Recent progress in sodium channel modulators for pain

Voltage-gated sodium channels (Na_vs) are an important family of transmembrane ion channel proteins and Na_v drug discovery is an exciting field. Pharmaceutical investment in Na_vs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Na_v structural biology pave the way for a future of fruitful prospective Na_v drug discovery.     

The identification of structurally novel,selective, orally bioavailable positive modulators of mGluR2

The optimisation of an HTS hit series (1) leading to the identification of structurally novel, selective, orally bioavailable mGluR2 positive modulators GSK1331258 and GSK1331268 is described. Structure–activity relationships, attenuation of dopaminergic activity, and potentiation of mGluR2 responses in rat hippocampal MPP-DG synapses are also reported.     

Structure-activity relationship studies on 2-heteroaryl-4-arylimidazoles NPY5 receptor antagonists

A series of 2-heteroaryl-4-arylimidazoles with potent in vitro activity at the NPY5 receptor was developed. Introduction of electron-withdrawing groups on the 4-aryl ring led to a significant improvement of in vitro potency. Several analogues from this series had anorectic activity in rodent feeding models, but were also found to have undesired behavioral effects in spontaneous locomotor activity.     

Cytoplasmic polyamines as permeant blockers and modulators of the voltage-gated sodium channel

We report that voltage-gated Na+ channels (Na(V)) from rat muscle (mu1) expressed in HEK293 cells exhibit anomalous rectification of whole-cell outward current under conditions of symmetrical Na+. This behavior gradually fades with time after membrane break-in, as if a diffusible blocking substance in the cytoplasm is slowly diluted by the pipette solution. The degree of such block and rectification is markedly altered by various mutations of the conserved Lys(III) residue in Domain III of the Na(V) channel selectivity filter (DEKA locus), a principal determinant of inorganic ion selectivity and organic cation permeation. Using whole-cell and macropatch recording techniques, we show that two ubiquitous polyamines, spermine and spermidine, are potent voltage-dependent cytoplasmic blockers of mu1 Na(V) current that exhibit relief of block at high positive voltage, a phenomenon that is also enhanced by certain mutations of the Lys(III) residue. In addition, we find that polyamines alter the apparent rate of macroscopic inactivation and exhibit a use-dependent blocking phenomenon reminiscent of the action of local anesthetics. In the presence of a physiological Na+/K+ gradient, spermine also inhibits inward Na(V) current and shifts the voltage dependence of activation and inactivation. Similarities between the endogenous blocking phenomenon observed in whole cells and polyamine block characterized in excised patches suggest that polyamines or related metabolites may function as endogenous modulators of Na(V) channel activity.     

Identification and characterization of novel TRPV4 modulators

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.     

Regulation of the epithelial sodium channel by N4WBP5A,a novel Nedd4/Nedd4-2-interacting protein

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, we propose that N4WBP5A functions by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it is a likely candidate to modulate ENaC function in vivo.     

Predicting novel disease mutations in the cardiac sodium channel

BackgroundVoltage-gated sodium channels Nav1.x mediate the rising phase of action potential in excitable cells. Variations in gene SCN5A, which encodes the hNav1.5 channel, are associated with arrhythmias and other heart diseases. About 1,400 SCN5A variants are listed in public databases, but for more than 30% of these the clinical significance is unknown and can currently only be derived by bioinformatics approaches.Methods and resultsWe used the ClinVar, SwissVar, Humsavar, gnomAD, and Ensembl databases to assemble a dataset of 1392 hNav1.5 variants (370 pathogenic variants, 602 benign variants and 420 variants of uncertain significance) as well as a dataset of 1766 damaging variants in 20 human sodium and calcium channel paralogs. Twelve in silico tools were tested for their ability to predict damaging mutations in hNav1.5. The best performing tool, MutPred, correctly predicted 93% of damaging variants in our hNav1.5 dataset. Among the 86 hNav1.5 variants for which electrophysiological data are also available, MutPred correctly predicted 82% of damaging variants. In the subset of 420 uncharacterized hNav1.5 variants MutPred predicted 196 new pathogenic variants. Among these, 74 variants are also annotated as damaging in at least one hNav1.5 paralog.ConclusionsUsing a combination of sequence-based bioinformatics techniques and paralogous annotation we have substantially expanded the knowledge on disease variants in the cardiac sodium channel and assigned a pathogenic status to a number of mutations that so far have been described as variants of uncertain significance. A list of reclassified hNav1.5 variants and their properties is provided.     

2-Alkyl-4-aryl-pyrimidine fused heterocycles as selective 5-HT2A antagonists

The synthesis and SAR for a novel series of 2-alkyl-4-aryl-tetrahydro-pyrido-pyrimidines and 2-alkyl-4-aryl-tetrahydro-pyrimido-azepines is described. Representative compounds were shown to be subtype selective 5-HT(2A) antagonists. Optimal placement of a basic nitrogen relative to the pyrimidine and the presence of a 4-fluorophenyl group in the pyrimidine 4-position was found to have a profound effect on affinity and selectivity.     

