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Novel degraded potassium channel-modulatory peptides were recently found in thermally processed scorpions, but their pharmacological properties remain unclear. Here, we identified a full-length scorpion toxin (i.e., BmKcug2) and its four truncated analogs (i.e., BmKcug2-P1, BmKcug2-P2, BmKcug2-P3 and BmKcug2-P4) with three conserved disulfide bonds in processed scorpion medicinal material by mass spectrometry. The pharmacological experiments revealed that the recombinant BmKcug2 and BmKcug2-P1 could selectively inhibit the human Kv1.2 and human Kv1.3 potassium channels, while the other three analogs showed a much weaker inhibitory effect on potassium channels. BmKcug2 inhibited hKv1.2 and hKv1.3 channels, with IC50 values of 45.6 ± 5.8 nM and 215.2 ± 39.7 nM, respectively, and BmKcug2-P1 inhibited hKv1.2 and hKv1.3, with IC50 values of 89.9 ± 9.6 nM and 1142.4 ± 64.5 nM, respectively. The chromatographic analysis and pharmacological properties of BmKcug2 and BmKcug2-P1 boiled in water for different times further strongly supported their good thermal stability. Structural and functional dissection indicated that one amino acid, i.e., Tyr36, determined the differential affinities of BmKcug2 and four BmKcug2 analogs. Altogether, this research investigated the different pharmacological properties of BmKcug2 and its truncated analogs, and the findings highlighted the diversity of K+ channel blockers from various scorpion species through thermal processing.

Increasing number of resistant bacteria have emerged with the overuse of antibiotics, which indicates that the bacterial infection has become a global challenge. Furthermore, the pollution of antibiotics to the environment has become a serious threat to public health. It is known that toxins produced by bacteria are the main cause of bacterial infections. Photothermal therapy is an effective antibacterial approach. However, the photothermal reagents cannot eliminate bacterial toxins, and even some anti-bacterial materials are toxic. Here, we synthesized a biomimetic recycled nanoparticle, red blood cell (RBC) membrane-coated Fe3O4 nanoparticles (RBC@Fe3O4), as an antibacterial agent. The RBC@Fe3O4 nanoparticles act as nano-sponges to trap toxins and then kill them all with a photothermal effect. We can describe this process simply as a battle between two armies. Our strategy is to disarm the "enemy" so that we can easily kill the "enemy" who has no power, which results in enhancing the bactericidal efficacy. The toxin of methicillin-resistant Staphylococcus aureus (MRSA) was absorbed by RBC@Fe3O4in vitro. In addition, in vivo studies proved that the RBC@Fe3O4 nanoparticles confer obvious survival benefits against toxin-induced lethality by absorbing the toxin of MRSA. Furthermore, using a mouse model of MRSA wound infection, the RBC@Fe3O4 nanoparticles with laser irradiation were found to have a superior wound-healing effect. Simultaneously, the RBC@Fe3O4 nanoparticles could be recycled in a simple way without affecting the bactericidal efficacy. The highly biocompatible and recyclable RBC@Fe3O4 biomimetic nanoparticles based on photothermal therapy and bacterial toxin adsorption strategy are promising for treating bacterial infections.

Phosphorus is a necessary component of all living organisms. This nutrient is mainly transported from the maternal blood to the fetus via the placenta, and insufficient phosphorus availability via the placenta disturbs the normal development of the fetus, especially fetal bone formation in late gestation. Key proteins (phosphate transporters and exporters) that are responsible for the maintenance of placental-fetal phosphorus homeostasis have been identified. A deficiency in the phosphate transporter Pit2 has been shown to result in placental calcification and the retardation of fetal development in mice. What roles does XPR1 (the only known phosphate exporter) play in maintaining placental-fetal phosphorus homeostasis? In this study, we found that Xpr1 expression is strong in the murine placenta and increases with age during gestation. We generated a global Xpr1 knockout mouse and found that heterozygous (Xpr1+/- ) and homozygous (Xpr1-/- ) fetuses have lower inorganic phosphate (Pi) levels in amniotic fluid and serum and a decreased skeletal mineral content. Xpr1-deficient placentas show abnormal Pi exchange during gestation. Therefore, Xpr1 deficiency in the placenta disrupts placental-fetal Pi homeostasis. We also discovered that the placentas of the Xpr1+/- and Xpr1-/- embryos are severely calcified. Mendelian inheritance statistics for offspring outcomes indicated that Xpr1-deficient embryos are significantly reduced in late gestation. In addition, Xpr1-/- mice die perinatally and a small proportion of Xpr1+/- mice die neonatally. RNA sequence (RNA-Seq) analysis of placental mRNA revealed that many of the transcripts are significantly differentially expressed due to Xpr1 deficiency and are linked to dysfunction of the placenta. This study is the first to reveal that XPR1 plays an important role in maintaining placental-fetal Pi homeostasis, disruption of which causes severe placental calcification, delays normal placental function, and restricts fetal growth. © 2019 American Society for Bone and Mineral Research.

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.

Scorpion venom is a mixture of bioactive peptides, among which neurotoxins and antimicrobial peptides serve especially vital functions. Scorpion venom peptides in Buthidae species have been well described, but toxic peptides from non-Buthidae species have been under-investigated. Here, an antimicrobial peptide gene, Ctri9594, was cloned and functionally identified from the venom of the scorpion Chaerilus tricostatus. The precursor nucleotide sequence of Ctri9594 is 199 nt in length, including a 43 nt 5′ UTR, 115 nt 3′ UTR and 210 nt ORF. The ORF encodes 69 amino acid residues, containing a 21 aa signal peptide, 14 aa mature peptide, 3 aa C-terminal posttranslational processing signal and 31 aa propeptide. Multiple sequence alignment and evolutionary analyses show that Ctri9594 is an antimicrobial peptide in scorpion venom. The mature peptide of Ctri9594 was chemically synthesized with a purity greater than 95% and a molecular mass of 1484.4 Da. Minimum inhibitory concentrations (MICs) indicate that the synthesized mature peptide of Ctri9594 has inhibitory activity against Gram-positive bacteria (Bacillus thuringensis, Bacillus subtilis, Staphylococcus aureus and Micrococcus luteus) but not Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) or a fungus (Candida albicans). The antimicrobial mechanism of Ctri9594 is inferred to be related to its amphiphilic α-helix structure.

