Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels.

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.

Association of respiratory failure with inhibition of NaV1.6 in the phrenic nerve.

As part of a drug discovery effort to identify potent inhibitors of NaV1.7 for the treatment of pain, we observed that inhibitors produced unexpected cardiovascular and respiratory effects in vivo. Specifically, inhibitors administered to rodents produced changes in cardiovascular parameters and respiratory cessation. We sought to determine the mechanism of the in vivo adverse effects by studying the selectivity of the compounds on NaV1.5, NaV1.4, and NaV1.6 in in vitro and ex vivo assays. Inhibitors lacking sufficient NaV1.7 selectivity over NaV1.6 were associated with respiratory cessation after in vivo administration to rodents. Effects on respiratory rate in rats were consistent with effects in an ex vivo hemisected rat diaphragm model and in vitro NaV1.6 potency. Furthermore, direct blockade of the phrenic nerve signaling was observed at exposures known to cause respiratory cessation in rats. Collectively, these results support a significant role for NaV1.6 in phrenic nerve signaling and respiratory function.

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.

Guiding Chemically Synthesized Peptide Drug Lead Optimization by Derisking Mast Cell Degranulation-Related Toxicities of a NaV1.7 Peptide Inhibitor.

Studies have shown that some peptides and small molecules can induce non IgE-mediated anaphylactoid reactions through mast cell activation. Upon activation, mast cells degranulate and release vasoactive and proinflammatory mediators, from cytoplasmic granules into the extracellular environment which can induce a cascade of severe adverse reactions. This study describes a lead optimization strategy to select NaV1.7 inhibitor peptides that minimize acute mast cell degranulation (MCD) toxicities. Various in vitro, in vivo, and PKPD models were used to screen candidates and guide peptide chemical modifications to mitigate this risk. Anesthetized rats dosed with peptides demonstrated treatment-related decreases in blood pressure and increases in plasma histamine concentrations which were reversible with a mast cell stabilizer, supporting the MCD mechanism. In vitro testing in rat mast cells with NaV1.7 peptides demonstrated a concentration-dependent increase in histamine. Pharmacodynamic modeling facilitated establishing an in vitro to in vivo correlation for histamine as a biomarker for blood pressure decline via the MCD mechanism. These models enabled assessment of structure-activity relationship (SAR) to identify substructures that contribute to peptide-mediated MCD. Peptides with hydrophobic and cationic characteristics were determined to have an elevated risk for MCD, which could be reduced or avoided by incorporating anionic residues into the protoxin II scaffold. Our analyses support that in vitro MCD assessment in combination with PKPD modeling can guide SAR to improve peptide lead optimization and ensure an acceptable early in vivo tolerability profile with reduced resources, cycle time, and animal use.

Discovery of Arylsulfonamide Nav1.7 Inhibitors: IVIVC, MPO Methods, and Optimization of Selectivity Profile.

The voltage-gated sodium channel Nav1.7 continues to be a high-profile target for the treatment of various pain afflictions due to its strong human genetic validation. While isoform selective molecules have been discovered and advanced into the clinic, to date, this target has yet to bear fruit in the form of marketed therapeutics for the treatment of pain. Lead optimization efforts over the past decade have focused on selectivity over Nav1.5 due to its link to cardiac side effects as well as the translation of preclinical efficacy to man. Inhibition of Nav1.6 was recently reported to yield potential respiratory side effects preclinically, and this finding necessitated a modified target selectivity profile. Herein, we report the continued optimization of a novel series of arylsulfonamide Nav1.7 inhibitors to afford improved selectivity over Nav1.6 while maintaining rodent oral bioavailability through the use of a novel multiparameter optimization (MPO) paradigm. We also report in vitro-in vivo correlations from Nav1.7 electrophysiology protocols to preclinical models of efficacy to assist in projecting clinical doses. These efforts produced inhibitors such as compound 19 with potency against Nav1.7, selectivity over Nav1.5 and Nav1.6, and efficacy in behavioral models of pain in rodents as well as inhibition of rhesus olfactory response indicative of target modulation.

Nav1.7 target modulation and efficacy can be measured in nonhuman primate assays.

