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Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value

[Image: see text] Cystic fibrosis (CF) is a genetic disorder that affects multiple tissues and organs. CF is caused by mutations in the CFTR gene, resulting in insufficient or impaired cystic fibrosis transmembrane conductance regulator (CFTR) protein. The deletion of phenylalanine at position 508 of the protein (F508del-CFTR) is the most common mutation observed in CF patients. The most effective treatments of these patients employ two CFTR modulator classes, correctors and potentiators. CFTR correctors increase protein levels at the cell surface; CFTR potentiators enable the functional opening of CFTR channels at the cell surface. Triple-combination therapies utilize two distinct corrector molecules (C1 and C2) to further improve the overall efficacy. We identified the need to develop a C2 corrector series that had the potential to be used in conjunction with our existing C1 corrector series and provide robust clinical efficacy for CF patients. The identification of a pyrrolidine series of CFTR C2 correctors and the structure–activity relationship of this series is described. This work resulted in the discovery and selection of (2S,3R,4S,5S)-3-(tert-butyl)-4-((2-methoxy-5-(trifluoromethyl)pyridin-3-yl)methoxy)-1-((S)-tetrahydro-2H-pyran-2-carbonyl)-5-(o-tolyl)pyrrolidine-2-carboxylic acid (ABBV/GLPG-3221), which was advanced to clinical trials.

Cystic fibrosis (CF) is an autosomal recessive disease resulting from mutations on both copies of the CFTR gene. Phenylalanine deletion at position 508 of the CFTR protein (F508del-CFTR) is the most frequent mutation in CF patients. Currently, the most effective treatments of CF use a dual or triple combination of CFTR correctors and potentiators. In triple therapy, two correctors (C1 and C2) and a potentiator are employed. Herein, we describe the identification and exploration of the SAR of a series of 4-aminopyrrolidine-2-carboxylic acid C2 correctors of CFTR to be used in conjunction with our existing C1 corrector series for the treatment of CF.

Cystic fibrosis (CF) is a multiorgan disease of the lungs, sinuses, pancreas, and gastrointestinal tract that is caused by a dysfunction or deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an epithelial anion channel that regulates salt and water balance in the tissues in which it is expressed. To effectively treat the most prevalent patient population (F508del mutation), two biomolecular modulators are required: correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Despite approved potentiator and potentiator/corrector combination therapies, there remains a high need to develop more potent and efficacious correctors. Herein, we disclose the discovery of a highly potent series of CFTR correctors and the structure-activity relationship (SAR) studies that guided the discovery of ABBV/GLPG-2222 (22), which is currently in clinical trials in patients harboring the F508del CFTR mutation on at least one allele.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts., eLife digest Cells must be able to respond to their changing environment in order to survive. When cells encounter particularly unfavorable conditions, they often react by activating a so-called ‘stress’ response. A group of proteins collectively known as eIF2B helps to regulate this response. In a severe neurological condition called Vanishing White Matter (VWM), the genes that produce the eIF2B proteins contain mutations that make eIF2B less active. As a result, certain cells in people with VWM are always stressed. Six years ago, researchers discovered a molecule that boosts the activity of eIF2B. In 2018, they found that it also works on various mutant forms of eIF2B found in VWM. The molecule had so far only been tested in biochemical laboratory experiments. Now, Wong et al. – including some of the researchers involved in the 2018 study – have tested whether an improved version of the molecule treats VWM in mice. The trial treatment successfully halted all signs of the disease in the mice. The molecule blunted the persistent stress response of the cells in the brain and spinal cord, primarily in a cell type that is severely affected by the human form of VWM. Cells in other parts of the body were spared. Overall, the results of the experiments suggest that an eIF2B activator may prove to be an effective treatment for VWM in humans. It could similarly be used to treat other conditions that activate this abnormal cell stress response. The molecule Wong et al. used is not suitable for use in humans, so work is continuing to find a suitable variant.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

