Discovery of pyrrolo-benzo-1,4-diazines as potent Nav1.7 sodium channel blockers

A series of pyrrolo-benzo-1,4-diazine analogs have been synthesized and displayed potent Na_v1.7 inhibitory activity and moderate selectivity over Na_v1.5. The syntheses, structure–activity relationships, and selected pharmacokinetic data of these analogs are described. Compound 41 displayed anti-nociceptive efficacy in the rat CFA pain model at 100 mpk oral dosing.     

海南捕鸟蛛毒素-IV对大鼠背根神经节细胞钠通道的抑制影响(英文)

海南捕鸟蛛毒素 IV(HNTX IV)是从中国捕鸟蛛Seleconosmiahainana粗毒中分离得到的一种肽类神经毒素 ,在成年大鼠背根神经节 (DRG)细胞上观察了该毒素对电压门控钠通道的影响。在全细胞膜片钳条件下 ,HNTX IV能明显抑制哺乳动物神经性河豚毒敏感型 (TTX S)钠电流 ,但不影响河豚毒不敏感型 (TTX R)钠电流。HNTX IV对DRG细胞TTX S钠电流的抑制作用具有浓度依从性 ,其有效半抑制浓度 (IC50 )为 44 .6nmol/L。该毒素不影响DRG钠电流的激活与失活时间特征 ,但能导致钠通道的半数稳态失活电压向超极化方向漂移约 10 .1mV。结果表明HNTX IV是一种新型的蜘蛛毒素 ,其影响电压门控钠通道的机制可能有别于那些结合于通道位点 3来延缓钠电流失活时间特征的蜘蛛毒素如δ 澳洲漏斗网蛛毒素、μ 美洲漏斗网蛛毒素I VI等。     

Modulation of human Nav1.7 channel gating by synthetic α-scorpion toxin OD1 and its analogs

Nine different voltage-gated sodium channel isoforms are responsible for inducing and propagating action potentials in the mammalian nervous system. The Na_v1.7 channel isoform plays an important role in conducting nociceptive signals. Specific mutations of this isoform may impair gating behavior of the channel resulting in several pain syndromes. In addition to channel mutations, similar or opposite changes in gating may be produced by spider and scorpion toxins binding to different parts of the voltage-gated sodium channel. In the present study, we analyzed the effects of the α-scorpion toxin OD1 and 2 synthetic toxin analogs on the gating properties of the Na_v1.7 sodium channel. All toxins potently inhibited channel inactivation, however, both toxin analogs showed substantially increased potency by more than one order of magnitude when compared with that of wild-type OD1. The decay phase of the whole-cell Na⁺ current was substantially slower in the presence of toxins than in their absence. Single-channel recordings in the presence of the toxins revealed that Na⁺ current inactivation slowed due to prolonged flickering of the channel between open and closed states. Our findings support the voltage-sensor trapping model of α-scorpion toxin action, in which the toxin prevents a conformational change in the domain IV voltage sensor that normally leads to fast channel inactivation.     

Membrane cholesterol depletion as a trigger of Nav1.9 channel‐mediated inflammatory pain

Cholesterol is a major lipid component of the mammalian plasma membrane. While much is known about its metabolism, its transport, and its role in atherosclerotic vascular disease, less is known about its role in neuronal pathophysiology. This study reveals an unexpected function of cholesterol in controlling pain transmission. We show that inflammation lowers cholesterol content in skin tissue and sensory DRG culture. Pharmacological depletion of cellular cholesterol entails sensitization of nociceptive neurons and promotes mechanical and thermal hyperalgesia through the activation of voltage‐gated Nav1.9 channels. Inflammatory mediators enhance the production of reactive oxygen species and induce partitioning of Nav1.9 channels from cholesterol‐rich lipid rafts to cholesterol‐poor non‐raft regions of the membrane. Low‐cholesterol environment enhances voltage‐dependent activation of Nav1.9 channels leading to enhanced neuronal excitability, whereas cholesterol replenishment reversed these effects. Consistently, we show that transcutaneous delivery of cholesterol alleviates hypersensitivity in animal models of acute and chronic inflammatory pain. In conclusion, our data establish that membrane cholesterol is a modulator of pain transmission and shed a new light on the relationship between cholesterol homeostasis, inflammation, and pain.     

Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.     

Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.     

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by the inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) has also been reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5–S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP, and control peptides followed by mass spectrometry of the digests, we demonstrated that the mutant sequence is severalfold more sensitive to MMP-9 proteolysis relative to the wild type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell surface levels of the Nav1.7, Nav1.6, and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.     

Three-Dimensional Structure of Selenocosmia huwena Lectin-I (SHL-I) from the Venom of the Spider Selenocosmia huwena by 2D-NMR

The three-dimensional structure of native SHL-I, a lectin from the venom of the Chinese bird spider Selenocosmia huwena, has been determined from two-dimensional ¹H NMR spectroscopy recorded at 500 and 600 MHz. The best 10 structures have NOE violation <0.3 Å, dihedral violation <2 deg, and average root-mean-square differences of 0.85 + 0.06 Å over backbone atoms. The structure consists of a three-stranded antiparallel -sheet and three turns. The three disulfide bridges and three-stranded antiparallel -sheet form a inhibitor cystine knot motif which is adopted by several other small proteins, such as huwentoxin-I, -conotoxin, and gurmarin. The C-terminal fragment from Leu28 to Trp32 adopts two sets of conformations corresponding to the cis and trans conformations of Pro31. The structure of SHL-I also has high similarity with that of the N-terminus of hevein, a lectin from rubber-tree latex.     

