Coagulation factor XIa is a new serine-protease family drug target for next-generation anticoagulants. With the snake venom Kunitz-type peptide BF9 as the scaffold, we obtained a highly active XIa inhibitor BF9-N17K in our previous work, but it also inhibited the hemostatic target plasmin. Here, in order to enhance the selectivity of BF9-N17K toward XIa, four mutants, BF9-N17K-L19A, BF9-N17K-L19S, BF9-N17K-L19D, and BF9-N17K-L19K, were further designed using the P2′ amino acid classification scanning strategy. The anticoagulation assay showed that the four P2′ single-point mutants still had apparent inhibitory anticoagulation activity that selectively inhibited the human intrinsic coagulation pathway and had no influence on the extrinsic coagulation pathway or common coagulation pathway, which indicated that the single-point mutants had minimal effects on the anticoagulation activity of BF9-N17K. Interestingly, the enzyme inhibitor assay experiments showed that the XIa and plasmin inhibitory activities were significantly changed by the P2′ amino acid replacements. The XIa inhibitory activity of BF9-N17K-L19D was apparently enhanced, with an IC50 of 19.28 ± 2.53 nM, and its plasmin inhibitory was significantly weakened, with an IC50 of 459.33 ± 337.40 nM. BF9-N17K-L19K was the opposite to BF9-N17K-L19D, which had enhanced plasmin inhibitory activity and reduced XIa inhibitory activity. For BF9-N17K-L19A and BF9-N17K-L19S, no apparent changes were found in the serine protease inhibitory activity, and they had similar XIa and plasmin inhibitory activities to the template peptide BF9-N17K. These results suggested that the characteristics of the charge of the P2′ site might be associated with the drug selectivity between the anticoagulant target XIa and hemostatic target plasmin. In addition, according to the molecular diversity and sequence conservation, a common motif GR/PCR/KA/SXIP-XYGGC is proposed in the XIa-inhibitory Kunitz-type peptides, which might provide a new clue for further peptide engineering. In conclusion, through P2′ amino acid classification scanning with the snake venom Kunitz-type peptide scaffold, a new potent and selective XIa inhibitor, BF9-N17K-L19D, was discovered, which provides a new XIa-targeting lead drug template for the treatment of thrombotic-related diseases.

Alzheimer's disease (AD) is a severe neurodegenerative disorder characterized by abnormal metabolism of (3-amyloid (A(3) precursor proteins and neuronal apoptosis, ultimately leading to cognitive dysfunction. The pathogenesis of AD is complex, and current single-target therapies are not effective in preventing the rapid progression of AD, which highlights the urgent need for developing multi-target drugs. In this study, a series of compounds were synthesized through a multi-targeting ligand strategy. After extensive screening and evaluation, we found a lead compound B14, which showed excellent dual targeting ability for effectively alleviating neuronal apoptosis and oxidative stress damage of AD. In our molecular and cellular level experiments, B14 could target and stabilize the i-motif structure formed on the BCL-2 promoter to upregulate BCL-2 expression, which could also bind to A(3 and inhibit its deposition. In the A(31-42-induced cell model, B14 could maintain mitochondrial function and number, regulate intracellular reactive oxygen species (ROS) and Ca2+ metabolism disorders, and effectively reduce A(31-42-induced apoptosis. Further studies showed that B14 also exhibited good ability to cross the blood-brain barrier (BBB), which significantly improved learning memory and cognitive deficits, reduced brain A(3 plaques, alleviated inflammation and restored oxidative stress markers in APP/PS1 mice. Our findings provide an innovative strategy of dual targeting BCL-2 promoter i-motif for transcriptional regulation and A(3 aggregation synergistically for mitigating AD pathologies. B14 represents a promising multitarget lead compound with a good potential for further development for AD treatment.