The Integrated Stress Response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a novel eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complexin vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of other disease contexts.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators. From a small screening campaign on F508del CFTR, hits were developed leading to the identification of pre-clinical candidates GLPG1837 and GLPG2451, each derived from a distinct chemical series. Both drug candidates enhance WT CFTR activity as well as low temperature or corrector rescued F508del CFTR, and are able to improve channel activity on a series of Class III, IV CFTR mutants. The observed activities in YFP halide assays translated well to primary cells derived from CF lungs when measured using Trans-epithelial clamp circuit (TECC). Both potentiators improve F508del CFTR channel opening in a similar manner, increasing the open time and reducing the closed time of the channel. When evaluating the potentiators in a chronic setting on corrected F508del CFTR, no reduction of channel activity in presence of potentiator was observed. The current work identifies and characterizes novel CFTR potentiators GLPG1837 and GLPG2451, which may offer new therapeutic options for CF patients.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

There is strong pharmacological, biological, and genetic evidence supporting the role of N-type calcium channels (Ca V 2.2) in nociception. There is also human validation data from ziconotide, the Ca V 2.2-selective peptidyl inhibitor used clinically to treat refractory pain. Unfortunately, ziconotide utility is limited by its narrow therapeutic window and required intrathecal route of administration. A major focus has been placed on identifying state-dependent Ca V 2.2 inhibitors to improve safety margins. Much less attention, however, has been given to characterizing the kinetics of Ca V 2.2 inhibitors as a means to further differentiate compounds and maximize therapeutic potential. Here we provide a detailed characterization of the Ca V 2.2 inhibitor T4 in terms of its state-dependence, use-dependence, kinetics, and mechanism of inhibition. Compound T4 displayed a >20-fold difference in potency when measured under inactivating conditions (IC 50 =1.1 μM) as compared to closed-state conditions (IC 50 =25 μM). At 3 μM, T4 produced a 15-fold hyperpolarizing shift in the inactivation curve for Ca V 2.2 while having no effect on channel activation. To assess the kinetic properties of T4 in a more physiological manner, its inhibition kinetics were assessed at 32 °C using 2 mM Ca 2+ as the charge carrier. Surprisingly, the repriming rate for Ca V 2.2 channels at hyperpolarized potentials was similar in both the presence and absence of T4. This was in contrast to other compounds which markedly delayed repriming. Furthermore, T4 inhibited Ca V 2.2 channels more potently when channel inactivation was driven through a tonic sub-threshold depolarization rather than through a use-dependent protocol, despite similar levels of inactivation.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

A novel series of N-type calcium channel inhibitors have been discovered. Optimization of potency and HT-ADME properties provides 4-aminocyclopentapyrrolidines with analgesic efficacy after oral dosing.

A novel 4-aminocyclopentapyrrolidine series of N-type Ca(2+) channel blockers have been discovered. Enantioselective synthesis of the 4-aminocyclopentapyrrolidines was enabled using N-tert-butyl sulfinamide chemistry. SAR studies demonstrate selectivity over L-type Ca(2+) channels. N-type Ca(2+) channel blockade was confirmed using electrophysiological recording techniques. Compound 25 is an N-type Ca(2+) channel blocker that produces antinociception in inflammatory and nociceptive pain models without exhibiting cardiovascular or motor liabilities.

Blockade of voltage-gated Ca²⁺ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²⁺ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC₅₀=0.8 μM and 1.4 μM, respectively) and T-type (IC₅₀=4.6 μM and 1.2 μM, respectively) Ca²⁺ channels in FLIPR based Ca²⁺ flux assays. A-686085 also potently blocked L-type Ca²⁺ channels (EC₅₀=0.6 μM), however, A-1048400 was much less active in blocking this channel (EC₅₀=28 μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC₅₀=300-365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²⁺ currents in rat dorsal root ganglion neurons (IC₅₀=3.0 μM and 1.6 μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²⁺ channels coupled with pharmacological selectivity vs. L-type Ca²⁺ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca V 2.2) as therapeutic targets for chronic pain. A state-dependent Ca V 2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca V 2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca V 2 inhibitor (3 R )-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1 H -1,2,4-triazol-3-yl)-1,3-dihydro-2 H -indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca V 2.2 channels with an IC 50 of 0.11 μM. The voltage dependence of Ca V 2.2 inhibition was examined using automated electrophysiology. TROX-1 IC 50 values were 4.2, 0.90, and 0.36 μM at −110, −90, and −70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca V 2.2 with a 10-fold IC 50 separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca V 2.2 channels with an IC 50 of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca V 2 subfamily. Depolarized IC 50 values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca V 2.1, Ca V 2.2, and Ca V 2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective Ca V 2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca V 2.2-selective inhibitor ziconotide in preclinical models of chronic pain.