Localization of Nav1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction,pheromone sensing and immune function

Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Na_v1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Na_v1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Na_v1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Na_v1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Na_v1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Na_v1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.     

The discovery of benzoxazine sulfonamide inhibitors of NaV1.7: Tools that bridge efficacy and target engagement

The voltage-gated sodium channel Na_V1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of Na_V1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat Na_V1.7 versus human Na_V1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse Na_V1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.     

Investigation of the selectivity of one type of small-molecule inhibitor for three Nav channel isoforms based on the method of computer simulation

Voltage-gated sodium (Na_v) channels play a pivotal role for the changes in membrane potential and belong to large membrane proteins that compose four voltage sensor domains (VSD1–4). In this study, we describe the binding mode and selectivity of one of the aryl sulfonamide sodium channel inhibitors, PF-04856264, for the VSD4s in Na_v1.4, Na_v1.5 and Na_v1.7, respectively, through molecular dynamics simulation and enhanced post-dynamics analyses. Our results show that there are three binding site regions (BSR1–3) in the combination of the ligand and receptors, of which BSR1 and BSR3 contribute to the selectivity and affinity of the ligand to the receptor. What’s more, the 39th residue (Y39 in VSD4^hNav1.4/ VSD4^hNav1.7 and A39 in VSD4^hNav1.5) and N42 in BSR1, the 84th residue (L84 in VSD4^hNav1.4, T84 in VSD4^hNav1.5, and M84 in VSD4^hNav1.7) in BSR2 and the conserved positive charged residues in BSR3 have major contributions to the interaction between the ligand and receptor. Further analysis reveals that if the 39th residue has a benzene ring structure, the connection of BSR1 and the ligand would be much stronger through π-stacking interaction. On the other hand, the strength and number of the hydrogen bonds formed by the ligand and the conserved arginines on S4 determine the contribution of BSR3 to the total free binding energy. We anticipate this study pave the way for the design of more effective and safe treatment for pain that selectively target Na_v1.7.     

Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins

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Mambalgin-1 Pain-relieving Peptide,Stepwise Solid-phase Synthesis,Crystal Structure,and Functional Domain for Acid-sensing Ion Channel 1a Inhibition

Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.     

Interstitial 2q24.3 deletion including SCN2A and SCN3A genes in a patient with autistic features,psychomotor delay,microcephaly and no history of seizures

Mutations in neuronal voltage-gated sodium channel genes SCN1A, SCN2A, and SCN3A may play an important role in the etiology of neurological diseases and psychiatric disorders, besides various types of epilepsy. Here we describe a 3-year-old boy with autistic features, language delay, microcephaly and no history of seizures. Array-CGH analysis revealed an interstitial deletion of ~ 291.9 kB at band 2q24.3 disrupting the entire SCN2A gene and part of SCN3A. We discuss the effects of haploinsufficiency of SCN2A and SCN3A on the genetic basis of neurodevelopmental and neurobehavioral disorders and we propose that this haploinsufficiency may be associated not only with epilepsy, but also with autistic features.     

Structural Insights into the Induced-fit Inhibition of Fascin by a Small-Molecule Inhibitor

Tumor metastasis is responsible for ~ 90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~ 35^o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.     

Neutralization of gating charges in domain II of the sodium channel alpha subunit enhances voltage-sensor trapping by a beta-scorpion toxin

beta-Scorpion toxins shift the voltage dependence of activation of sodium channels to more negative membrane potentials, but only after a strong depolarizing prepulse to fully activate the channels. Their receptor site includes the S3-S4 loop at the extracellular end of the S4 voltage sensor in domain II of the alpha subunit. Here, we probe the role of gating charges in the IIS4 segment in beta-scorpion toxin action by mutagenesis and functional analysis of the resulting mutant sodium channels. Neutralization of the positively charged amino acid residues in the IIS4 segment by mutation to glutamine shifts the voltage dependence of channel activation to more positive membrane potentials and reduces the steepness of voltage-dependent gating, which is consistent with the presumed role of these residues as gating charges. Surprisingly, neutralization of the gating charges at the outer end of the IIS4 segment by the mutations R850Q, R850C, R853Q, and R853C markedly enhances beta-scorpion toxin action, whereas mutations R856Q, K859Q, and K862Q have no effect. In contrast to wild-type, the beta-scorpion toxin Css IV causes a negative shift of the voltage dependence of activation of mutants R853Q and R853C without a depolarizing prepulse at holding potentials from -80 to -140 mV. Reaction of mutant R853C with 2-aminoethyl methanethiosulfonate causes a positive shift of the voltage dependence of activation and restores the requirement for a depolarizing prepulse for Css IV action. Enhancement of sodium channel activation by Css IV causes large tail currents upon repolarization, indicating slowed deactivation of the IIS4 voltage sensor by the bound toxin. Our results are consistent with a voltage-sensor-trapping model in which the beta-scorpion toxin traps the IIS4 voltage sensor in its activated position as it moves outward in response to depolarization and holds it there, slowing its inward movement on deactivation and enhancing subsequent channel activation. Evidently, neutralization of R850 and R853 removes kinetic barriers to binding of the IIS4 segment by Css IV, and thereby enhances toxin-induced channel activation.     

Structural Insights into the Inhibition of Zika Virus NS2B-NS3 Protease by a Small-Molecule Inhibitor

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Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.     

Two Alternative Conformations of a Voltage-Gated Sodium Channel

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9 Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4–S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.