Distinct modulating effects of TipE-homologs 2–4 on Drosophila sodium channel splice variants

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1–4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNa_v. However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNa_v channel variants. In this study, we found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNa_v splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNa_v variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNa_v variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, our results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. Our results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels.     

2-Alkyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles as novel 5-HT6 receptor agonists

A series of 2-alkyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indoles were synthesized and evaluated for their 5-HT6 activity. The most potent agonist in this series was 5-chloro-2-methyl-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole with an IC50=7.4 nM in 3H-LSD binding and an EC50=1.0 nM in a functional assay measuring production of cyclic AMP.     

Role of domain 4 in sodium channel slow inactivation

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.     

N-1-Alkyl-2-oxo-2-aryl amides as novel antagonists of the TRPA1 receptor

A series of potent antagonists of the ion channel transient receptor potential A1 (TRPA1) was developed by modifying lead structure 16 that was discovered by high-throughput screening. Based on lead compound 16, a SAR was established, showing a narrow region at the nitro-aromatic R(1) moiety and at the warhead, while the R(2) side had a much wider scope including ureas and carbamates. Compound 16 inhibits Ca(2+)-activated TRPA1 currents reversibly in whole cell patch clamp experiments, indicating that under in vivo conditions, it does not react covalently, despite its potentially electrophilic ketone.     

Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states

The TREK subfamily of two-pore domain (K2P) K⁺ channels exhibit polymodal gating by a wide range of physical and chemical stimuli. Crystal structures now exist for these channels in two main states referred to as the “up” and “down” conformations. However, recent studies have resulted in contradictory and mutually exclusive conclusions about the functional (i.e., conductive) status of these two conformations. To address this problem, we have used the state-dependent TREK-2 inhibitor norfluoxetine that can only bind to the down state, thereby allowing us to distinguish between these two conformations when activated by different stimuli. Our results reconcile these previously contradictory gating models by demonstrating that activation by pressure, temperature, voltage, and pH produce more than one structurally distinct open state and reveal that channel activation does not simply involve switching between the up and down conformations. These results also highlight the diversity of structural mechanisms that K2P channels use to integrate polymodal gating signals.     

2-Phenylspiroindenes: a novel class of selective estrogen receptor modulators (SERMs)

A series of 2-phenylspiroindenes was prepared. The most active analogue (2) was found to be comparable in potency to raloxifene (1) as an estrogen receptor ligand.     

Novel progesterone receptor modulators: 4-Aryl-phenylsulfonamides

We have developed a new series of progesterone receptor modulators based upon the 4-aryl-phenylsulfonamide. Initial work in the series afforded potent compounds with good properties, however an advanced intermediate proved to be genotoxic in a non-GLP Ames assay following metabolic activation. We subsequently solved this problem and identified advanced leads which demonstrated oral efficacy in rhesus monkey pharmacodynamic and kinetics models.     

Synthesis of 2-Alkyl-3-hydroxy-4-pyridinone-ribonucleosides,Potential Oral Iron Chelators

Abstract

Several ribonucleosides, named 2-alkyl-3-hydroxy-1-(β-D-ribofuranosyl or pyranosyl)-4-pyridinones, were synthesized in good yield. The method provides a useful means to obtain α-ketohydroxypyridin derivatives with different sugar moieties that, if used as drugs, might enhance their absorption from the intestine and certain other desirable pharmacological properties.     

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.     

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.     

Discovery of novel sodium channel inhibitors—A gene family-based approach

Voltage-gated sodium (Na_V) channel inhibitors are an important class of drugs that are used to treat a number of CNS indications including pain, local anaesthesia, epilepsy and bipolar disorder. These drugs all have their origins in traditional “empirical” pharmacology, and it was only some time after discovery that they were found to inhibit Na_V channels. The basis for therapeutic selectivity of these drugs within different disease indications is currently unknown. However, the subsequent discovery of a multi-gene family of Na_V channels suggests a possible mechanism and has opened the way for more targeted approaches to finding improved therapeutic inhibitors. This article describes some ongoing approaches to systematically clone, express and characterise the entire family of Na_V subtypes in order to better understand their properties and define their individual physiological and pathophysiological roles. As well as providing specific disease validation for individual subtypes, this also provides a panel of reagents for comprehensively exploring the efficacy, selectivity and potency relationships of existing Na_V-blocking drugs. In this way, a gene family-based approach to Na_V channels has enabled a “drug-to-target” approach, reversing the more usual “gene-to-target-to-drug” paradigm. Together with recent advances in assay technology, gene family-based approaches are increasing the tractability of these targets and are re-invigorating Na_V drug discovery within the pharmaceutical industry.