The scorpion venom system plays a critical role in capturing prey and defending against predators. In this study, the rapid developmental process of the first instar telson was first presented. The small amount of venom in the first instar could be stored well by the distorted and blocked venom ducts, which disappeared in the older scorpions. This special developmental process of the first instar telson revealed the notable survival ability of scorpions.

International audience; Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to more than 150 million infections and about 3.1 million deaths up to date. Currently, drugs screened are urgently aiming to block the infection of SARS-CoV-2. Here, we explored the interaction networks of kinase and COVID-19 crosstalk, and identified phosphoinositide 3-kinase (PI3K)/AKT pathway as the most important kinase signal pathway involving COVID-19. Further, we found a PI3K/AKT signal pathway inhibitor capivasertib restricted the entry of SARS-CoV-2 into cells under non-cytotoxic concentrations. Lastly, the signal axis PI3K/AKT/FYVE finger-containing phosphoinositide kinase (PIKfyve)/PtdIns(3,5)P2 was revealed to play a key role during the cellular entry of viruses including SARS-CoV-2, possibly providing potential antiviral targets. Altogether, our study suggests that the PI3K/AKT kinase inhibitor drugs may be a promising anti-SARS-CoV-2 strategy for clinical application, especially for managing cancer patients with COVID-19 in the pandemic era.

Author(s): Cao, Zhijian; Wang, Jing-Lin; McNutt, Patrick Michael; Utkin, Yuri N; Shahbazzadeh, Delavar; Wulff, Heike; Kovacic, Herve; Sabatier, Jean-Marc

Background Tick bites severely threaten human health because they allow the transmission of many deadly pathogens, including viruses, bacteria, protozoa, and helminths. Pruritus is a leading symptom of tick bites, but its molecular and neural bases remain elusive. Objectives This study sought to discover potent drugs and targets for the specific prevention and treatment of tick bite–induced pruritus and arthropod-related itch. Methods We used live-cell calcium imaging, patch-clamp recordings, and genetic ablation and evaluated mouse behavior to investigate the molecular and neural bases of tick bite–induced pruritus. Results We found that 2 tick salivary peptides, IP defensin 1 (IPDef1) and IR defensin 2 (IRDef2), induced itch in mice. IPDef1 was further revealed to have a stronger pruritogenic potential than IRDef2 and to induce pruritus in a histamine-independent manner. IPDef1 evoked itch by activating mouse MrgprC11 and human MRGPRX1 on dorsal root ganglion neurons. IPDef1-activated MrgprC11/X1 signaling sensitized downstream ion channel TRPV1 on dorsal root ganglion neurons. Moreover, IPDef1 also activated mouse MrgprB2 and its ortholog human MRGPRX2 selectively expressed on mast cells, inducing the release of inflammatory cytokines and driving acute inflammation in mice, although mast cell activation did not contribute to oxidated IPDef1–induced itch. Conclusions Our study identifies tick salivary peptides as a new class of pruritogens that initiate itch through MrgprC11/X1-TRPV1 signaling in pruritoceptors. Our work will provide potential drug targets for the prevention and treatment of pruritus induced by the bites or stings of tick and maybe other arthropods.

Interferon-inducible transmembrane proteins (IFITM1/2/3) have been reported to suppress the entry of a wide range of viruses. However, their antiviral functional residues and specific mechanisms are still unclear. Here, we firstly resolved the topology of IFITM1 on the plasma membrane where N-terminus points into the cytoplasm and C-terminus resides extracellularly. Further, KRRK basic residues of IFITM1 locating at 62&ndash, 67 of the conserved intracellular loop (CIL) were found to play a key role in the restriction on the Zika virus (ZIKV) and dengue virus (DENV). Similarly, KRRK basic residues of IFITM2/3 also contributed to suppressing ZIKV replication. Finally, IFITM1 was revealed to be capable of restricting the release of ZIKV particles from endosome to cytosol so as to impede the entry of ZIKV into host cells, which was tightly related with the inhibition of IFITM1 on the acidification of organelles. Overall, our study provided topology, antiviral functional residues and the mechanism of interferon-inducible transmembrane proteins.

Dengue virus (DENV) and Zika virus (ZIKV) have spread throughout many countries in the developing world and infect millions of people every year, causing severe harm to human health and the economy. Unfortunately, there are few effective vaccines and therapies available against these viruses. Therefore, the discovery of new antiviral agents is critical. Herein, a scorpion venom peptide (Smp76) characterized from Scorpio maurus palmatus was successfully expressed and purified in Escherichia coli BL21(DE3). The recombinant Smp76 (rSmp76) was found to effectively inhibit DENV and ZIKV infections in a dose-dependent manner in both cultured cell lines and primary mouse macrophages. Interestingly, rSmp76 did not inactivate the viral particles directly but suppressed the established viral infection, similar to the effect of interferon (IFN)-β. Mechanistically, rSmp76 was revealed to upregulate the expression of IFN-β by activating interferon regulatory transcription factor 3 (IRF3) phosphorylation, enhancing the type-I IFN response and inhibiting viral infection. This mechanism is significantly different from traditional virucidal antimicrobial peptides (AMPs). Overall, the scorpion venom peptide Smp76 is a potential new antiviral agent with a unique mechanism involving type-I IFN responses, demonstrating that natural AMPs can enhance immunity by functioning as immunomodulators. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-018-0068-4) contains supplementary material, which is available to authorized users.

Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain α-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7±6.6nM for rNav1.4, 454.2±50.1nM for hNav1.5 and 30.9±10.3μM for hNav1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNav1.4 and hNav1.5 channels toward a more negative direction and present the typical features of a β-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNav1.4 and hNav1.5 channels. The V1/2 values of the steady-state inactivation were altered to be more positive for rNav1.4 and more negative for hNav1.5. Moreover, the recovery of the hNav1.5 channel from inactivation was more significantly delayed than that of the rNav1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both α- and β-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.

