Discovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV1.2-Subtype-Selective Inhibitors

We previously reported that a lipophilic N-(4′-hydroxy-3′,5′-di-tert-butylbenzyl) derivative ( 1 ) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give ( 2 )) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human Na_V1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective Na_V1.2 inhibitors and >500 times less potent in inhibiting Na_V1.6 channels. On the other hand, a derivative (compound 4 ) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC₅₀ value of 49.9±1.6 mg kg⁻¹. As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.     

Photopharmacology of Ion Channels through the Light of the Computational Microscope

The optical control and investigation of neuronal activity can be achieved and carried out with photoswitchable ligands. Such compounds are designed in a modular fashion, combining a known ligand of the target protein and a photochromic group, as well as an additional electrophilic group for tethered ligands. Such a design strategy can be optimized by including structural data. In addition to experimental structures, computational methods (such as homology modeling, molecular docking, molecular dynamics and enhanced sampling techniques) can provide structural insights to guide photoswitch design and to understand the observed light-regulated effects. This review discusses the application of such structure-based computational methods to photoswitchable ligands targeting voltage- and ligand-gated ion channels. Structural mapping may help identify residues near the ligand binding pocket amenable for mutagenesis and covalent attachment. Modeling of the target protein in a complex with the photoswitchable ligand can shed light on the different activities of the two photoswitch isomers and the effect of site-directed mutations on photoswitch binding, as well as ion channel subtype selectivity. The examples presented here show how the integration of computational modeling with experimental data can greatly facilitate photoswitchable ligand design and optimization. Recent advances in structural biology, both experimental and computational, are expected to further strengthen this rational photopharmacology approach.     

Hypoxic Conditions Promote Rhythmic Contractile Oscillations Mediated by Voltage-Gated Sodium Channels Activation in Human Arteries

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Na_v), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Na_v in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent I_Na current. Various Na_v genes, encoding α-subunit isoforms sensitive (Na_v 1.2; 1.3; 1.7) and resistant (Na_v 1.5) to TTX, were detected both in arterial tissue and in SMCs. Na_v channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Na_v channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na⁺/Ca²⁺ exchanger, Na⁺/K⁺-ATPase and the cardiac Na_v channel. These results highlight the role of Na_v as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Na_v channels involved in the initiation of rhythmic oscillatory contractile activity.     

Chemical Tools for Studying the Biological Function of Glycolipids

Glycolipids play an important role in many biological processes and to this end, synthetic chemists have developed a variety of new techniques and “chemical tools” that allow for the study of glycolipids in vitro and in vivo. The types of probes prepared include fluorescent, radio‐labelled, biotinylated and photoreactive ones, as well as others based on liposomes, microarrays and other supramolecular constructs—each of which offers its own advantages, as is discussed. A number of more specialised probes, such as metabolically engineered glycolipids and photopolymerisable glycolipids, have also been prepared in order to investigate various processes including substrate specificities and binding interactions. The purpose of this review is to present the key approaches that can be used for the development of glycolipid probes, organised according to application, and also to discuss the limitations of such strategies, which include the nontrivial task of ensuring that the probe does not adversely influence the biological activity of the parent compound. On the whole, it is exciting to see what can be achieved through the development of chemical probes as tools to study biological processes, and it is envisioned that the reader will be inspired by the large number of superb studies highlighted here and will be encouraged to undertake further work in this research area.     

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels**

Voltage-gated sodium ion channels (Na_Vs) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of Na_Vs hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) – a potent and reversible inhibitor of multiple Na_V isoforms – and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or ‘click’ functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na⁺ conductance when applied to recombinant Na_Vs and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of Na_Vs expressed in the plasma membranes of cells.     

Cation Permeability of Voltage-Gated Hair Cell Ca2+ Channels of the Vertebrate Labyrinth

Some hearing, vestibular, and vision disorders are imputable to voltage-gated Ca²⁺ channels of the sensory cells. These channels convey a large Ca²⁺ influx despite extracellular Na⁺ being 70-fold more concentrated than Ca²⁺; such high selectivity is lost in low Ca²⁺, and Na⁺ can permeate. Since the permeation properties and molecular identity of sensory Ca²⁺ channels are debated, in this paper, we examine the Na⁺ current flowing through the L- and R-type Ca²⁺ channels of labyrinth hair cells. Ion currents and cytosolic free Ca²⁺ concentrations were simultaneously monitored in whole-cell recording synchronous to fast fluorescence imaging. L-type and R-type channels were present with different densities at selected sites. In 10 nM Ca²⁺, the activation and deactivation time constants of the L-type Na⁺ current were accelerated and its maximal amplitude increased by 6-fold compared to physiological Ca²⁺. The deactivation of the R-type Na⁺ current was not accelerated, and its current amplitude increased by 2.3-fold in low Ca²⁺; moreover, it was partially blocked by nifedipine in a voltage- and time-dependent manner. In conclusion, L channel gating is affected by the ion species permeating the channel, and its selectivity filter binds Ca²⁺ more strongly than that of R channel; furthermore, external Ca²⁺ prevents nifedipine from perturbing the R selectivity filter.     