Humans with loss-of-function mutations in the Nav1.7 channel gene (SCN9A) show profound insensitivity to pain, whereas those with gain-of-function mutations can have inherited pain syndromes. Therefore, inhibition of the Nav1.7 channel with a small molecule has been considered a promising approach for the treatment of various human pain conditions. To date, clinical studies conducted using selective Nav1.7 inhibitors have not provided analgesic efficacy sufficient to warrant further investment. Clinical studies to date used multiples of in vitro IC50 values derived from electrophysiological studies to calculate anticipated human doses. To increase the chance of clinical success, we developed rhesus macaque models of action potential propagation, nociception, and olfaction, to measure Nav1.7 target modulation in vivo. The potent and selective Nav1.7 inhibitors SSCI-1 and SSCI-2 dose-dependently blocked C-fiber nociceptor conduction in microneurography studies and inhibited withdrawal responses to noxious heat in rhesus monkeys. Pharmacological Nav1.7 inhibition also reduced odor-induced activation of the olfactory bulb (OB), measured by functional magnetic resonance imaging (fMRI) studies consistent with the anosmia reported in Nav1.7 loss-of-function patients. These data demonstrate that it is possible to measure Nav1.7 target modulation in rhesus macaques and determine the plasma concentration required to produce a predetermined level of inhibition. The calculated plasma concentration for preclinical efficacy could be used to guide human efficacious exposure estimates. Given the translatable nature of the assays used, it is anticipated that they can be also used in phase 1 clinical studies to measure target modulation and aid in the interpretation of phase 1 clinical data.

Translational Pharmacokinetic-Pharmacodynamic Modeling of NaV1.7 Inhibitor MK-2075 to Inform Human Efficacious Dose.

MK-2075 is a small-molecule selective inhibitor of the NaV1.7 channel investigated for the treatment of postoperative pain. A translational strategy was developed for MK-2075 to quantitatively interrelate drug exposure, target modulation, and the desired pharmacological response in preclinical animal models for the purpose of human translation. Analgesics used as a standard of care in postoperative pain were evaluated in preclinical animal models of nociceptive behavior (mouse tail flick latency and rhesus thermode heat withdrawal) to determine the magnitude of pharmacodynamic (PD) response at plasma concentrations associated with efficacy in the clinic. MK-2075 was evaluated in those same animal models to determine the concentration of MK-2075 required to achieve the desired level of response. Translation of MK-2075 efficacious concentrations in preclinical animal models to a clinical PKPD target in humans was achieved by accounting for species differences in plasma protein binding and in vitro potency against the NaV1.7 channel. Estimates of human pharmacokinetic (PK) parameters were obtained from allometric scaling of a PK model from preclinical species and used to predict the dose required to achieve the clinical exposure. MK-2075 exposure-response in a preclinical target modulation assay (rhesus olfaction) was characterized using a computational PKPD model which included a biophase compartment to account for the observed hysteresis. Translation of this model to humans was accomplished by correcting for species differences in PK NaV1.7 potency, and plasma protein binding while assuming that the kinetics of distribution to the target site is the same between humans and rhesus monkeys. This enabled prediction of the level of target modulation anticipated to be achieved over the dosing interval at the projected clinical efficacious human dose. Integration of these efforts into the early development plan informed clinical study design and decision criteria.

Benzoxazolinone aryl sulfonamides as potent, selective Nav1.7 inhibitors with in vivo efficacy in a preclinical pain model.

Studies on human genetics have suggested that inhibitors of the Nav1.7 voltage-gated sodium channel hold considerable promise as therapies for the treatment of chronic pain syndromes. Herein, we report novel, peripherally-restricted benzoxazolinone aryl sulfonamides as potent Nav1.7 inhibitors with excellent selectivity against the Nav1.5 isoform, which is expressed in the heart muscle. Elaboration of initial lead compound 3d afforded exemplar 13, which featured attractive physicochemical properties, outstanding lipophilic ligand efficiency and pharmacological selectivity against Nav1.5 exceeding 1000-fold. Key structure-activity relationships associated with oral bioavailability were leveraged to discover compound 17, which exhibited a comparable potency/selectivity profile as well as full efficacy following oral administration in a preclinical model indicative of antinociceptive behavior.

Discovery of selective, orally bioavailable, N-linked arylsulfonamide Nav1.7 inhibitors with pain efficacy in mice.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for the treatment of pain with gain-of-function mutations in man eliciting a variety of painful disorders and loss-of-function mutations affording insensitivity to pain. Unfortunately, drugs thought to garner efficacy via Nav1 inhibition have undesirable side effect profiles due to their lack of selectivity over channel isoforms. Herein we report the discovery of a novel series of orally bioavailable arylsulfonamide Nav1.7 inhibitors with high levels of selectivity over Nav1.5, the Nav isoform responsible for cardiovascular side effects, through judicious use of parallel medicinal chemistry and physicochemical property optimization. This effort produced inhibitors such as compound 5 with excellent potency, selectivity, behavioral efficacy in a rodent pain model, and efficacy in a mouse itch model suggestive of target modulation.

Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications.

