Functional effects of drugs and toxins interacting with NaV1.4.

NaV1.4 is a voltage-gated sodium channel subtype that is predominantly expressed in skeletal muscle cells. It is essential for producing action potentials and stimulating muscle contraction, and mutations in NaV1.4 can cause various muscle disorders. The discovery of the cryo-EM structure of NaV1.4 in complex with ß1 has opened new possibilities for designing drugs and toxins that target NaV1.4. In this review, we summarize the current understanding of channelopathies, the binding sites and functions of chemicals including medicine and toxins that interact with NaV1.4. These substances could be considered novel candidate compounds or tools to develop more potent and selective drugs targeting NaV1.4. Therefore, studying NaV1.4 pharmacology is both theoretically and practically meaningful.

The Inhibitory Effect of Magnolol on the Human TWIK1 Channel Is Related to G229 and T225 Sites.

TWIK1 (K2P1.1/KCNK1) belongs to the potassium channels of the two-pore domain. Its current is very small and difficult to measure. In this work, we used a 100 mM NH4+ extracellular solution to increase TWIK1 current in its stable cell line expressed in HEK293. Then, the inhibition of magnolol on TWIK1 was observed via a whole-cell patch clamp experiment, and it was found that magnolol had a significant inhibitory effect on TWIK1 (IC50 = 6.21 ± 0.13 µM). By molecular docking and alanine scanning mutagenesis, the IC50 of TWIK1 mutants G229A, T225A, I140A, L223A, and S224A was 20.77 ± 3.20, 21.81 ± 7.93, 10.22 ± 1.07, 9.55 ± 1.62, and 7.43 ± 3.20 µM, respectively. Thus, we conclude that the inhibition of the TWIK1 channel by magnolol is related to G229 and T225 on the P2- pore helix.

The Strength of hERG Inhibition by Erythromycin at Different Temperatures Might Be Due to Its Interacting Features with the Channels.

Erythromycin is one of the few compounds that remarkably increase ether-a-go-go-related gene (hERG) inhibition from room temperature (RT) to physiological temperature (PT). Understanding how erythromycin inhibits the hERG could help us to decide which compounds are needed for further studies. The whole-cell patch clamp technique was used to investigate the effects of erythromycin on hERG channels at different temperatures. While erythromycin caused a concentration-dependent inhibition of cardiac hERG channels, it also shifted the steady-state activation and steady-state inactivation of the channel to the left and significantly accelerated the onset of inactivation at both temperatures, although temperature itself caused a profound change in the dynamics of hERG channels. Our data also suggest that the binding pattern to S6 of the channels changes at PT. In contrast, cisapride, a well-known hERG blocker whose inhibition is not affected by temperature, does not change its critical binding sites after the temperature is raised to PT. Our data suggest that erythromycin is unique and that the shift in hERG inhibition may not apply to other compounds.

The intermolecular polar bond interaction and coupling induced spectral splitting phenomenon for a binary mixture.

To explain the polarization Raman noncoincidence effect of specific polar bonds and the noncoincidence phenomenon between FT-Raman and FT-IR spectra, aggregation-induced spectral splitting theory was proposed. In this paper, the vibration splitting theory was demonstrated using two strategies: improving the spectral resolution with cryogenic matrix isolation techniques and identifying cases where the coupling splitting is large enough to be distinguishable. The monomer and dimer splitting bands of acetone were detected when cryogenically isolated by the Ar matrix. Additionally, the polarization Raman and two-dimensional infrared spectra of a ß-propiolactone (PIL)/CCl4 binary mixture were collected at room temperature, and the spectral splitting phenomenon was clearly observed. The dynamic transformation between the monomer and dimer could be achieved and detected by adjusting the PIL concentration. The observed splitting phenomenon was further confirmed by theoretical DFT calculations based on the monomer and dimer of PIL, as well as the FT-IR and FT-Raman spectra of PIL. Concentration-triggered 2D-COS synchronous and asynchronous spectra also confirmed the splitting phenomenon and the dilution kinetics of PIL/CCl4.

Inhibitory Effects of 2-Aminoethoxydiphenyl Borate (2-APB) on Three KV1 Channel Currents.

