Structure-function relationship of new peptides activating human Nav1.1.

Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.

NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats.

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

Identification of aryl sulfonamides as novel and potent inhibitors of NaV1.5.

We describe the synthesis and biological evaluation of a series of novel aryl sulfonamides that exhibit potent inhibition of NaV1.5. Unlike local anesthetics that are currently used for treatment of Long QT Syndrome 3 (LQT-3), the most potent compound (-)-6 in this series shows high selectivity over hERG and other cardiac ion channels and has a low brain to plasma ratio to minimize CNS side effects. Compound (-)-6 is also effective inshortening prolonged action potential durations (APDs) in a pharmacological model of LQT-3 syndrome in pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Unlike most aryl sulfonamide NaV inhibitors that bind to the channel voltage sensors, these NaV1.5 inhibitors bind to the local anesthetic binding site in the central pore of the channel.

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Synthetic Analogues of Huwentoxin-IV Spider Peptide With Altered Human NaV1.7/NaV1.6 Selectivity Ratios.

Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R 26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Identification of Selective Acyl Sulfonamide-Cycloalkylether Inhibitors of the Voltage-Gated Sodium Channel (NaV) 1.7 with Potent Analgesic Activity.

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested that acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the NaV1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.

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.

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.

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.

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

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 triazolone derivatives as novel, potent stearoyl-CoA desaturase-1 (SCD1) inhibitors.

Stearoyl-CoA desaturase-1 (SCD1) plays an important role in lipid metabolism. Inhibition of SCD1 activity represents a potential novel approach for the treatment of metabolic diseases such as obesity, type 2 diabetes and dyslipidemia, as well as skin diseases, acne and cancer. Herein, we report the synthesis and structure-activity relationships (SAR) of a series of novel triazolone derivatives, culminating in the identification of pyrazolyltriazolone 17a, a potent SCD1 inhibitor, which reduced plasma C16:1/C16:0 triglycerides desaturation index (DI) in an acute Lewis rat model in a dose dependent manner, with an ED50 of 4.6 mg/kg. In preliminary safety studies, compound 17a did not demonstrate adverse effects related to SCD1 inhibition after repeat dosing at 100mg/kg. Together, these data suggest that sufficient safety margins can be achieved with certain SCD1 inhibitors, thus allowing exploration of clinical utility in metabolic disease settings.

Discovery of thiazolylpyridinone SCD1 inhibitors with preferential liver distribution and reduced mechanism-based adverse effects.

We discovered a series of novel and potent thiazolylpyridinone-based SCD1 inhibitors based on a 2-aminothiazole HTS hit by replacing the amide bond with a pyridinone moiety. Compound 19 demonstrated good potency against SCD1 in vitro and in vivo. The mouse liver microsomal SCD1 in vitro potency for 19 was improved by more than 240-fold compared to the original HTS hit. Furthermore, 19 demonstrated a dose-dependent reduction of plasma desaturation index with an ED50 of 6.3 mg/kg. Compound 19 demonstrated high liver to plasma and liver to eyelid exposures, indicating preferential liver distribution. The preliminary toxicology study with compound 19 did not demonstrate adverse effects related to SCD1 inhibition, suggesting a wide safety margin with respect to other known SCD1 inhibitors with wider distribution profiles.

Discovery of piperazin-1-ylpyridazine-based potent and selective stearoyl-CoA desaturase-1 inhibitors for the treatment of obesity and metabolic syndrome.

Stearoyl-CoA desaturase-1 (SCD1) catalyzes de novo synthesis of monounsaturated fatty acids from saturated fatty acids. Studies have demonstrated that rodents lacking a functional SCD1 gene have an improved metabolic profile, including reduced weight gain, lower triglycerides, and improved insulin response. In this study, we discovered a series of piperazinylpyridazine-based highly potent, selective, and orally bioavailable compounds. Particularly, compound 49 (XEN103) was highly active in vitro (mSCD1 IC(50) = 14 nM and HepG2 IC(50) = 12 nM) and efficacious in vivo (ED(50) = 0.8 mg/kg). It also demonstrated striking reduction of weight gain in a rodent model. Our findings with small-molecule SCD1 inhibitors confirm the importance of this target in metabolic regulation, describe novel models for assessing SCD1 inhibitors for efficacy and tolerability and demonstrate an opportunity to develop a novel therapy for metabolic disease.