An increasing number of scorpion fossils indicate that the venomous telson developed from the sharp telson in sea scorpions into the extant scorpion-like telson in aquatic scorpions in the Paleozoic Era and then further evolved into the fetal venom system. This hypothesis led us to evaluate the inhibition of scorpion venom-sensitive potassium channels by hemolymph from the scorpion Mesobuthus martensii. Scorpion hemolymph diluted 1:10 inhibited Kv1.1, Kv1.2, Kv1.3 and SK3 potassium channel currents by 76.4%, 90.2%, 85.8%, and 52.8%, respectively. These discoveries encouraged us to investigate the functional similarity between the more ancient defensin ingredients in hemolymph and the evolved neurotoxins in the venom. In addition to the expression of the representative defensin BmKDfsin3 and BmKDfsin5 in both venomous and non-venomous tissues, NMR analysis revealed structural similarities between scorpion defensin and neurotoxin. Functional experiments further indicated that scorpion defensin used the same mechanism as classical neurotoxin to block the neurotoxin-sensitive Kv1.1, Kv1.2, Kv1.3 and SK3 channels. These findings emphasize the likelihood that scorpion defensins evolved into neurotoxins that were adapted to the emergence of the scorpion telson from the sharp telson of sea scorpions into the extant scorpion-like telson in aquatic scorpions in the Paleozoic Era.

Scorpion toxins are well-known as the largest potassium channel peptide blocker family. They have been successfully proven to be valuable molecular probes for structural research on diverse potassium channels. The potassium channel pore region, including the turret and filter regions, is the binding interface for scorpion toxins, and structural features from different potassium channels have been identified using different scorpion toxins. According to the spatial orientation of channel turrets with differential sequence lengths and identities, conformational changes and molecular surface properties, the potassium channel turrets can be divided into the following three states: open state with less hindering effects on toxin binding, half-open state or half-closed state with certain effects on toxin binding, and closed state with remarkable effects on toxin binding. In this review, we summarized the diverse structural features of potassium channels explored using scorpion toxin tools and discuss future work in the field of scorpion toxin-potassium channel interactions.

Scorpions are insensitive to their own venoms, which contain various neurotoxins specific for mammalian or insect ion channels, whose molecular mechanism remains unsolved. Using MmKv1, a potassium channel identified from the genome of the scorpion Mesobuthus martensii, channel kinetic experiments showed that MmKv1 was a classical voltage-gated potassium channel with a voltage-dependent fast activation and slow inactivation. Compared with the human Kv1.3 channel (hKv1.3), the MmKv1 channel exhibited a remarkable insensitivity to both scorpion venom and toxin. The chimaeric channels of MmKv1 and hKv1.3 revealed that both turret and filter regions of the MmKv1 channel were critical for the toxin insensitivity of MmKv1. Furthermore, mutagenesis of the chimaeric channel indicated that two basic residues (Arg399 and Lys403) in the MmKv1 turret region and Arg425 in the MmKv1 filter region significantly affected its toxin insensitivity. Moreover, when these three basic residues of MmKv1 were simultaneously substituted with the corresponding residues from hKv1.3, the MmKv1-R399T/K403S/R425H mutant channels exhibited similar sensitivity to both scorpion venom and toxin to hKv1.3, which revealed the determining role of these three basic residues in the toxin insensitivity of the MmKv1 channel. More strikingly, a similar triad sequence structure is present in all Shaker-like channels from venomous invertebrates, which suggested a possible convergent functional evolution of these channels to enable them to resist their own venoms. Together, these findings first illustrate the mechanism by which scorpions are insensitive to their own venoms at the ion channel receptor level and enrich our knowledge of the insensitivity of scorpions and other venomous animals to their own venoms.

The discovery of human β-defensin 2 (hBD2), as a Kv1.3 channel inhibitor with the unique molecular mechanism and novel immune modulatory function, suggests that human β-defensins are a novel class of channel ligands. Here, the function and mechanism of the human β-defensin 1 (hBD1) binding to potassium channels was investigated. Based on the structural similarity between hBD1 and Kv1.3 channel-sensitive hBD2, hBD1 was found to selectively inhibit human and mouse Kv1.3 channels with IC50 values of 11.8 ± 3.1 μM and 13.2 ± 4.0 μM, respectively. Different from hBD2 modifying Kv1.3 channel activation and increasing activation time constant, hBD1 did not affect the activation feature of both human and mouse Kv1.3 channels. In comparison with hBD2 simultaneously interacting with the extracellular S1-S2 linker and pore region of Kv1.3 channel, the chimeric channel and mutagenesis experiments showed that hBD1 only bound to the extracellular pore region of Kv1.3 channel instead of extracellular S1-S2 linker or S3-S4 linker. Together, these findings enhance knowledge of hBD1 as a new immune-related Kv1.3 channel blocker and highlight the major functional differences between hBD1 and hBD2 to explore in future research.

As an alternative class of antimicrobial agents used to overcome drug-resistant infections, antimicrobial peptides (AMPs) have recently gained significant attention. In this study, we designed an improved antimicrobial peptide, K1K8, based on the molecular template of Hp1404. Compared to the wild-type Hp1404, K1K8 showed an improved antibacterial spectrum in vitro, a lower hemolytic activity, and an enhanced serum stability. Importantly, K1K8 also decreased methicillin-resistant Staphylococcus aureus (MRSA) bacterial counts in the wounded region in a mouse skin infection model. Interestingly, K1K8 did not induce bacterial resistance or non-specific immune response reactions. Moreover, the peptide killed bacterial cells mainly by disrupting the bacterial membrane. In summary, K1K8 has the potential to be used as an improved anti-infection agent for topical use, which opens an avenue that potential anti-infection drugs may be designed and developed from the molecular templates of AMPs.

The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensii Karsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus as well as methicillin-resistant Staphylococcus aureus. Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50 value for the Kv1.3 channel was 510.2 nm. Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins.

Arboviruses, especially Dengue virus (DENV) and Zika virus (ZIKV), have been a severe threat to human health in the last few years due to uncontrollable transmission. There are no approved vaccines or clinical drugs available for use to prevent and treat their infections. Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3) were reported to modulate viral entry, but the antiviral function of these modulators was unknown. Here, we reported that ML-SA1, a TRPML agonist, inhibited DENV2 and ZIKV in vitro in a dose-dependent manner. Time-of-drug-addition experiments showed that ML-SA1 mainly restricted viral entry. Moreover, the selective TRPML3 activator SN-2 was found to share a similar antiviral effect against DENV2 and ZIKV, but the specific TRPML1 agonist MK6-83 was not effective. Although ML-SA1 was further revealed to induce autophagy, its antiviral role was independent of autophagy induction. Finally, ML-SA1 was found to inhibit DENV2 and ZIKV by promoting lysosome acidification and protease activity to cause viral degradation. Together, our study identifies two TRPML agonists, ML-SA1 and SN-2, as potent inhibitors of DENV2 and ZIKV, which may lead to the discovery of new candidates against viruses.