The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays.The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology.N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable.The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type 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.

Voltage-gated calcium channel (Ca(v))2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca(v)2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca(v)2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca(v)2.2 channels under depolarized conditions (IC(50) = 0.27 microM) compared with hyperpolarized conditions (IC(50)20 microM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited omega-conotoxin GVIA-sensitive calcium currents (Ca(v)2.2 channel currents), with greater potency under depolarized conditions (IC(50) = 0.4 microM) than under hyperpolarized conditions (IC(50) = 2.6 microM), indicating state-dependent Ca(v)2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca(v)2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca(v)2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca(v)2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca(v)2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

Cav2.2 channels play a critical role in pain signaling by controlling synaptic transmission between dorsal root ganglion neurons and dorsal horn neurons. The Cav2.2-selective peptide blocker ziconotide (Prialt, Elan Pharmaceuticals, Dublin, Ireland) has proven efficacious in pain relief, but has a poor therapeutic index and requires intrathecal administration. This has provided impetus for finding an orally active, state-dependent Cav2.2 inhibitor with an improved safety profile. Members of the Cav2 subfamily of calcium channels are the main contributors to central and peripheral synaptic transmission, but the pharmacological effects of blocking each subtype is not yet defined. Here we describe a high-throughput fluorescent assay using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA]) designed to quickly evaluate the state dependence and selectivity of inhibitors across the Cav2 subfamily. Stable cell lines expressing functional Cav2 channels (Ca(V)alpha, beta(3), and alpha(2)delta subunits) were co-transfected with an inward rectifier (Kir2.3) so that membrane potential, and therefore channel state, could be controlled by external potassium concentration. Following cell incubation in drug with varying concentrations of potassium, a high potassium trigger was added to elicit calcium influx through available, unblocked channels. State-dependent inhibitors that preferentially bind to channels in the open or inactivated state can be identified by their increased potency at higher potassium concentrations, where cells are depolarized and channels are biased towards these states. Although the Cav2 channel subtypes differ in their voltage dependence of inactivation, by adjusting pre-trigger potassium concentrations, the degree of steady-state inactivation can be more closely matched across Cav2 subtypes to assess molecular selectivity.

The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Cerebellar Purkinje neurons often generate all-or-none burst firing in response to depolarizing stimuli. Voltage-clamp experiments using action potential waveforms show that burst firing depends on small net inward currents that flow after spikes and reflect the net balance between multiple large currents. Given this, burst firing is surprisingly robust in the face of changes in the magnitude of the underlying currents from cell to cell. We explored the basis of this robustness by examining the effects of reducing the sodium current, the major contributor to the postspike inward current. Burst firing persisted in concentrations of tetrodotoxin that produced half-block of sodium current. This robustness of bursting reflects an acute feedback mechanism whereby waveform changes from the reduced sodium current (reduced spike height and a hyperpolarizing shift in postspike voltage) cause compensatory decreases in postspike potassium currents. In particular, reduced spike height reduces calcium entry and subsequent calcium-activated potassium current, and the hyperpolarizing shift in postspike voltage speeds deactivation of Kv3-like potassium channels. Other experiments examined bursting in Nav1.6-/-mice, in which sodium current density is reduced in the long term. Under these circumstances, there was upregulation of both T-type and P-type calcium current and a change in the balance of calcium current and calcium-activated potassium current such that their net influence shifted from being inhibitory during bursts in wild-type neurons to excitatory during bursts from Nav1.6-/-mutant neurons. Thus, Purkinje neurons have both acute and long-term feedback mechanisms that serve to maintain burst firing when voltage-dependent sodium conductance is reduced.

