Thermally processed Buthus martensii Karsch scorpions are a traditional Chinese medical material for treating various diseases. However, their pharmacological foundation remains unclear. Here, a new degraded peptide of scorpion toxin was identified in Chinese scorpion medicinal material by proteomics. It was named BmK86-P1 and has six conserved cysteine residues. Homology modeling and circular dichroism spectra experiments revealed that BmK86-P1 not only contained representative disulfide bond-stabilized α-helical and β-sheet motifs but also showed remarkable stability at test temperatures from 20–95 °C. Electrophysiology experiments indicated that BmK86-P1 was a highly potent and selective inhibitor of the hKv1.2 channel with IC₅₀ values of 28.5 ± 6.3 nM. Structural and functional dissection revealed that two residues of BmK86-P1 (i.e., Lys¹⁹ and Ile²¹) were the key residues that interacted with the hKv1.2 channel. In addition, channel chimeras and mutagenesis experiments revealed that three amino acids (i.e., Gln³⁵⁷, Val³⁸¹ and Thr³⁸³) of the hKv1.2 channel were responsible for BmK86-P1 selectivity. This research uncovered a new bioactive peptide from traditional Chinese scorpion medicinal material that has desirable thermostability and Kv1.2 channel-specific activity, which strongly suggests that thermally processed scorpions are novel peptide resources for new drug discovery for the Kv1.2 channel-related ataxia and epilepsy diseases.

Defensins constitute a family of cationic antimicrobial peptides that act against different bacteria; however, global information regarding their antibacterial mechanisms from omics-based analyses is highly limited. In this study, transcriptomics and proteomics were used to explore the antibacterial mechanisms of defensin (BmKDfsin4) originally isolated from a scorpion on a common Gram-positive bacterium. Staphylococcus aureus (AB94004) was treated with BmKDfsin4 for 15, 30, or 45 min based on its ability to moderately inhibit bacterial growth for one hour. Compared with those in the control group, more than 1000 genes and nearly 500 proteins in S. aureus were significantly differentially expressed after BmKDfsin4 treatment. In-depth analysis revealed that BmKDfsin4 significantly upregulated bacterial ribosome-related pathways and ribosomal components. In contrast, BmKDfsin4 also significantly downregulated the synthesis and metabolism pathways of bacterial amino acids. Moreover, BmKDfsin4 inhibited the synthesis pathways of teichoic acid and peptidoglycan, which are the key components of the cell wall in S. aureus. Furthermore, glycolysis and other metabolic processes in S. aureus were markedly reduced by BmKDfsin4. Overall, the global information detected from S. aureus revealed the multiple antibacterial mechanisms of BmKDfsin4, which could encourage the exploration of global bacterial information from the defensin family with high degrees of sequence variability and accelerate the research and development of defensins as new antibacterial agents.

Thermally processed Buthus martensii Karsch scorpion is an important traditional Chinese medical material that has been widely used to treat various diseases in China for over one thousand years. Our recent work showed that thermally processed Buthus martensii Karsch scorpions contain many degraded peptides; however, the pharmacological activities of these peptides remain to be studied. Here, a new degraded peptide, BmTX4-P1, was identified from processed Buthus martensii Karsch scorpions. Compared with the venom-derived wild-type toxin peptide BmTX4, BmTX4-P1 missed some amino acids at the N-terminal and C-terminal regions, while containing six conserved cysteine residues, which could be used to form disulfide bond-stabilized α-helical and β-sheet motifs. Two methods (chemical synthesis and recombinant expression) were used to obtain the BmTX4-P1 peptide, named sBmTX4-P1 and rBmTX4-P1. Electrophysiological experimental results showed that sBmTX4-P1 and rBmTX4-P1 exhibited similar activities to inhibit the currents of hKv1.2 and hKv1.3 channels. In addition, the experimental electrophysiological results of recombinant mutant peptides of BmTX4-P1 indicated that the two residues of BmTX4-P1 (Lys²² and Tyr³¹) were the key residues for its potassium channel inhibitory activity. In addition to identifying a new degraded peptide, BmTX4-P1, from traditional Chinese scorpion medicinal material with high inhibitory activities against the hKv1.2 and hKv1.3 channels, this study also provided a useful method to obtain the detailed degraded peptides from processed Buthus martensii Karsch scorpions. Thus, the study laid a solid foundation for further research on the medicinal function of these degraded peptides.

The molecular interactions between venomous peptides and potassium channels have extensively enriched the knowledge of diverse peptide pharmacology, and the in-depth understanding of general features of the various peptide functions remains a formidable challenge. In this work, the role of peptide basic residues in peptide pharmacology was first investigated. Although the venomous BmK-NSPK peptide had the critically conserved functional residues occurring in its similar and potent potassium channel-inhibiting peptides, it was a remarkably weak inhibitor of potassium channels due to fewer basic residues. Additionally, 1 μM BmK-NSPK only inhibited 1.2 ± 1.0%, 1.7 ± 0.70%, 2.3 ± 0.49% and 5.4 ± 0.70% of hKv1.1, hKv1.2, hKv1.3 and hKv1.6 channel currents. The introduction of one or two basic residues in BmK-NSPK-I15K, BmK-NSPK-I18K, BmK-NSPK-I26K and BmK-NSPK-I18K/I26K could not improve BmK-NSPK activity. The modifications of more than three basic residues were found to continuously improve BmK-NSPK activity, and the corresponding BmK-NSPK-7K and BmK-NSPK-8K mutants could effectively inhibit hKv1.3 channel with IC₅₀ values of 2.04 ± 0.68 nM and 21.5 ± 1.99 nM, respectively. Also, 1 μM BmK-NSPK-7K and BmK-NSPK-8K mutants could inhibit 84.1 ± 7.0% and 84.3 ± 1.8% of hKv1.1 channel currents. In addition, BmK-NSPK-7K and BmK-NSPK-8K mutants were found to differentially inhibit hKv1.6 and chimeric hKv1.3 channels. These findings first highlight the critical role of basic residues in the activity of potassium channel peptide inhibitors and provide novel insight into the diverse peptide pharmacology.