Four immobilized polysaccharide-based chiral stationary phases have been examined for their enantioselectivity on azole analytes using normal phase liquid chromatography. The resolving ability of the individual CSP differed in different mobile phase compositions and separated analytes. This article focuses on the comparison of the enantioselectivity on these columns. The effects of the side chain as well as the polymer backbone of the CSP on chiral discrimination are discussed. To gain a better understanding of the chiral recognition mechanism, detailed computational simulations of Chiralpak IC interacting with eight pairs of enantiomers were carried out using the molecular docking software AutoDock. The docking studies provided information about the binding energies and the conformations of chiral stationary phase–solute complexes. The chirality of an analyte molecule determines the number and strength of the intermolecular interactions which led to the differences in binding energies between the complexes formed by (R)- and (S)-isomers. These differences were in agreement with the observed enantioselectivity in HPLC experiments. It was found that the enantioselective separation of azole analytes on Chiralpak IC was the result of a mixture of hydrogen bond, π–π, hydrophobic and some special interactions while chromatographic retention was mainly affected by the polarity of the mobile phase. Furthermore, the chiral separation was also correlated to the molecular features, as the enantioselectivity of a more rigid molecule was relatively poor. Analytes having hydrophobic chlorine atoms, phenyl groups and hydrogen bond acceptors close to the chiral center were expected to be well resolved.

Buthus martensii Karsch (BmK), is a kind of traditional Chinese medicine, which has been used for a long history for the treatment of many diseases, such as inflammation, pain and cancer. In this study, DKK-SP1/2/3 genes were screened and extracted from the cDNA library of BmK. The DKK-SP1/2/3 were expressed by using plasmid pSYPU-1b in E. coli BL21, and recombinant proteins were obtained by column chromatography. In the xylene-induced mouse ear swelling and carrageenan-induced rat paw swelling model, DKK-SP1 exerted a significant anti-inflammatory effect by inhibiting the expression of Nav1.8 channel. Meanwhile, the release of pro-inflammatory cytokines (COX-2, IL-6) was decreased significantly and the release of anti-inflammatory cytokines (IL-10) were elevated significantly. Moreover, DKK-SP1 could significantly decrease the Nav1.8 current in acutely isolated rat DRG neurons. In the acetic acid-writhing and ION-CCI model, DKK-SP2 displayed significant analgesic activity by inhibiting the expression of the Nav1.7 channel. Moreover, DKK-SP2 could significantly inhibit the Nav1.7 current in the hNav1.7-CHO cells.

The depressant β toxin anti-neuroexcitation peptide (ANEP) from the Chinese scorpion Buthus martensii Karsch has analgesic activity by interacting with receptor site 4 of the voltage-gated sodium channels (VGSCs). Here, with molecular dynamics simulations, we examined the binding modes between ANEP and the site 4 of mice sodium channel 1.7 (mNa_v1.7), a subtype of VGSCs related to peripheral pain. Homology modeling, molecular mechanics, and molecular dynamics in the biomembrane environment were adopted. The results suggested that ANEP bound to the resting site 4 mainly by amino acid residues in the β2–β3 loop and the ‘NC’ domains, and the activate site 4 mainly by amino acid residues in the hydrophobic domain of N-groove and residues in the ‘pharmacophore’. Effects analysis of 14 mutants in the predicted functional domains of ANEP on mouse twisting models showed that the analgesic activity of mutants L15 and E24 of the ‘pharmacophore’, W36, T37, W38, and T39 forming the loop between the β2- and β3-strands and N8, V12, C60, and K64 in the NC domain increased distinctly after these residues were substituted for Ala, respectively. The binding modes and the active sites predicted were consistent with available mutagenesis data, and which is meaningful to understand the related mechanisms of ANEP for Na_v1.7.

Trigeminal neuralgia (TN) is a common type of peripheral neuralgia in clinical practice, which is usually difficult to cure. Common analgesic drugs are difficult for achieving the desired analgesic effect. Syb-prII-1 is a β-type scorpion neurotoxin isolated from the scorpion venom of Buthus martensi Karsch (BmK). It has an important influence on the voltage-gated sodium channel (VGSCs), especially closely related to Nav1.8 and Nav1.9. To explore whether Syb-prII-1 has a good analgesic effect on TN, we established the Sprague Dawley (SD) rats’ chronic constriction injury of the infraorbital nerve (IoN-CCI) model. Behavioral, electrophysiological, Western blot, and other methods were used to verify the model. It was found that Syb-prII-1 could significantly relieve the pain behavior of IoN-CCI rats. After Syb-prII-1 was given, the phosphorylation level of the mitogen-activated protein kinases (MAPKs) pathway showed a dose-dependent decrease after IoN-CCI injury. Moreover, Syb-prII-1(4.0 mg/kg) could significantly change the steady-state activation and inactivation curves of Nav1.8. The steady-state activation and inactivation curves of Nav1.9 were similar to those of Nav1.8, but there was no significant difference. It was speculated that it might play an auxiliary role. The binding mode, critical residues, and specific interaction type of Syb-prII-1 and VSD2^rNav1.8 were clarified with computational simulation methods. Our results indicated that Syb-prII-1 could provide a potential treatment for TN by acting on the Nav1.8 target.