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisostere of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.     

Block of Voltage-Gated Sodium Channels by Aripiprazole in a State-Dependent Manner

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNa_v1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNa_v1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNa_v1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated K_r of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5–1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.     

Exploring the pharmacology and action spectra of photochromic open-channel blockers

Light control of voltage-gated ion channels: We have developed red-shifted derivatives of QAQ, a powerful doubly charged photochromic blocker. These derivatives allow for remote control of K(v) and Na(v) channel conductance with light and offer the opportunity to silence neuronal activity reversibly.     

Receptor-Dependent and Independent Regulation of Voltage-Gated Ca2+ Channels and Ca2+-Permeable Channels by Endocannabinoids in the Brain

The activity of specific populations of neurons in different brain areas makes decisions regarding proper synaptic transmission, the ability to make adaptations in response to different external signals, as well as the triggering of specific regulatory pathways to sustain neural function. The endocannabinoid system (ECS) appears to be a very important, highly expressed, and active system of control in the central nervous system (CNS). Functionally, it allows the cells to respond quickly to processes that occur during synaptic transmission, but can also induce long-term changes. The endocannabinoids (eCBs) belong to a large family of bioactive lipid mediators that includes amides, esters, and ethers of long-chain polyunsaturated fatty acids. They are produced “on demand” from the precursors located in the membranes, exhibit a short half-life, and play a key role as retrograde messengers. eCBs act mainly through two receptors, CB1R and CB2R, which belong to the G-protein coupled receptor superfamily (GPCRs), but can also exert their action via multiple non-receptor pathways. The action of eCBs depends on Ca²⁺, but eCBs can also regulate downstream Ca²⁺ signaling. In this short review, we focus on the regulation of neuronal calcium channels by the most effective members of eCBs-2-arachidonoylglycerol (2-AG), anandamide (AEA) and originating from AEA-N-arachidonoylglycine (NAGly), to better understand the contribution of ECS to brain function under physiological conditions.     

Role of L-Type Voltage-Gated Calcium Channels in Epileptiform Activity of Neurons

Epileptic discharges manifest in individual neurons as abnormal membrane potential fluctuations called paroxysmal depolarization shift (PDS). PDSs can combine into clusters that are accompanied by synchronous oscillations of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in neurons. Here, we investigate the contribution of L-type voltage-gated calcium channels (VGCC) to epileptiform activity induced in cultured hippocampal neurons by GABA(A)R antagonist, bicuculline. Using KCl-induced depolarization, we determined the optimal effective doses of the blockers. Dihydropyridines (nifedipine and isradipine) at concentrations ≤ 10 μM demonstrate greater selectivity than the blockers from other groups (phenylalkylamines and benzothiazepines). However, high doses of dihydropyridines evoke an irreversible increase in [Ca²⁺]_i in neurons and astrocytes. In turn, verapamil and diltiazem selectively block L-type VGCC in the range of 1–10 μM, whereas high doses of these drugs block other types of VGCC. We show that L-type VGCC blockade decreases the half-width and amplitude of bicuculline-induced [Ca²⁺]_i oscillations. We also observe a decrease in the number of PDSs in a cluster and cluster duration. However, the pattern of individual PDSs and the frequency of the cluster occurrence change insignificantly. Thus, our results demonstrate that L-type VGCC contributes to maintaining the required [Ca²⁺]_i level during oscillations, which appears to determine the number of PDSs in the cluster.     

A Personal Perspective on Chemical Biology: Before the Beginning

This perspective represents a brief personal account of early days before “chemical biology” emerged as a field of inquiry. Imagine a time when oligomers of DNA could not be synthesized and the order of the TACG letters in DNA could not be sequenced. Even the high resolution structure of the DNA double helix was not yet determined. 1975 was a time when there was a deep chasm between chemistry and biology. Chemists with precise knowledge of all the atoms in natural product architectures looked with dismay at the imprecise messy world of biology. Water was to be avoided! My view was that the power of synthetic organic chemistry should be used to create function, synthesis with a purpose. Our organic group at Caltech would embrace molecular recognition of biologics in water as a frontier for chemistry. We dreamed of inventing small molecules that would control the activity of macromolecules such as DNA, proteins and carbohydrates in living cells. We chemists would sky dive into the messy world of biology.     