Cerebellar Purkinje neurons have intrinsic membrane properties that favor burst firing, seen not only during complex spikes elicited by climbing fiber input but also with direct electrical stimulation of cell bodies. We examined the ionic conductances that underlie all-or-none burst firing elicited in acutely dissociated mouse Purkinje neurons by short depolarizing current injections. Blocking voltage-dependent calcium entry by cadmium or replacement of external calcium by magnesium enhanced burst firing, but it was blocked by cobalt replacement of calcium, probably reflecting block of sodium channels. In voltage-clamp experiments, we used the burst waveform of each cell as a voltage command and used ionic substitutions and pharmacological manipulations to isolate tetrodotoxin (TTX)-sensitive sodium current, P-type and T-type calcium current, hyperpolarization-activated cation current (Ih), voltage-activated potassium current, large-conductance calcium-activated potassium current, and small-conductance calcium-activated potassium (SK) current. Measured near the middle of the first interspike interval, TTX-sensitive sodium current carried the largest inward current, and T-type calcium current was also substantial. Current through P-type channels was large immediately after a spike but decayed rapidly. These inward currents were opposed by substantial components of voltage-dependent and calcium-dependent potassium current. Termination of the burst is caused partly by decay of sodium current, together with a progressive buildup of SK current after the first interspike interval. Although burst firing depends on the net balance between multiple large currents flowing after a spike, it is surprisingly robust, probably reflecting complex interactions between the exact voltage waveform and voltage and calcium dependence of the various currents.

Neuromodulation provides considerable flexibility to the output of neural networks. In spite of the extensive literature documenting the presence of modulatory peptide co-transmitters in many neurons, considerably less is known about the specific roles of co-transmission in circuit function. This review describes some of the potential consequences of peptide co-transmission in functional circuits, using specific examples from recent work on the actions of identified peptidergic projection neurons acting on the multifunctional neural network within the crustacean stomatogastric ganglion. This system reveals that co-transmission provides projection neurons with a rich assortment of strategies for eliciting multiple outputs from a multifunctional network.

N-, T- and P/Q-type voltage-gated Ca2+ channels are critical for regulating neurotransmitter release and cellular excitability and have been implicated in mediating pathological nociception. A-1264087 is a novel state-dependent blocker of N-, T- and P/Q-type channels. In the present studies, A-1264087 blocked (IC50 = 1.6 μM) rat dorsal root ganglia N-type Ca2+ in a state-dependent fashion. A-1264087 (1, 3 and 10 mg/kg po) dose-dependently reduced mechanical allodynia in rats with a spinal nerve ligation (SNL) injury. A-1264087 (4 mg/kg iv) inhibited both spontaneous and mechanically evoked activity of spinal wide dynamic range (WDR) neurons in SNL rats but had no effect in uninjured rats. The inhibitory effect on WDR neurons remained in spinally transected SNL rats. Injection of A-1264087 (10 nmol/0.5 μl) into the spinal cord reduced both spontaneous and evoked WDR activity in SNL rats. Application of A-1264087 (300 nmol/20 μl) into the receptive field on the hindpaw attenuated evoked but not spontaneous firing of WDR neurons. Using electrical stimulation, A-1264087 (4 mg/kg iv) inhibited Aδ- and C-fiber evoked responses and after-discharge of WDR neurons in SNL rats. These effects by A-1264087 were not present in uninjured rats. A-1264087 moderately attenuated WDR neuron windup in both uninjured and SNL rats. In summary, these results indicate that A-1264087 selectively inhibited spinal nociceptive transmission in sensitized states through both peripheral and central mechanisms.

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that 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. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

We report the investigation of sulfonamide-derived Cav2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Cav2.2 potency without the liability of the circulating sulfonamide metabolite.

Voltage-gated Ca 2+ channels play an important role in nociceptive transmission. There is significant evidence supporting a role for N-, T- and P/Q-type Ca 2+ channels in chronic pain. Here, we report that A-1264087, a structurally novel state-dependent blocker, inhibits each of these human Ca 2+ channels with similar potency (IC 50 = 1–2 μM). A-1264087 was also shown to inhibit the release of the pronociceptive calcitonin gene–related peptide from rat dorsal root ganglion neurons. Oral administration of A-1264087 produces robust antinociceptive efficacy in monoiodoacetate-induced osteoarthritic, complete Freund adjuvant–induced inflammatory, and chronic constrictive injury of sciatic nerve—induced, neuropathic pain models with ED 50 values of 3.0, 5.7, and 7.8 mg/kg (95% confidence interval=2.2–3.5, 3.7–10, and 5.5–12.8 mg/kg), respectively. Further analysis revealed that A-1264087 also suppressed nociceptive-induced p38 and extracellular signal–regulated kinase 1/2 phosphorylation, which are biochemical markers of engagement of pain circuitry in chronic pain states. Additionally, A-1264087 inhibited both spontaneous and evoked neuronal activity in the spinal cord dorsal horn in complete Freund adjuvant–inflamed rats, providing a neurophysiological basis for the observed antihyperalgesia. A-1264087 produced no alteration of body temperature or motor coordination and no learning impairment at therapeutic plasma concentrations. Perspective The present results demonstrate that the neuronal Ca 2+ channel blocker A-1264087 exhibits broad-spectrum efficacy through engagement of nociceptive signaling pathways in preclinical pain models in the absence of effects on psychomotor and cognitive function.