Viral infections still threaten human health all over the world, and many people die from viral diseases every year. However, there are no effective vaccines or drugs for preventing or managing most viral diseases. Thus, the discovery and development of broad-spectrum antiviral agents remain urgent. Here, we expressed and purified a venom peptide, Ev37, from the scorpion Euscorpiops validus in a prokaryotic system. We found that rEv37 can inhibit dengue virus type 2 (DENV-2), hepatitis C virus (HCV), Zika virus (ZIKV), and herpes simplex virus type 1 (HSV-1) infections in a dose-dependent manner at noncytotoxic concentrations, but that it has no effect on Sendai virus (SeV) and adenovirus (AdV) infections in vitro. Furthermore, rEv37 alkalized acidic organelles to prevent low pH–dependent fusion of the viral membrane–endosomal membrane, which mainly blocks the release of the viral genome from the endosome to the cytoplasm and then restricts viral late entry. Taken together, our results indicate that the scorpion venom peptide Ev37 is a broad-spectrum antiviral agent with a specific molecular mechanism against viruses undergoing low pH–dependent fusion activation during entry into host cells. We conclude that Ev37 is a potential candidate for development as an antiviral drug.

The alarming rise in the prevalence of antibiotic resistance among pathogenic bacteria poses a unique challenge for the development of effective therapeutic agents. Antimicrobial peptides (AMPs) have attracted a great deal of attention as a possible solution to the increasing problem of antibiotic-resistant bacteria. Marcin-18 was identified from the scorpion Mesobuthus martensii at both DNA and protein levels. The genomic sequence revealed that the marcin-18 coding gene contains a phase-I intron with a GT-AG splice junction located in the DNA region encoding the N-terminal part of signal peptide. The peptide marcin-18 was also isolated from scorpion venom. A protein sequence homology search revealed that marcin-18 shares extremely high sequence identity to the AMPs meucin-18 and megicin-18. In vitro, chemically synthetic marcin-18 and its homologs (meucin-18 and megicin-18) showed highly potent inhibitory activity against Gram-positive bacteria, including some clinical antibiotic-resistant strains. Importantly, in a mouse acute peritonitis model, these peptides significantly decreased the bacterial load in ascites and rescued nearly all mice heavily infected with clinical methicillin-resistant Staphylococcus aureus from lethal bacteremia. Peptides exerted antimicrobial activity via a bactericidal mechanism and killed bacteria through membrane disruption. Taken together, marcin-18 and its homologs have potential for development as therapeutic agents for treating antibiotic-resistant, Gram-positive bacterial infections.

Since there is a symbiotic and competitive relationship between microorganisms in the same ecological niche, fungal defensins have been found to be important resources for antimicrobial peptides. Here, a fungal defensin, triintsin, was characterized in a clinical isolate of Trichophyton interdigitale from a patient with onychomycosis. The comparison of its genomic and mRNA sequences showed the gene organization and structure of three coding exons separated by two introns. The precursor peptide of triintsin contained 85 amino acid residues, which were composed of three parts including an N-terminal signal domain of 21 residues, a pro-peptide of 47 residues that ended at lysine-arginine and a mature peptide of 38 residues at the C-terminus. The 3D-structure established by homology modeling revealed that triintsin presented a representative typical cysteine-stabilized α-helical and β-sheet fold. The reductive linear peptide of triintsin was obtained by chemical synthesis. After cyclization to form three pairs of disulfide bonds, the oxidative-type peptide displayed broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria but also showed anti-fungal activity. Moreover, triintsin can effectively inhibit the growth of clinical strains. Altogether, the peptide is a human pathogenic fungus-derived defensin with broad-spectrum antimicrobial activity.

Kunitz-type peptides from venomous animals are an important source of lead drug candidates towards human plasmin, a target of protease-associated diseases. However, no Kunitz-type plasmin inhibitor from venomous scorpion has been characterized. Here, we first investigated plasmin inhibiting activities of eight known Kunitz-type scorpion toxins Hg1, BmKTT-1, BmKTT-2, BmKTT-3, LmKTT-1a, LmKTT-1b, LmKTT-1c and BmKPI, and found a new plasmin inhibitor BmKTT-2, a Kunitz-type toxin peptide from the scorpion Buthus martensi karch. Protease inhibitory activity assay showed that BmKTT-2 potently inhibited plasmin with a Ki value of 8.75 ± 2.05 nM. Structure-function relationship studies between BmKTT-2 and plasmin showed that BmKTT-2 is a classical Kunitz-type plasmin inhibitor: Lys13 in BmKTT-2 is the P1 site, and Ala14 in BmKTT-2 is the P1' site. Interestingly, BmKTT-2 has potent inhibiting activities towards three important digestive serine proteases trypsin, chymotrypsin and elastase, suggesting a good stability for administering oral medications. To the best of our knowledge, BmKTT-2 is the first Kunitz-type human plasmin inhibitor from scorpion venom, providing novel insights into drug developments targeting human plasmin protease.

Viral infection is an early stage of its life cycle and represents a promising target for antiviral drug development. Here we designed and characterized three peptide inhibitors of hepatitis C virus (HCV) infection based on the structural features of the membrane-associated p7 polypeptide of HCV. The three peptides exhibited low toxicity and high stability while potently inhibiting initial HCV infection and suppressed established HCV infection at non-cytotoxic concentrations in vitro. The most efficient peptide (designated H2-3), which is derived from the H2 helical region of HCV p7 ion channel, inhibited HCV infection by inactivating both intracellular and extracellular viral particles. The H2-3 peptide inactivated free HCV with an EC50 (50% effective concentration) of 82.11 nm, which is >1000-fold lower than the CC50 (50% cytotoxic concentration) of Huh7.5.1 cells. H2-3 peptide also bound to cell membrane and protected host cells from viral infection. The peptide H2-3 did not alter the normal electrophysiological profile of the p7 ion channel or block viral release from Huh7.5.1 cells. Our work highlights a new anti-viral peptide design strategy based on ion channel, giving the possibility that ion channels are potential resources to generate antiviral peptides.

Peptides with Ascaris-type fold are a new kind of toxins founded from venomous animals recently. Functionally, these unique toxin peptides had been identified as potent protease inhibitors, which was similar to other known Ascaris-type peptides from non-venomous animals. Whether Ascaris-type peptides from venom animals have neurotoxin activities remains unclear. Here, a scorpion toxin SjAPI-2 with Ascaris-type fold was characterized to have a neurotoxin activity, which can selectively inhibit KCNQ1 potassium channel. SjAPI-2 had 62 amino acid residues, including 10 cysteine residues. Charged residue analyses showed that two acidic residues of SjAPI-2 were regionally distributed, and 10 basic residues were distributed widely throughout the whole peptide, which was similar to classical potassium channel toxins. Pharmacological studies confirmed that SjAPI-2 was a selective KCNQ1 potassium channel inhibitor with weak effects on other potassium channels, such as Kv1.1, Kv1.2, Kv1.3, SKCa2, SKCa3, and IKCa channels. Concentration-dependent studies showed that SjAPI-2 inhibited the KCNQ1 potassium channel with an IC 50 of 771.5 ± 169.9 nM. To the best of our knowledge, SjAPI-2 is the first neurotoxin with a unique Ascaris-type fold, providing novel insights into the divergent evolution of neurotoxins from venomous animals.

Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Serine protease inhibitors have been widely discovered from different animal venoms, but most of them belong to Kunitz-type toxin subfamily. Here, by screening scorpion venom gland cDNA libraries, we identified four new non-Kunitz serine protease inhibitors with a conserved Ascaris-type structural fold: Ascaris-type toxins Lychas mucronatus Ascaris-type protease inhibitor (LmAPI), Pandinus cavimanus Ascaris-type protease inhibitor (PcAPI), Pandinus cavimanus Ascaris-type protease inhibitor 2 (PcAPI-2), and Hottentotta judaicus Ascaris-type protease inhibitor (HjAPI). The detailed characterization of one Ascaris-type toxin LmAPI was further carried out, which contains 60 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges. Enzyme and inhibitor reaction kinetics experiments showed that recombinant LmAPI inhibits the activity of chymotrypsin potently with a Ki value of 15.5 nM, but has little effect on trypsin and elastase. Bioinformatics analyses suggested that LmAPI contains unique functional residues "TQD" and might be a useful template to produce specific protease inhibitors. Our results indicated that animal venoms are a natural source of new type of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

Defensins are important components of innate host defence system against bacteria, fungi, parasites and viruses. Here, we predicted six potential defensin genes from the genome of the scorpion Mesobuthus martensii and then validated four genes from them via the combination of PCR and genomic sequence analysis. These four scorpion defensin genes share the same gene organization and structure of two exons and one phase-I intron with the GT-AG rule. Conserved motif and phylogenetic analysis showed that they belonged to the members of the invertebrate cysteine-stabilized α-helix/β-sheet motif defensin (CSαβ) defensin family. All these four CSαβ defensin genes have the expression feature of constitutive transcription (CON) by the whole scorpion infection model, promoter sequence analysis and dual luciferase assays. Further evolution and comparison analysis found that the invertebrate CSαβ defensin genes from most of arachnids and mollusks appear to share the expression pattern of CON, but those from insects and lower invertebrates (nematodes, annelids, cnidarians and sponges) seem to have identical inducible transcription (IND) after being challenged by microorganisms. Together, we identified four scorpion CSαβ defensin genes with the expression feature of CON, and characterized the diversified expression patterns of the invertebrate CSαβ defensin genes, which will shed insights into the evolution of the invertebrate CSαβ defensin genes and their expression patterns.

The effective treatment of autoimmune diseases remains a challenge. Voltage-gated potassium Kv1.3 channels, which are expressed in lymphocytes, are a new therapeutic target for treating autoimmune disease. Consequently, Kv1.3 channel-inhibiting venom-derived peptides are a prospective resource for new drug discovery and clinical application. Area covered: Preclinical and clinical studies have produced a wealth of information on Kv1.3 channel-inhibiting venom-derived peptides, especially from venomous scorpions and sea anemones. This review highlights the advances in screening and design of these peptides with diverse structures and potencies. It focuses on representative strategies for improving peptide selectivity and discusses the preclinical research on those venom-derived peptides as well as their clinical developmental status. Expert opinion: Encouraging results indicate that peptides isolated from the venom of venomous animals are a large resource for discovering immunomodulators that act on Kv1.3 channels. Since the structural diversity of venom-derived peptides determines the variety of their pharmacological activities, the design and optimization of venom-peptides for improved Kv1.3 channel-specificity has been advanced through some representative strategies, such as peptide chemical modification, amino acid residue truncation and binding interface modulation. These advances should further accelerate research, development and the future clinical application of venom-derived peptides selectively targeting Kv1.3 channels.

Hepatitis C virus (HCV) infection is a major worldwide health problem which can cause chronic hepatitis, liver fibrosis and hepatocellular carcinoma (HCC). There is still no vaccine to prevent HCV infection. Currently, the clinical treatment of HCV infection mainly relies on the use of direct-acting antivirals (DAAs) which are expensive and have side effects. Here, BmKDfsin3, a scorpion defensin from the venom of Mesobuthus martensii Karsch, is found to dose-dependently inhibit HCV infection at noncytotoxic concentrations and affect viral attachment and post-entry in HCV life cycle. Further experimental results show that BmKDfsin3 not only suppresses p38 mitogen-activated protein kinase (MAPK) activation of HCV-infected Huh7.5.1 cells, but also inhibits p38 activation of Huh7.5.1 cells stimulated by tumor necrosis factor-&alpha, (TNF-&alpha, ), interleukin-1&beta, (IL-1&beta, ) or lipopolysaccharide (LPS). BmKDfsin3 is also revealed to enter into cells. Using an upstream MyD88 dimerization inhibitor ST2345 or kinase IRAK-1/4 inhibitor I, the inhibition of p38 activation represses HCV replication in vitro. Taken together, a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.

The diverse α-scorpion toxins are invaluable pharmacological tools and potential drugs targeting sodium channels, but the pharmacological profiles of most toxins remains unknown so far. Here, we reported pharmacological activities of two novel α-scorpion toxins LmαTX3 and LmαTX5 from the Lychas mucronatus . Using the expression vector pET-28a, the recombinant LmαTX3 and LmαTX5 were separated by RP-HPLC and identified by MALDI-TOF-MS. Subsequently, sodium channels rNa v 1.2, mNa v 1.4, hNa v 1.5 and hNa v 1.7 were used for evaluating the pharmacological activities of LmαTX3 and LmαTX5 toxins. The electrophysiological experiments showed that both 10 μM recombinant LmαTX3 and LmαTX5 seriously inhibited the fast inactivation of mNa v 1.4 and hNa v 1.5 channels, moderately affected hNa v 1.7 channel, and hardly modulated rNa v 1.2 channel. The dose–response experiments further indicated the EC 50 values of LmαTX3 were 4.98 ± 0.79 μM for mNa v 1.4, 1.23 ± 0.31 μM for hNa v 1.5 and 31.46 ± 2.32 μM for hNa v 1.7 channels, respectively. Similar pharmacological profiles of recombinant LmαTX5 were also observed, and its EC 50 values were 4.53 ± 1.38 μM, 1.03 ± 0.43 μM and 67.62 ± 2.31 μM for mNa v 1.4, hNa v 1.5 and hNa v 1.7, respectively. In addition, the recombinant LmαTX3 from the vector pET-14b had much less effect on the fast inactivation of mNa v 1.4, hNa v 1.5 and hNa v 1.7 channels, which indicated that the expression vector pET-14b likely played a critical role in toxin function. Together, these findings first highlighted that scorpion toxins from L. mucronatus were a new molecular resource of discovering pharmacological probes and prospective drugs targeting sodium channels in the future.

Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.

Scorpions are one of the most ancient groups of terrestrial animals. They have maintained a steady morphology over more than 400 million years of evolution. Their venom arsenals for capturing prey and defending against predators may play a critical role in their ancient and conservative appearance. In the current review, we present the scorpion fauna of China: 53 species covering five families and 12 genera. We also systematically list toxins or genes from Chinese scorpion species, involving eight species covering four families. Furthermore, we review the diverse functions of typical toxins from Chinese scorpion species, involving Na+ channel modulators, K+ channel blockers, antimicrobial peptides and protease inhibitors. Using scorpion species and their toxins from China as an example, we build the bridge between scorpion species and their toxins, which helps us to understand the molecular and functional diversity of scorpion venom arsenal, the dynamic and functional evolution of scorpion toxins, and the potential relationships of scorpion species and their toxins.

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first “map” of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. Biological significance Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known “Combat poison with poison” strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of “combat poison with poison to cure diseases”.

Highlights • Hp1036 and Hp1239 are two new cationic host defense peptides from scorpion venom. • They inhibitory effect on multiple steps of HSV-1 life cycle. • They adopted α-helix structure in approximate membrane environment. • They are virucidal of HSV-1 and destroyed the morphology of HSV-1. • They easily entered Vero cells and reduced the intracellular viral infectivity., Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen that causes severe diseases, but there are not effective and safe drugs in clinical therapy besides acyclovir (ACV) and related nucleoside analogs. In this study, two new venom peptides from the scorpion Heterometrus petersii were identified with effective inhibitory effect on HSV-1 infection in vitro. Both Hp1036 and Hp1239 peptides exhibited potent virucidal activities against HSV-1 (EC50 = 0.43 ± 0.09 and 0.41 ± 0.06 μM, respectively) and effective inhibitory effects when added at the viral attachment (EC50 = 2.87 ± 0.16 and 5.73 ± 0.61 μM, respectively), entry (EC50 = 4.29 ± 0.35 and 4.32 ± 0.47 μM, respectively) and postentry (EC50 = 7.86 ± 0.80 and 8.41 ± 0.73 μM, respectively) steps. Both Hp1036 and Hp1239 peptides adopted α-helix structure in approximate membrane environment and resulted in the destruction of the viral morphology. Moreover, Hp1036 and Hp1239 peptides entered Vero cells and reduced the intracellular viral infectivity. Taken together, Hp1036 and Hp1239 peptides are two anti-viral peptides with effective inhibitory effect on multiple steps of HSV-1 life cycle and therefore are good candidate for development as virucides.

Although numerous Kunitz-type toxins were isolated from snake venom, no bifunctional Kunitz-type snake toxins with protease and potassium channel inhibiting properties have been reported till now. With the help of bioinformatics analyses and biological experiments, we characterized Kunitz-type snake toxin BF9 as a bifunctional peptide. Enzyme and inhibitor reaction kinetics experiments showed that BF9 inhibited α-chymotrypsin with Ki value of 1.8 × 10−8 M. Electrophysiological experiments showed that BF9 inhibited the Kv1.3 potassium channel with an IC50 of 120.0 nM, which demonstrated that serine protease inhibitor BF9 could also inhibit potassium channels. In addition, the key amino acids of BF9 responsible for the unique bifunctional mechanism are further investigated. To the best of our knowledge, BF9 is the first Kunitz-type snake peptide with the unique bifunctionality of potassium channel and serine protease inhibiting properties, providing novel insights into divergent evolution and functional applications of snake Kunitz-type peptides.

Kunitz-type proteins from animal venom are good tools for understanding structure-function relationships between serine proteases and their inhibitors. We used a cDNA library to clone and characterize the Buthus martensi Kunitz-type protease inhibitor (BmKPI) present in the venom gland of the scorpion B. martensi. The gene codes for a signal peptide of 19 residues and a mature peptide of 64 residues. The mature BmKPI peptide possesses a unique cysteine framework reticulated by four disulfide bridges, unlike many other Kunitz-type proteins with three disulfide bridges. The recombinant BmKPI peptide was functionally expressed and showed strong inhibitory activity toward trypsin (Ki 1.8 × 10⁻⁶ M), chymotrypsin (Ki 3.2 × 10⁻⁸ M), and elastase (Ki 1.6 × 10⁻⁷ M). Structure-functional relationship between elastase and BmKPI was further studied. Cysteine mutagenesis experiment showed that the unique disulfide bridge Cys53-Cys61 had little effect on its inhibiting elastase. Molecular dynamics simulation revealed that BmKPI possesses elastase inhibitory active sites similar to the classical Kunitz-type venom peptides, although their cysteine frameworks were different. These results showed that BmKPI is a new multifunctional serine protease inhibitor. To the best of our knowledge, BmKPI is the first functionally characterized Kunitz-type elastase inhibitor derived from scorpion venoms.

The scorpion family Chaerilidae is phylogenetically differentiated from Buthidae. Their venom components are not known, and the evolution of the venom components is not well understood. Here, we performed a transcriptome analysis of the venom glands from two scorpion species, Chaerilus tricostatus and Chaerilus tryznai. Fourteen types of venom peptides were discovered from two species, 10 of which were shared by both C. tricostatus and C. tryznai. Notably, the venom components of Chaerilidae were also found to contain four toxin types (NaTx, β-KTx, Scamp and bpp-like peptides), previously considered to be specific to Buthidae. Moreover, cytolytic peptides were the most abundant toxin type in C. tricostatus, C. tryznai and the family Euscorpiidae. Furthermore, 39 and 35 novel atypical venom molecules were identified from C. tricostatus and C. tryznai, respectively. Finally, the evolutionary analysis showed that the NaTx, β-KTx, and bpp-like toxin types were recruited into the venom before the lineage split between Buthidae and non-Buthidae families. This study provides an integrated understanding of the venom components of the scorpion family Chaerilidae. The family Chaerilidae has a specific venom arsenal that is intermediate between Buthidae and non-Buthidae, which suggests the dynamic evolution of scorpion venom components from Buthidae to non-Buthidae species. Biological Significance This work gave a first overview of the venom components of Chaerilidae scorpions, and discovered large numbers of new toxin molecules, which significantly enriches the molecular diversity of scorpion venom peptides/proteins components. Based on phylogenetic analysis we speculated that the NaTx, β-KTx and bpp-like toxin type genes were recruited into venom before the lineage split between Buthidae and non-Buthidae. By Comparing the toxin types and abundance of the Buthidae, Chaerilidae and non-Buthidae families, we found that the family Chaerilidae has a specific venom arsenal that is intermediate Buthidae and non-Buthidae, which suggests the dynamic evolution of scorpion venom components from Buthidae to non-Buthidae species.

Until now, there are 26 scorpion species of 7 genera of 5 families recorded in Xizang (China). Xizang Autonomous Region (Tibet) is the scorpion biodiversity richest area in China (53 scorpion species of 12 genera of 5 families), also the highest altitude habitat of scorpions in the world. We present information of type specimens, an identification key of the scorpion species from Xizang, the distribution, updated feature pictures, and discussion on the disputed species. The redescriptions of Scorpiops jendeki Kovařík, 2000 (Yunnan) and S. tibetanus Hirst, 1911 (Xizang), comments and feature figures of species of genus Scorpiops are provided for identification.

Recently, peptide drugs have evolved into mainstream therapeutics, representing a significant portion of the pharmaceutical market. However, their bioavailability remains to be improved compared with that of chemical drugs. Here, we screened and identified a new peptide, Ctry2459, from a scorpion venom peptide library that was proven to inhibit hepatitis C virus (HCV) infection via inactivating infectious viral particles. However, Ctry2459 cannot suppress established infection of HCV because of the poor cellular uptake and restriction of endosomes. Based on the molecular template of the Ctry2459 peptide, we designed two histidine-rich peptides (Ctry2459-H2 and Ctry2459-H3) with significantly enhanced cellular uptake and improved intracellular distribution. Moreover, the two mutated peptides, as well as the wild-type peptide Ctry2459, exhibited virucidal activities against HCV. In distinct contrast to the Ctry2459 peptide, the mutated peptides significantly suppressed the established HCV infection at the cellular level but demonstrated lower cytotoxic and hemolytic activities. Our work presents an effective design strategy for optimizing natural antiviral peptides and opens a new avenue for enhancing the bioavailability of peptide drugs.

Non-Buthidae venomous scorpions are huge natural sources of toxin peptides; however, only a few studies have been done to understand their toxin peptides. Herein, we describe three new potential immunomodulating toxin peptides, Ctri18, Ctry68 and Ctry2908, from two non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai. Sequence alignment analyses showed that Ctri18, Ctry68 and Ctry2908 are three new members of the scorpion toxin α-KTx15 subfamily. Electrophysiological experiments showed that Ctri18, Ctry68 and Ctry2908 blocked the Kv1.3 channel at micromole to nanomole levels, but had weak effects on potassium channel KCNQ1 and sodium channel Nav1.4, which indicated that Ctri18, Ctry68 and Ctry2908 might have specific inhibiting effects on the Kv1.3 channel. ELISA experiments showed that Ctri18, Ctry68 and Ctry2908 inhibited IL-2 cytokine secretions of activated T lymphocyte in human PBMCs. Excitingly, consistent with the good Kv1.3 channel inhibitory activity, Ctry2908 inhibited cytokine IL-2 secretion in nanomole level, which indicated that Ctry2908 might be a new lead drug template toward Kv1.3 channels. Together, these studies discovered three new toxin peptides, Ctri18, Ctry68 and Ctry2908, with Kv1.3 channel and IL-2 cytokine-inhibiting activities from two scorpions, C. tricostatus and C. tryznai, and highlighted that non-Buthidae venomous scorpions are new natural toxin peptide sources.

Peptide toxins from venomous animals are natural resources with diverse biological functions and therapeutic potential towards human diseases. For venomous scorpions, many valuable peptide toxins have been discovered from Buthidae scorpions, but few works were done about non-buthidae scorpions. Here, we cloned and characterized the first disulfide-bridged toxin peptide St20 from the non-buthidae scorpion Scorpiops tibetanus. St20 has a putative 23-residue signal peptide, followed by a presumed 34-residue mature peptide including 8 cysteines. Sequence alignments and structural analysis suggested that St20 is a new member of α-KTx23 scorpion toxin subfamily with a conserved CSα/β structural fold. Functional studies showed that St20 inhibited human T lymphocyte surface marker CD69 expression and cytokine IL-2 secretion. Beside this, St20 inhibited two important pro-inflammatory factors TNF-α and IFN-γ secretion in the activated human T lymphocyte. Animal experiments showed that the delayed-type hypersensitivity response in rat autoimmune disease model was ameliorated in the present of peptide toxin St20. Together, our results showed that St20 is the first disulfide-bridged toxin peptide from the non-buthidae scorpion Scorpiops tibetanus with immunosuppressive and anti-inflammatory activities, suggesting that toxins from non-buthidae scorpions might be a new source of peptide drug discovery towards human diseases.