2-Aminoethoxydiphenyl borate (2-APB), a boron-containing compound, is a multitarget compound with potential as a drug precursor and exerts various effects in systems of the human body. Ion channels are among the reported targets of 2-APB. The effects of 2-APB on voltage-gated potassium channels (KV) have been reported, but the types of KV channels that 2-APB inhibits and the inhibitory mechanism remain unknown. In this paper, we discovered that 2-APB acted as an inhibitor of three representative human KV1 channels. 2-APB significantly blocked A-type Kv channel KV1.4 in a concentration-dependent manner, with an IC50 of 67.3 µM, while it inhibited the delayed outward rectifier channels KV1.2 and KV1.3, with IC50s of 310.4 µM and 454.9 µM, respectively. Further studies on KV1.4 showed that V549, T551, A553, and L554 at the cavity region and N-terminal played significant roles in 2-APB's effects on the KV1.4 channel. The results also indicated the importance of fast inactivation gating in determining the different effects of 2-APB on three channels. Interestingly, a current facilitation phenomenon by a short prepulse after 2-APB application was discovered for the first time. The docked modeling revealed that 2-APB could form hydrogen bonds with different sites in the cavity region of three channels, and the inhibition constants showed a similar trend to the experimental results. These findings revealed new molecular targets of 2-APB and demonstrated that 2-APB's effects on KV1 channels might be part of the reason for the diverse bioactivities of 2-APB in the human body and in animal models of human disease.

The Mechanism of Crocetin Targeting Cardiovascular Disease Based on Network Pharmacology Constrained by Spectral Experiments.

The network pharmacology under conditions is a recent development trend. We use network pharmacology methods to analyze the mechanism of crocetin (CRO) that regulates cardiovascular diseases. In this work, the spectral experimental data of CRO-Protein interaction is first time combined with constraint conditions to solve the problems of targeting redundancy and lack of verification. CRO targets and cardiovascular disease targets were obtained by the target database. The STRING platform was used for PPI analysis. The GO and KEGG pathways of the target were analyzed using the Metascape platform; The core functional targets of CRO were screened by molecular docking techniques and the spectra of CRO and human serum albumin (HSA). Under the collaborative constraint conditions, the core targets of CRO that regulate cardiovascular diseases are ADRA1A, ADRA1B, CHRM1, CHRM2, GABRA1, and PTGS2; This study incorporates spectroscopy and molecular docking as constraints into the network pharmacological analysis, which significantly improves the credibility of network pharmacological analysis compared with unconstrained conditions. This method provides theoretical references for the in-depth study of the mechanism between active substances and protein targets for other medicines in network pharmacology.

Delivery Mechanism of the Pharmaceutical Complex of Genistein-Adenine Based on Spectroscopic and Molecular Modelling at Atomic Scale.

Genistein (GS) exhibits various biological activities, but its clinical application is limited because of the low bioavailability. In this study, a GS-adenine pharmaceutical complex was prepared through solvent evaporation to improve the bioavailability of GS, and a molecular model of a two-component supramolecular pharmacological transport mechanism was established. The structure of GS-adenine was characterized, in addition, interaction patterns between GS and adenine were investigated using density functional theory. The results showed that the solubility of GS-adenine was five times higher than that of GS, and the cumulative release rate of GS-adenine was 86 %. The results of fluorescence spectroscopy and molecular dynamic simulations showed that GS-adenine bound to the Sudlow's site I of HSA mainly through hydrophobic interactions. This study provides a useful reference for synthesizing pharmaceutical complexes to improve solubility and for exploring the mechanism of multiple pharmaceutical components in vivo.

Cardiac toxicity of Triptergium wilfordii Hook F. may correlate with its inhibition to hERG channel.

Tripterygium wilfordii Hook F. (TWHF) is a Chinese traditional medicine with cardiac toxicities. However, the mechanism of acute cardiac toxicity is not very clear. By using patch clamp techniques, we found that 0.05 mg/ml and 0.1 mg/ml of the aqueous crude extract of TWHF inhibit 21.4 ± 1.6% and 86.7 ± 5.7% (n = 5) of hERG current Amplitudes (IhERG) respectively. We further found that Celastrol, one of main components of TWHF, inhibits hERG with an IC50 of 0.83 µM. Additional mutagenesis studies show that mutations of T623A, S624A and F656A significantly alter the inhibition and S624A has the strongest effect, supported by our docking model. Our data suggest that inhibition of hERG channel activity by Celastrol contributed to TWHF cardiotoxicity.

