Charles J. Cohen

Ric-3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7 Subunit in Mammalian Cells

Expression of functional, recombinant α7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor α7 subunit. In addition to confirming the previously reported effect on α7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional α7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface α-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the α7 subunit. In contrast, co-expression of α7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous α7 receptors. Immunoprecipitation studies demonstrate that α7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of α7 protein on the plasma membrane of cells lacking ric-3, but surface α-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of α7 receptors in HEK293 cells.

Maximal upstroke velocity as an index of available sodium conductance. Comparison of maximal upstroke velocity and voltage clamp measurements of sodium current in rabbit Purkinje fibers.

We compared the maximal upstroke velocity of action potentials in short rabbit Purkinje fibers with sodium currents measured with a two-microelecrrode voltage clamp. The number of sodium channels available to open during a sudden depolarization was varied either by blockade with tetrodotoxin or by inactivation with steady depolarizations. In both cases, the maximal upstroke velocity was found to be a very nonlinear measure of the number of available sodium channels. For example, 3 μM tetrodotoxin blocks 85% of the sodium channels, but reduces the maximal upstroke velocity by only 33%. Voltage clamp and upstroke velocity experiments were reconstructed with a computer model of the rabbit Purkinje fiber preparation that was closely based on experimental measurements of passive cable properties and sodium channel characteristics. The simulations indicate that our voltage clamp measurements of sodium current accurately report changes in channel availability, but they also show that the maximal upstroke velocity is a strongly nonlinear index of available sodium conductance. Most of the nonlinearity arises from the activation kinetics of the sodium channels: as the pool of available channels decreases, a greater percentage of those channels activate and contribute inward current at the time of the maximal upstroke velocity. Simulations predict that the maximal upstroke velocity-available sodium conductance relationship would still remain nonlinear at 37°C or under different stimulus conditions that give uniform or continuously propagated action potentials. The nonlin earity may invalidate inferences based on earlier maximal upstroke velocity experiments: the existence of two types of sodium channels with different tetrodotoxin sensitivity, steady state voltage dependence of tetrodotoxin block, voltage range over which sodium channels inactivate, and rapid, then slow recovery of sodium channel availability following a sudden repolarization. All of these conclusions need to be reevaluated.

Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

A channel involved in pain perception Voltage-gated sodium (Nav) channels propagate electrical signals in muscle cells and neurons. In humans, Nav1.7 plays a key role in pain perception. It is challenging to target a particular Nav isoform; however, arylsulfonamide antagonists selective for Nav1.7 have been reported recently. Ahuja et al. characterized the binding of these small molecules to human Nav channels. To further investigate the mechanism, they engineered a bacterial Nav channel to contain features of the Nav1.7 voltage-sensing domain that is targeted by the antagonist and determined the crystal structure of the chimera bound to an inhibitor. The structure gives insight into the mechanism of voltage sensing and will enable the design of more-selective Nav channel antagonists. Science, this issue p. 10.1126/science.aac5464 Structural studies give insight into how a human sodium channel involved in pain perception can be selectively inhibited. INTRODUCTION Voltage-gated sodium (Nav) channels open and close ion-selective pores in response to changes in membrane potential, and this gating underlies the generation of action potentials. Nav channels are large membrane proteins that contain four peripheral voltage-sensor domains (VSD1–4) that influence the functional state of the central ion-conducting pore. Mutations within the nine human Nav channel isoforms are associated with migraine (Nav1.1), epilepsy (Nav1.1–Nav1.3, Nav1.6), pain (Nav1.7–Nav1.9), cardiac (Nav1.5), and muscle paralysis (Nav1.4) syndromes. Accordingly, Nav channel blockers are used for the treatment of many neurological and cardiovascular disorders. These drugs bind within the central pore domain and generally lack isoform selectivity owing to the high sequence conservation found among Nav channels, limiting their therapeutic utility. In this study, we focused on a recently identified class of isoform-selective small-molecule antagonists that target a unique binding site on the fourth voltage-sensor domain, VSD4. Here we report the structural determination of such small-molecule aryl sulfonamide antagonists in complex with human Nav1.7 VSD4. Our studies demonstrate how this important new class of gating modifier engages VSD4 to inhibit Nav channel activity through a “voltage-sensor trapping” mechanism. RATIONALE For structural studies, we devised a novel protein-engineering strategy that overcomes the technical complexities of producing full-length human Nav channels. Exploiting the evolutionary relationship between human and bacterial Nav channels, we fused portions of Nav1.7 VSD4 onto the bacterial channel NavAb. Using ligand-binding assays and alanine-scanning mutagenesis, we demonstrated that the antagonist binding site present in the human Nav1.7 channel is preserved within this human VSD4-NavAb chimeric channel. This chimeric construct allowed purification, crystallization, and structure determination of potent aryl sulfonamide antagonists in complex with the human Nav1.7 VSD4 binding site. RESULTS Functional studies using patch-clamp electrophysiology revealed that aryl sulfonamide inhibitors bind with high affinity to an isoform-selective and extracellularly accessible site on VSD4. These inhibitors show a high level of state dependence, potently blocking human Nav1.7 only when VSD4 is in its activated conformation. Our crystallographic studies revealed that the anionic warhead from the aryl sulfonamide inhibitors directly engages the fourth gating charge residue (R4) on the voltage-sensing S4 helix, effectively trapping VSD4 in its activated state. Isoform selectivity is achieved by inhibitor interactions with nonconserved residues found on the S2 and S3 transmembrane helices. The drug receptor site is partially submerged within the membrane bilayer, and a peripherally bound phospholipid was observed to form a tripartite complex with the antagonist and channel. CONCLUSION A new crystallization strategy has enabled the structural determination of VSD4 from human Nav1.7 in complex with potent, state-dependent, isoform-selective small-molecule antagonists. Mechanistically, inhibitor binding traps VSD4 in an activated conformation, which stabilizes a nonconductive state of the channel, and likely prevents recovery from inactivation. Unique phospholipid interactions and an exposed inhibitor binding site expand the importance of the membrane bilayer in ion channel biology. We anticipate that these structures will enable drug design efforts aimed at other voltage-gated ion channels and may accelerate the development of new treatments for pain that selectively target Nav1.7. Drug binding sites in sodium channels. (Left) Top-view model of human Nav1.7. When open, sodium passes through the channel. Blocking drugs lacking isoform selectivity bind to a conserved site within the central pore. Isoform-selective inhibitors bind to a distinct site on VSD4. (Right) Strategy for Nav1.7 crystallography. Portions of Nav1.7 VSD4 were grafted onto a tetrameric channel (NavAb) and crystallized. (Inset) Side view of aryl sulfonamide binding site with the S4 helix and arginine gating charges highlighted pink. Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.

Discovery of Aryl Sulfonamides as Isoform-Selective Inhibitors of NaV1.7 with Efficacy in Rodent Pain Models

We report on a novel series of aryl sulfonamides that act as nanomolar potent, isoform-selective inhibitors of the human sodium channel hNaV1.7. The optimization of these inhibitors is described. We aimed to improve potency against hNaV1.7 while minimizing off-target safety concerns and generated compound 3. This agent displayed significant analgesic effects in rodent models of acute and inflammatory pain and demonstrated that binding to the voltage sensor domain 4 site of NaV1.7 leads to an analgesic effect in vivo. Our findings corroborate the importance of hNaV1.7 as a drug target for the treatment of pain.

The discovery of benzenesulfonamide-based potent and selective inhibitors of voltage-gated sodium channel Na v 1.7

The voltage gated sodium channel Nav1.7 represents an interesting target for the treatment of pain. Human genetic studies have identified the crucial role of Nav1.7 in pain signaling. Herein, we report the design and synthesis of a novel series of benzenesulfonamide-based Nav1.7 inhibitors. Structural-activity relationship (SAR) studies were undertaken towards improving Nav1.7 activity and minimizing CYP inhibition. These efforts resulted in the identification of compound 12k, a highly potent Nav1.7 inhibitor with a thousand-fold selectivity over Nav1.5 and negligible CYP inhibition.