The voltage-gated calcium channel Ca(v)2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca(v)2.2. In particular, 19 is an inhibitor of Ca(v)2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca(v)2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

Development of calcium channel blockers is attractive, but has in the past been hampered by lack of high throughput electrophysiological technology. This limitation has been overcome by the implementation of automated patch clamp systems that allow identification of state-dependent compounds, which preferentially target pathologically overactive channels. We recently presented a fluorescence-based high-throughput screen for P/Q-type calcium channels followed by automated electrophysiology. Here, we provide a detailed description of the development of the secondary screen, and show the full analysis of the inactivation kinetics of the recombinant P/Q channel that served as a basis for the automated patch clamp protocol. Increasing the length of pre-depolarization shifted the inactivation to more hyperpolarized potentials. No steadystate inactivation was reached up to pre-depolarization durations of 3 min, while stability of the recordings progressively declined. As a compromise, a 3s pre-depolarization protocol was proposed for functional screening. In order to validate the electrophysiological screening, we compared kinetics and pharmacology of recombinant P/Q-type channels between automated and manual patch clamp measurements. Channel activation was similar under both conditions. By contrast, inactivation occurred at more hyperpolarized potentials in the automated system. Therefore, P/Q-type calcium channel inactivation is sensitive to the applied technological platform and needs to be adjusted when performing automated patch clamp recordings. Our results indicate that a thorough analysis of the inactivation kinetics is mandatory, when establishing an electrophysiological screening protocol for calcium channel blockers. As some data obtained by automated recordings may not be identical to manual patch clamp analysis, we recommend a proper initial validation of the screening assay and – if necessary – a posthoc adjustment of automated patch clamp values. The protocol presented here supports hit-to-lead and lead optimization efforts during the development of novel P/Q-type calcium channel blockers, and may be valuable for the generation of assays in other ion channel programs.

Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory β1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 μM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Secondary active glucose transport is mediated by at least four members of the solute-linked carrier 5 gene family (sodium/glucose transporter [SGLT] 1-4). Human genetic disorders of SGLTs including glucose-galactose malabsorption and familial renal glucosuria have increased attention on members of this family of transporters as putative drug targets. Using human SGLT1 (hSGLT1) as a paradigm, we developed a functional assay that should be adaptable to ultra-high-throughput operation and to other SGLTs. Human embryonic kidney (HEK) 293 cells stably expressing hSGLT1 (hSGLT1/HEK293 cells) display a Na(+)-dependent, phlorizin-sensitive alpha-methyl-D-[(14)C]glucopyranoside flux with expected kinetic parameters. In electrophysiological studies with hSGLT1/HEK293 cells, substrate-dependent changes in membrane potential were observed, consistent with the electrogenic operation of hSGLT1. With the use of voltage-sensitive dyes, a membrane potential, fluorescence resonance energy transfer-based functional assay on a voltage/ion probe reader platform has been established for SGLT1. This high-capacity functional assay displays similar characteristics in terms of substrate specificity and phlorizin sensitivity to those determined by more traditional approaches and should provide a means to identify novel and selective SGLT inhibitors.

The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.

Many neurons contain multiple cotransmitters, including neuropeptides. In the stomatogastric nervous system a number of different neuropeptides are found colocalized with small molecule neurotransmitters. Three proctolin-containing projection neurons contain different cotransmitters, and modulate the stomatogastric ganglion motor patterns differently. A number of neuropeptides, including proctolin, found in inputs to the stomatogastric ganglion, converge onto the same membrane current. This includes colocalized peptides. Studying the peptidergic modulation of the stomatogastric ganglion provides a unique opportunity to uncover general principles of organization of peptidergic control systems.

