channel drug targets in both heterologous expression systems and associated disease relevant cell types. Despite the potential utility and growing adoption of optogenetics, there remains a critical need for compatible platform technologies with the speed, sensitivity, and throughput to enable their application to broader drug screening applications. To address this challenge, we developed the SwarmTM, a custom designed optical instrument for highly parallelized, multicolor measurements in excitable cells, simultaneously recording changes in voltage and calcium activities at high temporal resolution under optical stimulation. The compact design featuring high power LEDs, large numerical aperture optics, and fast photodiode detection enables all-optical individual well readout of 24-wells simultaneously from multi-well plates while maintaining sufficient temporal resolution to probe millisecond response dynamics. The Swarm delivers variable intensity blue-light optogenetic stimulation to enable membrane depolarization and red or lime-light excitation to enable fluorescence detection of the resulting changes in membrane potential or calcium levels, respectively. The Swarm can screen ~10,000 wells/day in 384-well format, probing complex pharmacological interactions via a wide array of stimulation protocols. To evaluate the Swarm screening system, we optimized a series of heterologous optogenetic spiking HEK293 cell assays for several voltage-gated sodium channel subtypes including Nav1.2, Nav1.5, and Nav1.7. The Swarm was able to record pseudo-action potentials stably across all 24 objectives and provided pharmacological characterization of diverse sodium channel blockers. We performed a Nav1.7 screen of 200,000 small molecules in a 384-well plate format with all 560 plates reaching a Z' > 0.5. As a demonstration of the versatility of the Swarm, we also developed an assay measuring cardiac action potential and calcium waveform properties simultaneously under paced conditions using human induced pluripotent stem (iPS) cell-derived cardiomyocytes as an additional counter screen for cardiac toxicity. In summary, the Swarm is a novel high-throughput all-optical system capable of collecting information-dense data from optogenetic assays in both heterologous and iPS cell-derived models, which can be leveraged to drive diverse therapeutic discovery programs for nervous system disorders and other disease areas involving excitable cells.

'Channeling' therapeutic discovery for epileptic encephalopathy through iPSC technologies.

Induced pluripotent stem cell (iPSC) and gene editing technologies have revolutionized the field of in vitro disease modeling, granting us access to disease-pertinent human cells of the central nervous system. These technologies are particularly well suited for the study of diseases with strong monogenic etiologies. Epilepsy is one of the most common neurological disorders in children, with approximately half of all genetic cases caused by mutations in ion channel genes. These channelopathy-associated epilepsies are clinically diverse, mechanistically complex, and hard to treat. Here, we review the genetic links to epilepsy, the opportunities and challenges of iPSC-based approaches for developing in vitro models of channelopathy-associated disorders, the available tools for effective phenotyping of iPSC-derived neurons, and discuss the potential therapeutic approaches for these devastating diseases.

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

Suppression of Kv3.3 channels by antisense oligonucleotides reverses biochemical effects and motor impairment in spinocerebellar ataxia type 13 mice.

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.

Probing Synaptic Signaling with Optogenetic Stimulation and Genetically Encoded Calcium Reporters.

Optogenetics provides a powerful approach for investigating neuronal electrophysiology at the scale required for drug discovery applications. Probing synaptic function with high throughput using optogenetics requires robust tools that enable both precise stimulation of and facile readout of synaptic activity. Here we describe two functional assays to achieve this end: (1) a pre-synaptic calcium assay that utilizes the channelrhodopsin, CheRiff, patterned optogenetic stimulus, and the pre-synaptically targeted calcium reporter jRGECO1a to monitor pre-synaptic changes in calcium influx and (2) a synaptic transmission assay in which CheRiff and cytosolic jRGECO1a are expressed in non-overlapping sets of neurons, enabling pre-synaptic stimulation and post-synaptic readout of activity. This chapter describes the methodology and practical considerations for implementation of these two assays.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

The Opioid Crisis and the Future of Addiction and Pain Therapeutics.

Opioid misuse and addiction are a public health crisis resulting in debilitation, deaths, and significant social and economic impact. Curbing this crisis requires collaboration among academic, government, and industrial partners toward the development of effective nonaddictive pain medications, interventions for opioid overdose, and addiction treatments. A 2-day meeting, The Opioid Crisis and the Future of Addiction and Pain Therapeutics: Opportunities, Tools, and Technologies Symposium, was held at the National Institutes of Health (NIH) to address these concerns and to chart a collaborative path forward. The meeting was supported by the NIH Helping to End Addiction Long-TermSM (HEAL) Initiative, an aggressive, trans-agency effort to speed scientific solutions to stem the national opioid crisis. The event was unique in bringing together two research disciplines, addiction and pain, in order to create a forum for crosscommunication and collaboration. The output from the symposium will be considered by the HEAL Initiative; this article summarizes the scientific presentations and key takeaways. Improved understanding of the etiology of acute and chronic pain will enable the discovery of novel targets and regulatable pain circuits for safe and effective therapeutics, as well as relevant biomarkers to ensure adequate testing in clinical trials. Applications of improved technologies including reagents, assays, model systems, and validated probe compounds will likely increase the delivery of testable hypotheses and therapeutics to enable better health outcomes for patients. The symposium goals were achieved by increasing interdisciplinary collaboration to accelerate solutions for this pressing public health challenge and provide a framework for focused efforts within the research community. SIGNIFICANCE STATEMENT: This article summarizes key messages and discussions resulting from a 2-day symposium focused on challenges and opportunities in developing addiction- and pain-related medications. Speakers and attendees came from 40 states in the United States and 15 countries, bringing perspectives from academia, industry, government, and healthcare by researchers, clinicians, regulatory experts, and patient advocates.

Simultaneous voltage and calcium imaging and optogenetic stimulation with high sensitivity and a wide field of view.

Transmembrane voltage and intracellular calcium concentration are coupled parameters essential to the function of neurons, cardiomyocytes, and other excitable cells. Here we introduce the Firefly-HR microscope for simultaneous optogenetic stimulation and voltage and calcium imaging with fluorescent proteins using three spectrally distinct visible color bands. Firefly-HR combines patterned stimulation, near-total internal reflection laser excitation through a prism located between the sample and a water-immersion objective, and concurrent imaging of three color channels. The microscope has efficient light collection, low fluorescent background, and a large field of view (0.24 x 1.2 mm @ 1000 frames/sec). We characterize optical crosstalk and demonstrate capabilities with three applications: (1) probing synaptically connected neuronal microcircuits, (2) examining the coupling between neuronal action potentials and calcium influx, and (3) studying the pharmacology of paced human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via simultaneous recordings of voltage, calcium, and contraction.

All-Optical Electrophysiology for Disease Modeling and Pharmacological Characterization of Neurons.

A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single-cell sensitivity and millisecond temporal resolution. Neuronal activity is elicited using blue light activation of the channelrhodopsin variant 'CheRiff'. Action potentials and synaptic signals are measured with 'QuasAr', a rapid and sensitive voltage-indicating protein with near-infrared fluorescence that scales proportionately with transmembrane potential. This integrated technology of optical stimulation and recording of electrical signals enables investigation of neuronal electrical function with unprecedented scale and precision. © 2017 by John Wiley & Sons, Inc.

Pyrazolopyrimidines as Potent Stimulators for Transient Receptor Potential Canonical 3/6/7 Channels.

Transient receptor potential canonical 3/6/7 (TRPC3/6/7) are highly homologous receptor-operated nonselective cation channels. Despite their physiological significance, very few selective and potent agonists are available for functional examination of these channels. Using a cell-based high throughput screening approach, a lead compound with the pyrazolopyrimidine skeleton was identified as a TRPC6 agonist. Synthetic schemes for the lead and its analogues were established, and structural-activity relationship studies were carried out. A series of potent and direct agonists of TRPC3/6/7 channels were identified, and among them, 4m-4p have a potency order of TRPC3 > C7 > C6, with 4n being the most potent with an EC50 of <20 nM on TRPC3. Importantly, these compounds exhibited no stimulatory activity on related TRP channels. The potent and selective compounds described here should be suitable for evaluation of the roles of TRPC channels in the physiology and pathogenesis of diseases, including glomerulosclerosis and cancer.

Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels.

BACKGROUND AND PURPOSE: Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated. EXPERIMENTAL APPROACH: Using fluorescence membrane potential and Ca(2+) assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening. KEY RESULTS: The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na(+) , K(+) and Ca(2) (+) channels in dissociated neurons. CONCLUSIONS AND IMPLICATIONS: The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.

Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.

Discovery and characterization of 2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382: a potent and selective positive allosteric modulator of MrgX1.

Previous studies have shown that the activation of mouse MrgC11, a G-protein-coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large-scale screen has been carried out to isolate small-molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of positive allosteric modulators (PAMs) with therapeutic implications in combating chronic pain. Herein we report an iterative parallel synthesis effort and a structure-activity relationship study of a series of arylsulfonamides which led to the discovery of the first PAM of MrgX1, ML382.

Potent and selective inhibitors of the TASK-1 potassium channel through chemical optimization of a bis-amide scaffold.

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC50=16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.

HTS assays for developing the molecular pharmacology of ion channels.

Ion channels remain important targets for drug discovery. Improvements in ion channel screening technologies over the past ten years have provided opportunities for drug discovery on novel and known targets by enabling efficient, high throughput screening and improved compound profiling. Fluorescent assays using ion-specific or membrane potential sensors are the most commonly used methods in high throughput campaigns due to a favorable combination of throughput, cost and information content. Automated electrophysiology instruments provide a valuable complement to fluorescent assays for many channel types and are emerging as alternative approaches for high throughput screening.

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.

Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.