The tremendous therapeutic potential of voltage-gated sodium channels (Na(v)s) has been the subject of many studies in the past and is of intense interest today. Na(v)1.7 channels in particular have received much attention recently because of strong genetic validation of their involvement in nociception. Here we summarize the current status of research in the Na(v) field and present the most relevant recent developments with respect to the molecular structure, general physiology, and pharmacology of distinct Na(v) channel subtypes. We discuss Na(v) channel ligands such as small molecules, toxins isolated from animal venoms, and the recently identified Na(v)1.7-selective antibody. Furthermore, we review eight characterized ligand binding sites on the Na(v) channel α subunit. Finally, we examine possible therapeutic applications of Na(v) ligands and provide an update on current clinical studies.

Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3).

TWIK-related acid-sensitive K(+) (K(2P) 9.1, TASK-3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK-3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK-3 channel modulation exists, there are only a few documented examples of potent and selective small-molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK-3 channel antagonists based on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine high-throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.

TASK-3 as a potential antidepressant target.

Modulation of TASK-3 (Kcnk9) potassium channels affect neurotransmitter release in thalamocortical centers and other sleep-related nuclei having the capacity to regulate arousal cycles and REM sleep changes associated with mood disorders and antidepressant action. Circumstantial evidence from this and previous studies suggest the potential for TASK-3 to be a novel antidepressant therapeutic target; TASK-3 knock-out mice display augmented circadian amplitude and exhibit sleep architecture characterized by suppressed REM activity. Detailed analysis of locomotor activity indicates that the amplitudes of activity bout duration and bout number are augmented in TASK-3 mutants well beyond that seen in wildtypes, findings substantiated by amplitude increases in body temperature and EEG recordings of sleep stage bouts. Polysomnographic analysis of TASK-3 mutants reveals increases in nocturnal active wake and suppressed REM sleep time while increased slow wave sleep typifies the inactive phase, findings that have implications for the cognitive impact of reduced TASK-3 activity. In direct measures of their resistance to despair behavior, TASK-3 knock-outs displayed significant decreases in immobility relative to wildtype controls in both tail suspension and forced swim tests. Treatment of wildtype animals with the antidepressant Fluoxetine markedly reduced REM sleep, while leaving active wake and slow wave sleep relatively intact. Remarkably, these effects were absent in TASK-3 mutants indicating that TASK-3 is either directly involved in the mechanism of this drug's action, or participates in parallel pathways that achieve the same effect. Together, these results support the TASK-3 channel to act as a therapeutic target for antidepressant action.

Pyridyl amides as potent inhibitors of T-type calcium channels.

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.

Discovery and expanded SAR of 4,4-disubstituted quinazolin-2-ones as potent T-type calcium channel antagonists.

The discovery and synthesis of 4,4-disubstituted quinazolinones as T-type calcium channel antagonists is reported. Based on lead compounds 2 and 3, a focused SAR campaign driven by the optimization of potency, metabolic stability, and pharmacokinetic profile identified 45 as a potent T-type Ca(2+) channel antagonist with minimized PXR activation. In vivo, 45 suppressed seizure frequency in a rat model of absence epilepsy and showed significant alterations of sleep architecture after oral dosing to rats as measured by EEG.

In vitro characterization of T-type calcium channel antagonist TTA-A2 and in vivo effects on arousal in mice.

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinson's disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Ca(v)3.1, 3.2, 3.3) voltage dependently and with high potency (IC(50) ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Ca(v)3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Ca(v)3.1 and Ca(v)3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.

Discovery of 4,4-Disubstituted Quinazolin-2-ones as T-Type Calcium Channel Antagonists.

A novel series of quinazolinone T-type calcium channel antagonists have been prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 by modifications of the 3- and 4-positions of the quinazolinone ring afforded potent and selective antagonists that displayed in vivo central nervous system efficacy in epilepsy and tremor models, as well as significant effects on rat active wake as measured by electrocorticogram.

Short-acting T-type calcium channel antagonists significantly modify sleep architecture in rodents.

A novel phenyl acetamide series of short-acting T-type calcium channel antagonists has been identified and evaluated using in vitro and in vivo assays. Heterocycle substitutions of the 4-position of the phenyl acetamides afforded potent and selective antagonists that exhibited desired short plasma half-lives across preclinical species. Lead compound TTA-A8 emerged as a compound with excellent in vivo efficacy as indicated by its significant modulation of rat sleep architecture in an EEG telemetry model, favorable pharmacokinetic properties, and excellent preclinical safety. TTA-A8 recently progressed into human clinical trials, and in line with our predictions, preliminary studies (n = 12) with a 20 mg oral dose afforded a high C max of 1.82 ± 0.274 µM with an apparent terminal half-life of 3.0 ± 1.1 h.

Positive allosteric interaction of structurally diverse T-type calcium channel antagonists.

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.

Antagonism of T-type calcium channels inhibits high-fat diet-induced weight gain in mice.

The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet-induced weight gain, without changes in food intake or sensitivity to high-fat diet-induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet-induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity.