Computer aided identification of sodium channel blockers in the clinical treatment of epilepsy using molecular docking tools

AbbreviationsAEDs - Antiepileptic drugs, BLAST - Basic Local Alignment Search Tool, CBZ - Carbamazepine, GEFS+ - Generalized Epilepsy with Febrile Seizures Plus, GPCR - G Protein Coupled Receptor, Nav - Sodium channel with specific voltage conduction, PDB - Protein Data Bank, PHT - Phenytoin, PIR - Protein Information resources, SAVES - Structural Analysis and Verification Server, VGSC - Voltage-gated Sodium channels.     

Mutations in the transmembrane helix S6 of domain IV confer cockroach sodium channel resistance to sodium channel blocker insecticides and local anesthetics

Indoxacarb and metaflumizone are two sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated non-conducting state, a mode of action similar to that of local anesthetics (LAs). Recently, two sodium channel mutations, F1845Y (F⁴ⁱ¹⁵Y) and V1848I (V⁴ⁱ¹⁸I), in the transmembrane segment 6 of domain IV (IVS6), were identified to be associated with indoxacarb resistance in Plutella xylostella. F⁴ⁱ¹⁵ is known to be critical for the action of LAs on mammalian sodium channels. Previously, mutation F⁴ⁱ¹⁵A in a cockroach sodium channel, BgNa_v1-1a, has been shown to reduce the action of lidocaine, a LA, but not the action of SCBIs. In this study, we introduced mutations F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸A/I individually into the cockroach sodium channel, BgNa_v1-1a, and conducted functional analysis of the three mutants in Xenopus oocytes. We found that both the F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations reduced the inhibition of sodium current by indoxacarb, DCJW (an active metabolite of indoxacarb) and metaflumizone. F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations also reduced the use-dependent block of sodium current by lidocaine. In contrast, substitution V⁴ⁱ¹⁸A enhanced the action metaflumizone and lidocaine. These results show that both F⁴ⁱ¹⁵Y and V⁴ⁱ¹⁸I mutations may contribute to target-site resistance to SCBIs, and provide the first molecular evidence for common amino acid determinants on insect sodium channels involved in action of SCBIs and LA.     

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke

A novel series of pyrrolidine derivatives as Na⁺ channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na⁺ channels. Structure–activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na⁺ channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke.     

Pore properties of rat brain II sodium channels mutated in the selectivity filter domain

Ion selectivity of voltage-activated sodium channels is determined by amino-acid residues in the pore regions of all four homologous repeats. The major determinants are the residues DEKA (for repeats I-IV) which form a putative ring structure in the pore; the homologous structure in Ca-channels consists of EEEE. By combining site-directed mutagenesis of a non-inactivating form of the rat brain sodium channel II with electrophysiological methods, we attempted to quantify the importance of charge, size, and side-chain position of the amino-acid residues within this ring structure on channel properties such as monovalent cation selectivity, single-channel conductance, permeation and selectivity of divalent cations, and channel block by extracellular Ca²⁺ and tetrodotoxin (TTX). In all mutant channels tested, even those with the same net charge in the ring structure as the wild type, the selectivity for Na⁺ and Li⁺ over K⁺, Rb⁺, Cs⁺, and NH₄ ⁺ was significantly reduced. The changes in charge did not correlate in a simple fashion with the single-channel conductances. Permeation of divalent ions (Ca²⁺, Ba²⁺, Sr²⁺, Mg²⁺, Mn²⁺) was introduced by some of the mutations. The IC₅₀ values for the Ca²⁺ block of Na⁺ currents decreased exponentially with increasing net negative charge of the selectivity ring. The sensitivity towards channel block by TTX was reduced in all investigated mutants. Mutations in repeat IV are an exception as they caused smaller effects on all investigated channel properties compared with the other repeats. Received: 24 July 1996 / Accepted: 12 September 1996     

The discovery of benzenesulfonamide-based potent and selective inhibitors of voltage-gated sodium channel Nav1.7

The voltage gated sodium channel Na_v1.7 represents an interesting target for the treatment of pain. Human genetic studies have identified the crucial role of Na_v1.7 in pain signaling. Herein, we report the design and synthesis of a novel series of benzenesulfonamide-based Na_v1.7 inhibitors. Structural–activity relationship (SAR) studies were undertaken towards improving Na_v1.7 activity and minimizing CYP inhibition. These efforts resulted in the identification of compound 12k, a highly potent Na_v1.7 inhibitor with a thousand-fold selectivity over Na_v1.5 and negligible CYP inhibition.     