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Narirutin via Block of Nav1.7 Voltage-Gated Sodium Channel

Neuropathic pain is a refractory chronic disease affecting millions of people worldwide. Given that present painkillers have poor efficacy or severe side effects, developing novel analgesics is badly needed. The multiplex structure of active ingredients isolated from natural products provides a new source for phytochemical compound synthesis. Here, we identified a natural product, Narirutin, a flavonoid compound isolated from the Citrus unshiu, showing antinociceptive effects in rodent models of neuropathic pain. Using calcium imaging, whole-cell electrophysiology, western blotting, and immunofluorescence, we uncovered a molecular target for Narirutin’s antinociceptive actions. We found that Narirutin (i) inhibits Veratridine-triggered nociceptor activities in L4-L6 rat dorsal root ganglion (DRG) neurons, (ii) blocks voltage-gated sodium (Na_V) channels subtype 1.7 in both small-diameter DRG nociceptive neurons and human embryonic kidney (HEK) 293 cell line, (iii) does not affect tetrodotoxin-resistant (TTX-R) Na_V channels, and (iv) blunts the upregulation of Na_v1.7 in calcitonin gene-related peptide (CGRP)-labeled DRG sensory neurons after spared nerve injury (SNI) surgery. Identifying Na_v1.7 as a molecular target of Narirutin may further clarify the analgesic mechanism of natural flavonoid compounds and provide an optimal idea to produce novel selective and efficient analgesic drugs.     

Visualizing and Manipulating Biological Processes by Using HaloTag and SNAP-Tag Technologies

Visualizing and manipulating the behavior of proteins is crucial to understanding the physiology of the cell. Methods of biorthogonal protein labeling are important tools to attain this goal. In this review, we discuss advances in probe technology specific for self-labeling protein tags, focusing mainly on the application of HaloTag and SNAP-tag systems. We describe the latest developments in small-molecule probes that enable fluorogenic (no wash) imaging and super-resolution fluorescence microscopy. In addition, we cover several methodologies that enable the perturbation or manipulation of protein behavior and function towards the control of biological pathways. Thus, current technical advances in the HaloTag and SNAP-tag systems means that they are becoming powerful tools to enable the visualization and manipulation of biological processes, providing invaluable scientific insights that are difficult to obtain by traditional methodologies. As the multiplex of self-labeling protein tag systems continues to be developed and expanded, the utility of these protein tags will allow researchers to address previously inaccessible questions at the forefront of biology.     

阿维菌素类杀虫剂与配体门控离子通道相互作用研究进展

介绍了受体对药物的选择性结合与受体中存在的一些关键位点的氨基酸残基有很大关系。氨基酸残基在通道内排成一个装满水的口袋似的结构,这个口袋结构充当着药物与哺乳类GABAA受体和甘氨酸受体的结合位点。阿维菌素的主要作用靶标谷氨酸门控氯离子通道与这两类受体具有密切的关系,因此,可推测阿维菌素与受体的作用方式也有相类似的模式。这对创制新的具有安全、高活性、高选择性的杀虫剂有重要意义。     

苯亚磺酸钠在聚乙烯醇为粘合剂的光敏热成像材料中的化学增感

以水溶性聚乙烯醇(PVA)为粘合剂，以异位法制备的颗粒尺寸为100nm的AgBr乳剂作光敏元，考察了苯亚磺酸钠(SBS)的用量、增感时间和增感温度对光敏热成像(PTG)材料照相性能的影响．实验结果表明：分别增感硬脂酸银(AgSt)、AgBr或者两者混合物，PTG材料均可取得明显的增感效果；灰雾随着苯亚磺酸钠用量增加而增加；增感温度以及增感时间不同，PTG材料的感光度不同．     

Roles of Voltage-Gated Tetrodotoxin-Sensitive Sodium Channels NaV1.3 and NaV1.7 in Diabetes and Painful Diabetic Neuropathy

Diabetes mellitus (DM) is a common chronic medical problem worldwide; one of its complications is painful peripheral neuropathy, which can substantially erode quality of life and increase the cost of management. Despite its clinical importance, the pathogenesis of painful diabetic neuropathy (PDN) is complex and incompletely understood. Voltage-gated sodium channels (VGSCs) link many physiological processes to electrical activity by controlling action potentials in all types of excitable cells. Two isoforms of VGSCs, Na_V1.3 and Na_V1.7, which are encoded by the sodium voltage-gated channel alpha subunit 3 and 9 (Scn3A and Scn9A) genes, respectively, have been identified in both peripheral nociceptive neurons of dorsal root ganglion (DRG) and pancreatic islet cells. Recent advances in our understanding of tetrodotoxin-sensitive (TTX-S) sodium channels Na_V1.3 and Na_V1.7 lead to the rational doubt about the cause–effect relation between diabetes and painful neuropathy. In this review, we summarize the roles of Na_V1.3 and Na_V1.7 in islet cells and DRG neurons, discuss the link between DM and painful neuropathy, and present a model, which may provide a starting point for further studies aimed at identifying the mechanisms underlying diabetes and painful neuropathy.     

5.25英寸玻璃基片化学增强工艺研究

玻璃基片是制作光盘的重要元件.本文介绍了玻璃光盘基片的离子交换增强原理和方法,给出了钠钙硅玻璃离子交换增强处理结果和直径130mm增强玻璃光盘基片的技术性能.