The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts.

Stellera chamaejasme L. is a traditional Chinese medicine with a long history to treat stubborn skin ulcer, and it also has antiviral and antitumor effects. Neochamaejasmine B (NCB), Neochamaejasmine A (NCA) and Chamaechromone (CMC) are the major components in dried roots of Stellera chamaejasme L.. Our studies suggested that NCB, NCA and CMC are inhibitors of Organic anion transporter 1 (OAT1). OAT1 is encoded by solute carrier family 22 member 6 gene (SLC22A6) in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney. Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments. The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L. and lamivudine via OAT1 both in vitro and in vivo. In vitro, the uptake studies in Madin-Darby canine kidney (MDCK) cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC. NCB was a noncompetitive and competitive inhibitor interaction with OAT1. IC50 values of NCB, NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46, 8.35 and 0.61 µmol·L-1, respectively. In vivo, the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve (AUC0-∞) and maximal plasma concentration (Cmax) of lamivudine after co-administration is increased 2.94-fold and 1.87-fold, respectively, compared to lamivudine administration alone. The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L. extracts via OAT1 in vivo are consistent with studies in vitro. The inhibition of OAT1-mediated uptake of lamivudine by NCB, NCA and CMC is the possible mechanism for Stellera chamaejasme L. extracts improving the oral bioavailability of lamivudine in rats.

Neochamaejasmin A inhibits KV1.4 channel activity via direct binding to the pore.

Stellera chamaejasme L. (Thymelaeaceae) is a toxic perennial herb and widespread in Mongolia and the northern parts of China. Previous studies have revealed that Neochamaejasmin A (NCA), one of the main active ingredients in the plant roots, has many bioactivities such as inhibiting the P-gp-mediated efflux. But whether NCA affects ion channels is unknown. Here the whole cell patch clamp technique was used to investigate whether NCA affects ion channels, especially how it inhibits KV1.4. Mutagenesis and structure-based molecular simulation were used for analysis of inhibition mechanism and identification of binding site. Among all the channels assayed, KV1.4 stood out as the one on which NCA showed strongest inhibition activity with IC50 of 7.55 µM. Compared with NCA's isomerides, neochamaejasmin B (NCB) and chamaechromone (CMC), NCA also exhibited superior inhibition ability on KV1.4. Three mutations, V549A, A553V and V560A, occurred inside the pore, were found to significantly alleviate the NCA blocking effects, suggesting that they are the important binding sites of NCA. Structure-based modelling showed that the phenolic hydroxyl group of NCA can form hydrogen bonds with main chains of Val549 and Ala553 in IS6 and IVS6 segment respectively, which support our in vitro results. In conclusion, data suggest that NCA might inhibit KV1.4 channels via direct binding to the pore domain.

Peimine inhibits hERG potassium channels through the channel inactivation states.

BACKGROUND AND OBJECTIVE: Fritillaria is a Chinese traditional herb. It has a long history and many medicinal usages including antitussive, anti-inflammatory and pain relieving actions. It is also used as food. However, its cardiac safety has not been tested. Peimine is one of the main active compounds of Fritillaria. To be listed as an herb in the Chinese Pharmacopoeia, a special minimal percentage of Peimine in the dry sample of Fritillaria is required. The main concern for cardiac safety determination is the possible inhibition of hERG ion channels. Thus, Peimine was chosen to investigate its inhibitory effects on hERG channels. METHODS: Whole cell patch clamp technique was used. RESULTS AND CONCLUSION: We found that Peimine inhibited the hERG peak tail currents in a concentration dependent manner with an IC50 value of 43.7µM (n=4) by whole cell patch clamp techniques. Multiple results suggest that the inhibition was related to the channel inactivation. First, Peimine inhibition was significantly increased when the prepulse voltage was increased from -30mV to +10mV. Second, increasing prepulse length also significantly increased blockade by Peimine. Third, our finding that the inhibition by Peimine was use-dependent is related to changes in the inactivated state of the channel. Finally, the result that Peimine significantly decreased inactivation constant also suggested that Peimine affect the channel inactivation state. Mutation of Y652 to Alanine reduced sensitivity to Peimine, suggesting that Y652 is an important hERG binding sites for Peimine.

Peimine, a main active ingredient of Fritillaria, exhibits anti-inflammatory and pain suppression properties at the cellular level.