The Kv1.3 channel-acting scorpion toxins usually adopt the conserved anti-parallel β-sheet domain as the binding interface, but it remains challenging to discover some highly selective Kv1.3 channel-acting toxins. In this work, we investigated the pharmacological profile of the Kv1.3 channel-acting BmKTX-D33H, a structural analogue of the BmKTX scorpion toxin. Interestingly, BmKTX-D33H, with its conserved anti-parallel β-sheet domain as a Kv1.3 channel-interacting interface, exhibited more than 1000-fold selectivity towards the Kv1.3 channel as compared to other K⁺ channels (including Kv1.1, Kv1.2, Kv1.7, Kv11.1, KCa2.2, KCa2.3, and KCa3.1). As expected, BmKTX-D33H was found to inhibit the cytokine production and proliferation of both Jurkat cells and human T cells in vitro. It also significantly improved the delayed-type hypersensitivity (DTH) responses, an autoreactive T cell-mediated inflammation in rats. Amino acid sequence alignment and structural analysis strongly suggest that the "evolutionary" Gly11 residue of BmKTX-D33H interacts with the turret domain of Kv1 channels; it appears to be a pivotal amino acid residue with regard to the selectivity of BmKTX-D33H towards the Kv1.3 channel (in comparison with the highly homologous scorpion toxins). Together, our data indicate that BmKTX-D33H is a Kv1.3 channel-specific blocker. Finally, the remarkable selectivity of BmKTX-D33H highlights the great potential of evolutionary-guided peptide drug design in future studies.

Hepatitis B virus (HBV) is a noncytopathic human hepadnavirus that causes acute, chronic hepatitis and hepatocellular carcinoma (HCC). As the clinical utility of current therapies is limited, new anti-HBV agents and sources for such agents are still highly sought after. Here, we report that Mucroporin-M1, a scorpion venom-derived peptide, reduces the amount of extracellular HBsAg, HBeAg, and HBV DNA productions of HepG2.2.15 cells in a dose-dependent manner and inhibits HBV capsid DNA, HBV intracellular RNA replication intermediates and the HBV Core protein in the cytoplasm of HepG2.2.15 cells. Using a mouse model of HBV infection, we found that HBV replication was significantly inhibited by intravenous injection of the Mucroporin-M1 peptide. This inhibitory activity was due to a reduction in HBV promoter activity caused by a decrease in the binding of HNF4α to the precore/core promoter region. Furthermore, we confirmed that Mucroporin-M1 could selectively activate mitogen-activated protein kinases (MAPKs) and lead to the down-regulation of HNF4α expression, which explains the decreased binding of HNF4α to the HBV promoter. Moreover, when the protein phosphorylation activity of the MAPK pathway was inhibited, both HNF4α expression and HBV replication recovered. Finally, we proved that treatment with the Mucroporin-M1 peptide increased phosphorylation of the MAPK proteins in HBV-harboring mice. These results implicate Mucroporin-M1 peptide can activate the MAPK pathway and then reduce the expression of HNF4α, resulting in the inhibition of HBV replication in vitro and in vivo. Our work also opens new doors to discovering novel anti-HBV agents or sources.

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was suc-cessfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5′ UTR and a 171 nt 3′ UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

Antibiotic-resistant microbes, such as methicillin-resistant Staphylococcus aureus , seriously threaten human health. The outbreak of “superbugs” in recent years emphasizes once again the need for the development of new antimicrobial agents or resources. Antimicrobial peptides have an evident bactericidal effect against multidrug-resistant pathogens. In the present study, a new antimicrobial peptide, ctriporin, was cloned and characterized from the venom of the scorpion Chaerilus tricostatus , an animal which has not yet been explored for toxic peptide resources. The MICs of ctriporin against Staphylococcus aureus , Bacillus thuringiensis , Bacillus subtilis , Micrococcus luteus , and Candida albicans are 5 to 20 μg/ml. Meanwhile, it MIC against clinical antibiotic-resistant bacterial strains is 10 μg/ml. Furthermore, the potential for ctriporin to be used as a topical antibiotic for treating staphylococcal skin infections was investigated. External use of the peptide ctriporin dramatically decreased the bacterial counts and cured skin infections in mice. In addition, ctriporin demonstrates antimicrobial efficacy via the bactericidal mechanism of rapid cell lysis. Together, these results suggest the potential of developing ctriporin as a new topical antibiotic.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. There is no vaccine available for HCV, and almost half of patients cannot be cured using standard combination therapy. Thus, new anti-HCV strategies and drugs are urgently needed. Here, the gene encoding a new α-helical peptide, Hp1090, was screened from the venomous gland cDNA library of the scorpion Heterometrus petersii. Structural analysis showed that Hp1090 is an amphipathic α-helical peptide. In vitro HCV RNA inhibitory assays indicated that Hp1090 peptide inhibited HCV infection with an IC(50) of 7.62 μg/ml (5.0 μM), whereas Hp1035 peptide, showing high homology to Hp1090, exhibited no anti-HCV activity. Hp1090 acted as a viricide against HCV particles in vitro and prevented the initiation of HCV infection. Furthermore, this peptide interacted with HCV particles directly and rapidly permeabilized phospholipid membranes. Collectively, it seems that Hp1090 is virocidal for HCV in vitro, directly interacting with the viral membrane and decreasing the virus infectivity. These results suggest that Hp1090 could be considered an anti-HCV lead compound with virocidal mechanism that offers a potential therapeutic approach to HCV infection. Our work opens a new avenue for antiviral drug discovery in natural scorpion venom.

Scorpion venoms contain a vast untapped reservoir of natural products, which have the potential for medicinal value in drug discovery. In this study, toxin components from the scorpion Heterometrus petersii venom were evaluated by transcriptome and proteome analysis. Ten known families of venom peptides and proteins were identified, which include: two families of potassium channel toxins, four families of antimicrobial and cytolytic peptides, and one family from each of the calcium channel toxins, La1-like peptides, phospholipase A2, and the serine proteases. In addition, we also identified 12 atypical families, which include the acid phosphatases, diuretic peptides, and ten orphan families. From the data presented here, the extreme diversity and convergence of toxic components in scorpion venom was uncovered. Our work demonstrates the power of combining transcriptomic and proteomic approaches in the study of animal venoms.