BACE1 modulates filopodia-like protrusions induced by sodium channel beta4 subunit

Processing of APP by BACE1 plays a crucial role in the pathogenesis of Alzheimer disease (AD). Recently, the voltage-gated sodium channel (Na_v) β4 subunit (β4), an auxiliary subunit of Na_v that is supposed to serve as a cell adhesion molecule, has been identified as a substrate for BACE1. However, the biological consequence of BACE1 processing of β4 remains illusive. Here, we report the biological effects of β4 processing by BACE1. Overexpression of β4 in Neuro2a cells promoted neurite extension and increased the number of F-actin rich filopodia-like protrusions. While coexpression of BACE1 together with β4 further accelerated neurite extension, the number of filopodia-like protrusions was reduced. Overexpression of C-terminal fragment of β4 that was generated by BACE1 (β4-CTF) partially recapitulated the results obtained with BACE1 overexpression. These results suggest that the processing of β4 by BACE1 regulates neurite length and filopodia-like protrusion density in neurons.     

The uptake and metabolism of urea by Chara australis: IV. Symport with sodium—A slip model for the high and low affinity systems

We have previously investigated the electrogenic influx of urea in Chara, and the urea- and sodium-dependent membrane current. We have shown that there is a sodium-stimulated component of urea influx and a urea-stimulated component of sodium influx, and that these are of the same size. We conclude that the electrogenic inward transport of urea, and of its analogues acetamide and acrylamide, is by sodium symport, with a stoichiometric ratio of 1∶1. The kinetics of the fluxes and currents show two different K _M values for sodium in different cells and two different kinds of kinetics for the effect of urea on membrane current, one of which fits the Michaelis-Menten equation, while the other shows a maximum and fits the difference of two Michaelis-Menten terms, suggesting a phenomenon like cis-inhibition. Similarities in kinetic characteristics between the inhibitory site and the electrically silent uptake site (System II) lead us to suggest that the same protein may be responsible for both the low-K _M, electrogenic influx of urea (System I) and the high-K _M, electrically silent influx by System II. We suggest a “slip” model for urea uptake in Chara.     

Structure and activity relationship in the (S)-N-chroman-3-ylcarboxamide series of voltage-gated sodium channel blockers

Recent findings showing a relation between mutations in the Na(V)1.7 channel in humans and altered pain sensation has contributed to increase the attractiveness of this ion channel as target for development of potential analgesics. Amido chromanes 1 and 2 were identified as blockers of the Na(V)1.7 channel and analogues with modifications of the 5-substituent and the carboxamide part of the molecule were prepared to establish the structure-activity relationship. Compounds 13 and 29 with good overall in vitro and in vivo rat PK profile were identified. Furthermore, 29 showed in vivo efficacy in a nociceptive pain model.     

A highly predictive 3D-QSAR model for binding to the voltage-gated sodium channel: Design of potent new ligands

A comprehensive comparative molecular field analysis (CoMFA) model for the binding of ligands to the neuronal voltage-gated sodium channel was generated based on 67 diverse compounds. Earlier published CoMFA models for this target provided μM ligands, but the improved model described here provided structurally novel compounds with low nM IC₅₀. For example, new compounds 94 and 95 had IC₅₀ values of 129 and 119 nM, respectively.     