Mechanism of Action of GHRP-6 and Nonpeptidyl Growth Hormone Secretagogues

Growth hormone (GH) secretion from the pituitary gland is regulated by the hypothalamic peptide hormones growth hormone releasing hormone (GHRH) and somatotropin release inhibiting factor (SRIF) (Scheme 11.1). The factor controlling the episodic nature of GH release is unknown but its effects are probably mediated by feedback loops involving the positive effector GHRH and the negative regulator SRIF (1). In 1984, Bowers and Momany and coworkers (2, 3) described the synthesis and properties of a series of small peptide GH secretagogues that were based on the structure of Leu and Met enkephalins. Growth hormone releasing peptide (GHRP)- 6 was the most potent of these peptides and was subsequently shown to be active in man (4, 5). Because of the limited oral bioavailability of peptides we sought a class of GH secretagogues more amenable to chemical modification so that oral bioavailability and pharmacokinetic properties could be optimized. Implicit in establishing assays to identify new small molecules was an understanding of the mechanisms regulating GH release from the anterior pituitary gland. Based on its size, GHRP-6 was considered a potential template for a small molecule peptide mimetic. Our approach was based on screening selected structures in functional and mechanism based assays. Following identification of a benzolactam lead structure, L-692,429 was synthesized and used as a prototype to investigate specificity and efficacy in clinically relevant target populations (6–11).

Design of Conformationally Constrained Acyl Sulfonamide Isosteres: Identification of N-([1,2,4]Triazolo[4,3-a]pyridin-3-yl)methane-sulfonamides as Potent and Selective hNaV1.7 Inhibitors for the Treatment of Pain

The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3- a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.

Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors

Significance Subtype-selective modulation of ion channels is often important, but extremely difficult to achieve for drug development. Using Nav1.7 as an example, we show that this challenge could be attributed to poor design in ion channel assays, which fail to detect most potent and selective compounds and are biased toward nonselective mechanisms. By exploiting different drug binding sites and modes of channel gating, we successfully direct a membrane potential assay toward non–pore-blocking mechanisms and identify Nav1.7-selective compounds. Our mechanistic approach to assay design addresses a significant hurdle in Nav1.7 drug discovery and is applicable to many other ion channels. Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non–pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Selective NaV1.7 Antagonists with Long Residence Time Show Improved Efficacy against Inflammatory and Neuropathic Pain

Selective block of NaV1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several NaV1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.

THE THERAPEUTIC UTILITY OF TARGETING Cav2 CHANNELS

The Cav2 family of voltage-gated calcium channels consists of 3 main subtypes (Cav2.1, Cav2.2 and Cav2.3), defined by their (α1 subunit (α12.1, α12.2 and α12.3, respectively; also known as α1A,α1B and αE). The use of selective peptide toxins has allowed the association of Cav2.1, 2.2 and 2.3 with P- or Q-, N-, and B-type calcium currents, respectively (Catterall, 2000; Ertel et al., 2000; Jarvis and Zamponi, 2001). These currents are found almost exclusively in the CNS, PNS and neuroendocrine cells and constitute the predominant forms of presynaptic voltage-gated calcium current. Presynaptic action potentials cause channel opening, and neurotransmitter release is steeply dependent upon the subsequent calcium entry (Meir et al., 1999; Sabatini and Regehr, 1999). Presynaptic calcium entry is modulated by many types of G-protein coupled receptors (GPCRs), and studies with agonists for these receptors indicate that modulation of Cav2 channels is a widespread and highly efficacious means of regulating neurotransmission. In this review, we describe the prospects for targeting Cav2 channels with novel therapeutic agents.