Fritillaria is one of the most important herbs in Chinese traditional medicine and represents an annual ï¿¥700 million industry. It is often used as an anti-inflammatory, pain relieving and antitussive medicine. However, the mechanisms of these effects are still unclear. Peimine is one of active ingredients of Fritillaria. Using the patch-clamp technique, we profiled the action of Peimine against selected ion channels stably expressed in HEK 293 cell lines. Our data indicated that Peimine was not only able to block the Nav1.7 ion channel but also preferably inhibited the Kv1.3 ion channel. Thus, the study suggested potential mechanisms of Fritillaria as a pain relieving and anti-inflammatory herb.

Neochamaejasmin B increases the bioavailability of chamaechromone coexisting in Stellera chamaejasme L. via inhibition of MRP2 and BCRP.

Chamaechromone and neochamaejasmin B (NCB) are the most abundant components in the dried roots of the toxic perennial herb Stellera chamaejasme L. and have pharmacological activities. The objective of this study was to investigate the transport mechanism of these two components in vivo and in vitro. The transport and cellular accumulation studies in Madin-Darby canine kidney (MDCK) cells overexpressing human multidrug resistance protein 2 (MRP2) or P-gp and LLC-PK1 cells overexpressing human breast cancer resistance protein (BCRP) were performed. The results showed that chamaechromone was a good substrate of MRP2 and BCRP but not a substrate of P-gp. NCB was found to be a MRP2 inhibitor in transfected cells and significantly enhanced the cellular accumulation of chamaechromone in MDCK cells overexpressing MRP2. Similar results were obtained in LLC-PK1-BCRP cells. In addition, the influence of NCB on the bioavailability of chamaechromone following their co-administration was also determined in rats. The results showed that the area under the plasma concentration-time curve and maximal plasma concentration of chamaechromone in rats were increased by 48.9% and 81.9%, respectively. The mechanism of improving the oral bioavailability of chamaechromone was attributable to the inhibition of the BCRP and MRP2-mediated efflux of chamaechromone by NCB.

Inhibitory effects of neochamaejasmin B on P-glycoprotein in MDCK-hMDR1 cells and molecular docking of NCB binding in P-glycoprotein.

Stellera chamaejasme L. (Thymelaeaceae) is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as "Langdu", which is embodied in the Pharmacopoeia of the P.R. China (2010) as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB) on P-glycoprotein (P-gp, ABCB1, MDR1). Rhodamine-123 (R-123) transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1) mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki' values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki', the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one).

Cyclosporin A affects the bioavailability of ginkgolic acids via inhibition of P-gp and BCRP.

Ginkgolic acids (GAs) in natural product Ginkgobiloba L. are the pharmacological active but also toxic components. Two compounds, GA (C15:1) and GA (C17:1) are the most abundant GAs. In this study, several in vitro and in vivo models were applied to investigate transport mechanism of GAs. A rapid and sensitive LC-MS/MS method for the simultaneous determination of GA (C15:1) and GA (C17:1) was applied to analyze the biological specimens. The Papp(AP→BL) values of GA (C15:1) and GA (C17:1) were 1.66-2.13×10(-)(6)cm/s and 1.34-1.85×10(-)(6)cm/s determined using MDCK and MDCK-MDR1 cell monolayers, respectively. The Papp(BL→AP) were remarkably greater in the MDCK-MDR1 cell line, which were 6.77-11.2×10(-)(6)cm/s for GA (C15:1) and 4.73-5.15×10(-)(6)cm/s for GA (C17:1). Similar results were obtained in LLC-PK1 and LLC-PK1-BCRP cell monolayers. The net efflux ratio of GA (C15:1) and GA (C17:1) in both cell models was greater than 2 and markedly reduced by the presence of Cyclosporin A (CsA) or GF120918, inhibitors of P-gp and BCRP, suggesting that GAs are P-gp and BCRP substrates. The results from a rat bioavailability study also showed that co-administrating CsA intravenously (20mg/kg) could significantly increase GA (C15:1) and GA (C17:1) AUC0-t by 1.46-fold and 1.53-fold and brain concentration levels of 1.43-fold and 1.51-fold, respectively, due to the inhibition of P-gp and BCRP efflux transporters by CsA.

Expression of recombinant human acetylcholinesterase and its application in screening its inhibitors.

This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.