Six neuromodulators [proctolin,Cancer borealistachykinin-related peptide Ia, crustacean cardioactive peptide (CCAP), red pigment-concentrating hormone, TNRNFLRFamide, and pilocarpine] converge onto the same voltage-dependent inward current in stomatogastric ganglion (STG) neurons of the crabC. borealis. We show here that each of these modulators acts on a distinct subset of pyloric network neurons in the STG. To ask whether the differences in cell targets could account for their differential effects on the pyloric rhythm, we systematically compared the motor patterns produced by proctolin and CCAP. The motor patterns produced in proctolin and CCAP differed quantitatively in a number of ways. Proctolin and CCAP both act on the lateral pyloric neuron and the inferior cardiac neuron. Proctolin additionally acts on the pyloric dilator (PD) neurons, the pyloric (PY) neurons, and the ventricular dilator neuron. Using the dynamic clamp, we introduced an artificial peptide-elicited current into the PD and PY neurons, in the presence of CCAP, and converted the CCAP rhythm into a rhythm that was statistically similar to that seen in proctolin. This suggests that the differences in the network effects of these two modulators can primarily be attributed to the known differential distributions of their receptors onto distinct subsets of neurons, despite the fact that they activate the same current.

The stomatogastric ganglion of the crab, Cancer borealis, is modulated by >20 different substances, including numerous neuropeptides. One of these peptides, proctolin, activates an inward current that shows strong outward rectification ([Golowasch and Marder, 1992][1]). Decreasing the extracellular Ca2+concentration linearizes the current–voltage curve of the proctolin-induced current. We used voltage clamp to study the currents evoked by proctolin and five additional modulators [ C. borealis tachykinin-related peptide Ia (CabTRP Ia), crustacean cardioactive peptide, red pigment-concentrating hormone, TNRNFLRFamide, and the muscarinic agonist pilocarpine] in stomatogastric ganglion neurons, both in the intact ganglion and in dissociated cell culture. Subtraction currents yielded proctolin-like current–voltage relationships for all six substances, and the current–voltage curves of all six substances showed linearization in low external Ca2+. The lateral pyloric neuron responded to all six modulators, but the ventricular dilator neuron only responded to a subset of them. Bath application of saturating concentrations of proctolin occluded the response to CabTRP and vice versa. N -(6-Aminohexyl)-5-chloro-1-napthalensulfonamide, a calmodulin inhibitor, increased the amplitude and altered the voltage dependence of the responses elicited by CabTRP and proctolin. Together, these data indicate that all six substances converge onto the same voltage-dependent current, although they activate different receptors. Therefore, differential network responses evoked by these substances may primarily depend on the receptor distribution on network neurons. [1]: #ref-22

The multifunctional neural circuits in the crustacean stomatogastric ganglion (STG) are influenced by many small-molecule transmitters and neuropeptides that are co-localized in identified projection neurons to the STG. We describe the pattern of gamma-aminobutyric acid (GABA) immunoreactivity in the stomatogastric nervous system of the crab Cancer borealis and demonstrate biochemically the presence of authentic GABA in C. borealis. No STG somata show GABA immunoreactivity but, within the stomatogastric nervous system, GABA immunoreactivity co-localizes with several neuropeptides in two identified projection neurons, the modulatory proctolin neuron (MPN) and modulatory commissural neuron 1 (MCN1). To determine which actions of these neurons are evoked by GABA, it is necessary to determine the physiological actions of GABA on STG neurons. We therefore characterized the response of each type of STG neuron to focally applied GABA. All STG neurons responded to GABA. In some neurons, GABA evoked a picrotoxin-sensitive depolarizing, excitatory response with a reversal potential of approximately −40 mV. This response was also activated by muscimol. In many STG neurons, GABA evoked inhibitory responses with both K(+)- and Cl(−)-dependent components. Muscimol and beta-guanidinopropionic acid weakly activated the inhibitory responses, but many other drugs, including bicuculline and phaclofen, that act on vertebrate GABA receptors were not effective. In summary, GABA is found in projection neurons to the crab STG and can evoke both excitatory and inhibitory actions on STG neurons.