Coagulation factor XIa is a drug target for new anticoagulants, but no XIa inhibitors have been found from schistosoma, a worm living in the bloodstream. In this study, by sequence blasting and structural fold searching of Kunitz-domain containing proteins from schistosoma, a specific Kunitz-domain Shp4 that appears in nine secreted proteins from four schistosoma species was identified successfully. Functional studies showed that Shp4 is a novel XIa inhibitor with a Ki value of 3.35 nM, which inhibited coagulation factor XIa activity in a non-heparin-dependent manner. His-pull-down assay further indicated the direct interaction between Shp4 and XIa. Animal experiments showed that Shp4 is a useful peptide lead drug with well antithrombotic effects in vivo. Alanine-scanning experiments showed that R18 is the key residue for XIa inhibitory and anticoagulation activities. Structural modelling of nine schistosoma-derived full-length secreted proteins suggested that the C-terminal non-Kunitz-domain fragments might play important roles in modulating the Kunitz-domain functions by steric hindrance effect and transmembrane helix structure. In conclusion, our work characterized the first XIa inhibitor from schistosoma with high anticoagulation activity and well antithrombotic effects, and highlights a potential strategy to modulate the Kunitz-domain, not only by the functional loop, but also by diverse C-terminal fragments.

Cronobacter sakazakii is a Gram-negative bacterium known for causing severe infections in neonates, particularly through contaminated infant formula. This study investigated the role of the outer membrane lipoprotein NlpD in the environmental tolerance of C. sakazakii. A nlpD knockout mutant was constructed, and its impact on desiccation resistance, biofilm formation, motility, and proteomic profiles was evaluated and compared with that of the wild-type strain. The nlpD mutant presented reduced desiccation tolerance, reduced ability to form a biofilm, and altered surface hydrophobicity and motility patterns. The complemented strain restored these phenotypic changes, confirming that the observed effects were specifically due to the deletion of nlpD. Proteomic analysis revealed significant differential expression of proteins involved in metabolic and biosynthetic pathways upon nlpD deletion. These findings emphasize the multifaceted role of NlpD in enhancing the environmental tolerance of C. sakazakii, suggesting its importance in the resilience and survival of the bacterium in adverse conditions.

The ESKAPE pathogen-associated antimicrobial resistance is a global public health issue, and novel therapeutic strategies are urgently needed. The short cationic antimicrobial peptide (AMP) family represents an important subfamily of scorpion-derived AMPs, but high hemolysis and poor antimicrobial activity hinder their therapeutic application. Here, we recomposed the hydrophilic face of Ctriporin through lysine substitution. We observed non-linear correlations between the physiochemical properties of the peptides and their activities, and significant deviations regarding the changes of antimicrobial activities against different bacterial species, as well as hemolytic activity. Most importantly, we obtained two Ctriporin analogs, CM5 and CM6, these two have significantly reduced hemolytic activity and more potent antimicrobial activities against all tested antibiotic-resistant ESKAPE pathogens. Fluorescence experiments indicated they may perform the bactericidal function through a membrane-lytic action model. Our work sheds light on the potential of CM5 and CM6 in developing novel antimicrobials and gives clues for optimizing peptides from the short cationic AMP family.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Viruses are one of the leading causes of human disease, and many highly pathogenic viruses still have no specific treatment drugs. Therefore, producing new antiviral drugs is an urgent matter. In our study, we first found that the natural wasp venom peptide Protopolybia-MP III had a significant inhibitory effect on herpes simplex virus type 1 (HSV-1) replication in vitro by using quantitative real-time PCR (qPCR), Western blotting, and plaque-forming assays. Immunofluorescence analysis showed Protopolybia-MP III could enter cells, and it inhibited multiple stages of the HSV-1 life cycle, including the attachment, entry/fusion, and post-entry stages. Furthermore, ultracentrifugation and electron microscopy detected that Protopolybia-MP III significantly suppressed HSV-1 virion infectivity at different temperatures by destroying the integrity of the HSV-1 virion. Finally, by comparing the antiviral activity of Protopolybia-MP III and its mutants, a series of peptides with better anti-HSV-1 activity were identified. Overall, this work found the function and mechanism of the antiviral wasp venom peptide Protopolybia-MP III and its derivatives against HSV-1 and laid the foundation for the research and development of wasp venom-derived antiviral candidate peptide drugs.