Structural determinants for the action of grayanotoxin in D1 S4-S5 and D4 S4-S5 intracellular linkers of sodium channel alpha-subunits

We located a novel binding site for grayanotoxin on the cytoplasmic linkers of voltage-dependent cardiac (rH1) or skeletal-muscle (mu 1) Na(+) channel isoforms (segments S4-S5 in domains D1 and D4), using the alanine scanning substitution method. GTX-modification of Na(+) channels, transiently expressed in HEK 293 cells, was evaluated under whole-cell voltage clamp, from the ratio of maximum chord conductance for modified and unmodified Na(+) channels. In mu 1, mutations K237A, L243A, S246A, K248A, K249A, L250A, S251A, or T1463A, caused a moderate, but statistically significant decrease in this ratio. On making corresponding mutations in rH1, only L244A dramatically reduced the ratio. Because in mu 1, the serine at position 251 is the only heterologous residue with respect to rH1 (Ala-252), we made a double mutant L243A&S251A to match the sequence of mu 1 and rH1 in S4-S5 linkers of both domains. This double mutation resulted in a significant decrease in the ratio, to the same extent as L244A substitution in rH1 did, indicating that the site at Leu-244 in rH1 or at Leu-243 in mu 1 is a novel one, exhibiting a synergistic effect of grayanotoxin.     

Phenyl isoxazole voltage-gated sodium channel blockers: structure and activity relationship

Blocking of certain sodium channels is considered to be an attractive mechanism to treat chronic pain conditions. Phenyl isoxazole carbamate 1 was identified as a potent and selective Na_V1.7 blocker. Structural analogues of 1, both carbamates, ureas and amides, were proven to be useful in establishing the structure-activity relationship and improving ADME related properties. Amide 24 showed a good overall in vitro profile, that translated well to rat in vivo PK.     

Crystal structure analysis of human serum albumin complexed with sodium 4-phenylbutyrate

Sodium 4-phenylbutyrate (PB) is an orphan drug for the treatment of urea cycle disorders. It also inhibits the development of endoplasmic reticulum stress, the action of histone deacetylases and as a regulator of the hepatocanalicular transporter. PB is generally considered to have the potential for use in the treatment of the diseases such as cancer, neurodegenerative diseases and metabolic diseases. In a previous study, we reported that PB is primarily bound to human serum albumin (HSA) in plasma and its binding site is drug site 2. However, details of the binding mode of PB to HSA remain unknown. To address this issue, we examined the crystal structure of HSA with PB bound to it. The structure of the HSA–PB complex indicates that the binding mode of PB to HSA is quite similar to that for octanoate or drugs that bind to drug site 2, as opposed to that for other medium-chain length of fatty acids. These findings provide useful basic information related to drug–HSA interactions. Moreover, the information presented herein is valuable in terms of providing safe and efficient treatment and diagnosis in clinical settings.     

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

δ-Atracotoxins (δ-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. We have isolated and determined the amino acid sequence of a novel δ-ACTX, designated δ-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with δ-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-clamp conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current amplitude accompanied by a slowing of sodium current inactivation similar to other δ-ACTXs. However, δ-ACTX-Hv1b is approximately 15–30-fold less potent than other δ-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between δ-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.     

Simultaneous use of sodium nitrate and urea as nitrogen sources improves biomass composition of Arthrospira platensis cultivated in a tubular photobioreactor

Arthrospira platensis is widely cultivated in open ponds for industrial purposes. However, high‐protein A. platensis biomass produced in photobioreactors (PBRs) is recommended for pharmaceutical and cosmetic formulations. A. platensis was cultivated in a 3.5 L tubular airlift PBR using both sodium nitrate and urea as nitrogen sources. Sodium nitrate was added from the start of the cultivation using a batch process. Urea was supplied daily at exponentially increasing feeding rate using a fed‐batch process. The simultaneous optimization of the independent variables, namely, total quantity of sodium nitrate (m_T1) and total quantity of urea (m_T2), led to an optimal condition of m_T1 = 15.0 mmol/L and m_T2 = 7.5 mmol/L. Maximum biomass concentration (5183 ± 94 mg/L) corresponding to the highest biomass productivity (683 ± 13 mg/L/day) was obtained under such condition. The addition protocol of both nitrogen sources resulted in high productivities of protein (6.2 ± 0.4 mg/L/day) as well as chlorophyll‐a (372.2 ± 7.7 mg/L/day). Such innovative process could be applied in the large‐scale production of A. platensis using tubular PBR for novel applications.     