Biologically active peptides have attracted increasing attention in research on the development of new drugs. Mastoparans, a group of wasp venom linear cationic α-helical peptides, have a variety of biological effects, including mast cell degranulation, activation of protein G, and antimicrobial and anticancer activities. However, the potential hemolytic activity of cationic α-helical peptides greatly limits the clinical applications of mastoparans. Here, we systematically and comprehensively studied the hemolytic activity of mastoparans based on our wasp venom mastoparan family peptide library. The results showed that among 55 mastoparans, 18 had strong hemolytic activity (EC50 ≤ 100 μM), 14 had modest hemolytic activity (100 μM < EC50 ≤ 400 μM) and 23 had little hemolytic activity (EC50 > 400 μM), suggesting functional variation in the molecular diversity of mastoparan family peptides from wasp venom. Based on these data, structure–function relationships were further explored, and, hydrophobicity, but not net charge and amphiphilicity, was found to play a critical role in the hemolytic activity of mastoparans. Combining the reported antimicrobial activity with the present hemolytic activity data, we found that four mastoparan peptides, Parapolybia-MP, Mastoparan-like peptide 12b, Dominulin A and Dominulin B, have promise for applications because of their high antimicrobial activity (MIC ≤ 10 μM) and low hemolytic activity (EC50 ≥ 400 μM). Our research not only identified new leads for the antimicrobial application of mastoparans but also provided a large chemical space to support the molecular design and optimization of mastoparan family peptides with low hemolytic activity regardless of net charge or amphiphilicity.

Cronobacter sakazakii (C. sakazakii) is a notorious pathogen responsible for infections in infants and newborns, often transmitted through contaminated infant formula. Despite the use of traditional pasteurization methods, which can reduce microbial contamination, there remains a significant risk of pathogenic C. sakazakii surviving due to its exceptional stress tolerance. In our study, we employed a comparative proteomic approach by comparing wild-type strains with gene knockout strains to identify the essential genes crucial for the successful survival of C. sakazakii during desiccation. Our investigation revealed the significance of envZ-ompR, recA, and flhD gene cassettes in contributing to desiccation tolerance in C. sakazakii. Furthermore, through our comparative proteomic profiling, we identified the maltodextrin-binding protein encoded by ESA_03421 as a potential factor influencing dry tolerance. This protein is regulated by EnvZ-OmpR, RecA, and FlhD. Notably, the knockout of ESA_03421 resulted in a 150% greater reduction in Log CFU compared to the wild-type C. sakazakii. Overall, our findings offer valuable insights into the mechanisms underlying C. sakazakii desiccation tolerance and provide potential targets for the development of new antimicrobial strategies aimed at reducing the risk of infections in infants and newborns.

Wasp stings have become an increasingly serious public health problem because of their high incidence and mortality rates in various countries and regions. Mastoparan family peptides are the most abundant natural peptides in hornet venoms and solitary wasp venom. However, there is a lack of systematic and comprehensive studies on mastoparan family peptides from wasp venoms. In our study, for the first time, we evaluated the molecular diversity of 55 wasp mastoparan family peptides from wasp venoms and divided them into four major subfamilies. Then, we established a wasp peptide library containing all 55 known mastoparan family peptides by chemical synthesis and C-terminal amidation modification, and we systematically evaluated their degranulation activities in two mast cell lines, namely the RBL-2H3 and P815 cell lines. The results showed that among the 55 mastoparans, 35 mastoparans could significantly induce mast cell degranulation, 7 mastoparans had modest mast cell degranulation activity, and 13 mastoparans had little mast cell degranulation activity, suggesting functional variation in mastoparan family peptides from wasp venoms. Structure–function relationship studies found that the composition of amino acids in the hydrophobic face and amidation in the C-terminal region are critical for the degranulation activity of mastoparan family peptides from wasp venoms. Our research will lay a theoretical foundation for studying the mechanism underlying the degranulation activity of wasp mastoparans and provide new evidence to support the molecular design and molecular optimization of natural mastoparan peptides from wasp venoms in the future.

Schistosoma japonicum is a parasitic worm that lives in the mesenteric vein of its host and feeds on blood, suggesting that it might be a natural resource of novel anticoagulants. Here, by comprehensive analyses of the genomic sequences of Schistosoma japonicum, a new Kunitz-type gene precursor was identified. The Kunitz-type gene precursor codes for an 18-residue signal peptide and a 60-residue mature peptide. The Kunitz peptide was functionally expressed, and it had apparent inhibitory activity towards the intrinsic coagulation pathway but no effect on the extrinsic coagulation pathway even at the high concentration of 3 μM. Enzyme and inhibitor experiments further showed that the Kunitz domain peptide was a potent and selective FXa inhibitor, so it was named Schixator (Schistosoma FXa inhibitor). Schixator inhibits coagulation factor FXa with a Ki of 2.66 nM, but had weak inhibitory activity towards chymotrypsin, FXIa, plasma kallikrein, and plasmin, and no inhibitory activity towards trypsin, elastase, FIIa or FXIIa. In vivo, the intravenous administration of Schixator into mice dramatically decreased the number of thrombi in the carotid artery in an FeCl3-induced thrombus formation model without producing bleeding complications. To the best of our knowledge, Schixator is the first potent and selective FXa inhibitor from parasitic worms with antithrombotic effects and a low bleeding risk that provides a new clue for lead drug discovery against thrombosis-associated human diseases.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is becoming a troublesome issue worldwide, and anti-CRAB drug research and development is urgently needed. To identify new anti-CRAB drug leads, we investigated seven scorpion venom-derived α-helical peptides that differ in their sequence composition and length. Three peptides, Hp1404, ctriporin and Im5, showed antimicrobial activities against Acinetobacter baumannii. Further antimicrobial assays revealed that Hp1404 exhibited the best cell selectivity with high anti-CRAB and low hemolytic activities. Fluorescence assays demonstrated that Hp1404 can induce dose-dependent disruptions of the bacterial cell membrane, implying a membrane-lytic mode of action. Taken together, our work sheds light on the potential of the scorpion venom-derived peptide Hp1404 for the development of novel antimicrobial agents against CRAB infections.

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

Methicillin-resistant staphylococci have become growing threats to human health, and novel antimicrobials are urgently needed. Natural antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. Here, two novel cationic α-helical antimicrobial peptides, Lausporin-1 and Lausporin-2, were identified from the venom gland of the scorpion L. australasiae through a cDNA library screening strategy. Biochemical analyses demonstrated that Lausporin-1 and Lausporin-2 are cationic α-helical amphipathic molecules. Antimicrobial assays demonstrated that the two peptides possess antibacterial activities against several species of antibiotic-resistant staphylococci. Importantly, they are active against methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus capitis, with the minimum inhibitory concentrations ranging from 2.5 to 10 μg/ml. Moreover, both peptides can induce dose-dependent plasma membrane disruptions of the bacteria. In short, our work expands the knowledge of the scorpion L. australasiae venom-derived AMPs and sheds light on the potential of Lausporin-1 and Lausporin-2 in the development of novel drugs against methicillin-resistant staphylococci.

Scorpion venoms are rich resources of antimicrobial peptides (AMPs). While the short-chain noncysteine-containing AMPs have attracted much attention as templates for drug development, the antimicrobial potential of long-chain noncysteine-containing AMPs has been largely overlooked. Here, by using the online HeliQuest server, we designed and analyzed a series of 14-residue fragments of Smp43, a 43-residue long-chain noncysteine-containing AMP identified from the venom of Scorpio maurus palmatus. We found that Smp43(1-14) shows high antimicrobial activity against both Gram-positive and Gram-negative bacteria and is nontoxic to mammalian cells at the antimicrobial dosage. Sequence alignments showed that the designed Smp43(1-14) displays a unique primary structure that is different from other natural short-chain noncysteine-containing AMPs from scorpions, such as Uy17, Uy192 and IsCT. Moreover, the peptide Smp43(1-14) caused concentration-dependent fluorescence increases in the bacteria for all of the tested dyes, propidium iodide, SYTOXTM Green and DiSC3-5, suggesting that the peptide may kill the bacteria through the formation of pore structures in the plasma membrane. Taken together, our work sheds light on a new avenue for the design of novel short-chain noncysteine-containing AMPs and provides a good peptide template with a unique sequence for the development of novel drugs for use against bacterial infectious diseases.

Only a few work have been done for peptides from non-venom gland tissues of venomous animals. Here, with the help of the whole body transcriptomic and the hemolymph proteomic data of the Chinese scorpion Buthus martensii Karsch, we identified the first Ascaris-type peptide BmHDP from scorpion hemolymph. The precursor of BmHDP has 80 residues, including a 16 residue signal peptide and a 64 residue mature peptide. The mature peptide has 10 conserved cysteines and adopts a conserved Ascaris-type fold. Using combined inclusion body refolding and biochemical identification strategies, recombinant BmHDP was obtained successfully. Protease inhibitory assays showed that BmHDP inhibited chymotrypsin apparently at a concentration of 8 nM. Patch-clamp experiments showed that BmHDP inhibited the Kv1.3 potassium channel apparently at a concentration of 1000 nM. Coagulation experiment assays showed that BmHDP inhibited intrinsic coagulation pathway apparently at a concentration of 500 nM. To the best of our knowledge, BmHDP is the first Ascaris-type peptide from scorpion hemolymph. Our work highlighted a functional link between scorpion non-venom gland peptides and venom gland toxin peptides, and suggested that scorpion hemolymph might be a new source of bioactive peptides.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

The Kv1.3 channel plays potential roles in immune, inflammation and coagulation system. Many studies showed that Kv1.3 channel inhibitors have immunosuppressive and anti-inflammatory activities, but no Kv1.3 channel inhibitors have been found to have anticoagulation activities. Here, based on our previous work about Kv1.3 channel toxin peptide inhibitors, we first attempt to test anticoagulation activities of four known venom-derived Kv1.3 channel inhibitors with different structural folds: BmKTX with CSα/β structural fold, OmTx3 with CSα/α structural fold, BF9 with Kuntz-type structural fold, and SjAPI-2 with Ascaris-type structural fold. Our results showed that BmKTX and OmTx3 have no activities towards both intrinsic and extrinsic coagulation pathway, SjAPI-2 just has weak activity towards intrinsic coagulation pathway, and BF9 has potent activity towards intrinsic coagulation pathway with no apparent effect on extrinsic coagulation pathway. Enzyme and inhibitor reaction kinetics experiments further showed that BF9 inhibited intrinsic coagulation pathway-associated coagulation factor XIa, but have no apparent effects on common coagulation pathway coagulation factor IIa. Structure-activity relationship showed that Gly14, Asn17, Ala18 and Ile20 of BF9 are main residues involved in the inhibiting effect on factor XIa. To the best of our knowledge, BF9 is the first anticoagulant with Kv1.3 channel inhibitory activity. Together, our present studies found the first dual functional peptides with Kv1.3 channel and coagulation factor XIa inhibitory activities, and provided a new molecular template for the lead drug discovery towards immune and thrombosis-associated human diseases.

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST-Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram-positive Staphylococcus aureus, but had no effect on Gram-negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease-associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus-derived defensin peptides.

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.