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins

Scorpion toxins have been found lacking effect on Na(+) current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3-S4 constituting receptor site-3, and for receptor site-4, two residues (Leu-Pro) are inserted near IIS4 of scorpion sodium channel.     

Synthesis and decoloring properties of sodium humate/poly (N-isopropylacrylamide) hydrogels

A series of novel sodium humate/poly(N-isopropylacrylamide) (SH/PNIPA) hydrogels were synthesized by solution polymerization. The swelling and decoloring properties of SH/PNIPA hydrogels were also examined. Experiment results show that there exist hydrogen-bonding interactions between SH and PNIPA in the SH/PNIPA hydrogels network, which are not strong enough to disrupt the aggregation of dehydrated PNIPA chains at phase transition temperature, leading to the same volume phase transition temperature as pure PNIPA hydrogel. The adsorption and desorption of methylene blue (MB) for the hydrogels were influenced by temperature, initial MB concentration and SH amount. Low temperature favors the adsorption and desorption of MB. Appropriate SH amount of the hydrogels is crucial for the adsorption and desorption of MB. The maximum adsorption capacity was 10.8 mg MB per gram of SH/PNIPA gel.     

Formation and properties of polyelectrolyte complexes of chitosan hydrochloride and sodium dextransulfate

Methods of potentiometry, turbidimetry, colorimetry, IR spectroscopy, and element analysis were used to investigate the conditions of formation and the properties of non-stoichiometric polyelectrolyte complexes of chitosan hydrochloride (CHC) and sodium dextransulfate (the molecules of both polysaccharides appear as semirigid chains, but their charges are opposite). It was determined that the complexes' formation of polyelectrolytes studied is predominantly electrostatic in the presence of urea. As was also found turbidity and stability of the polycomplexes solutions depended markedly on pH value of CHC and a nature of the low-molecular-weight salts added. The complexes obtained were soluble in water, aqueous urea, and water-organic mixtures. The extent of solubility depended on the composition of the complexes and could be influenced by addition of appropriate concentrations of certain low-molecular-weight salts.     

Synthesis and biological evaluation of 4-piperidinecarboxylate and 4-piperidinecyanide derivatives for T-type calcium channel blockers

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-piperidinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and in vivo activity against α(1G) calcium channel. Among them, several compounds showed good T-type calcium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at 10 μM=61.85-71.99, hERG channel IC(50)=1.57 ± 0.14-4.98 ± 0.36 μM). Selected compound 31a was evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia.     

4-Thia-trans-2-alkenoyl-CoA derivatives: properties and enzymatic reactions

4-Thiaacyl-CoA analogues, in which the 4-methylene group is replaced by a thioether sulfur atom, represent new chromophoric substrates of acyl-CoA dehydrogenases and oxidase. The corresponding 4-thia-trans-2-enoyl-CoA products exhibit a strong new absorption band (extinction coefficient 22 mM-1 cm-1) that is red shifted from 312 to 338 nm upon binding to the medium-chain acyl-CoA dehydrogenase. 4-Thiaoctanoyl-CoA reduces the dehydrogenase several-fold slower than octanoyl-CoA, although in turnover it is dehydrogenated 1.5-fold faster. The redox potential of 4-thia analogues is some 30 mV more negative than that of their unsubstituted counterparts. 4-Thia-trans-2-enoyl-CoA derivatives are slowly hydrated by enoyl-CoA hydratase (EC 4.2.1.17) to the corresponding thiohemiacetal which fragments nonenzymatically to 1 equiv each of malonylsemialdehyde-CoA and alkanethiol. This fragmentation reaction might explain the release of methanethiol during the transamination pathway of methionine degradation. 4-Oxaoctanoyl-CoA is a much poorer substrate and kinetic reductant of acyl-CoA dehydrogenase and oxidase than the 4-thia analogue. The corresponding enoyl-CoA product is also fragmented by the hydratase, yielding butanol and malonylsemialdehyde-CoA. Thus, 4-heterosubstituted acyl-CoA derivatives provide new tools for the study of beta-oxidation enzymes.