The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 µM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

FS145, the first flea-derived disintegrin, inhibits angiogenesis through specifically binding integrin αvß3.

FS145, a protein containing a WGD motif, was previously described from the salivary transcriptome of the flea Xenopsylla cheopis. Nevertheless, its biological function and complete structure are still uncertain. Herein, FS145 was confirmed to adopt a common αßß structure with the WGD motif exposed on its surface and located right at the top of a loop composed of residues 72-81. Furthermore, FS145 dose-dependently inhibited the proliferation, adhesion, migration, and tube formation of HUVECs by not only binding to integrin αvß3 but also by subsequently inactivating the FAK/Src/MAPK pathway along with the reduction of the expression of MMP-2, MMP-9, VEGFA, bFGF, Ang2, Tie2, HIF-1α, and FAK. Moreover, FS145 also inhibited aortic vessel sprout and showed strong anti-angiogenic activities as assessed ex vivo, by employing the rat aortic ring assay, chick embryo chorioallantoic membrane, and zebrafish embryo models. Altogether, our results suggest that FS145 suppresses angiogenesis ex vivo and in vitro by blocking integrin αvß3. The current study reveals the first anti-angiogenesis disintegrin with WGD motif from invertebrates and provides a beneficial pharmacological activity to inhibit abnormal angiogenesis.

Cathelicidin-HG Alleviates Sepsis-Induced Platelet Dysfunction by Inhibiting GPVI-Mediated Platelet Activation.

Platelet activation contributes to sepsis development, leading to microthrombosis and increased inflammation, which results in disseminated intravascular coagulation and multiple organ dysfunction. Although Cathelicidin can alleviate sepsis, its role in sepsis regulation remains largely unexplored. In this study, we identified Cath-HG, a novel Cathelicidin from Hylarana guentheri skin, and analyzed its structure using nuclear magnetic resonance spectroscopy. The modulatory effect of Cath-HG on the symptoms of mice with sepsis induced by cecal ligation and puncture was evaluated in vivo, and the platelet count, degree of organ damage, and microthrombosis were measured. The antiplatelet aggregation activity of Cath-HG was studied in vitro, and its target was verified. Finally, we further investigated whether Cath-HG could regulate thrombosis in vivo in a FeCl3 injury-induced carotid artery model. The results showed that Cath-HG exhibited an α-helical structure in sodium dodecyl sulfate solution and effectively reduced organ inflammation and damage, improving survival in septic mice. It alleviated sepsis-induced thrombocytopenia and microthrombosis. In vitro, Cath-HG specifically inhibited collagen-induced platelet aggregation and modulated glycoprotein VI (GPVI) signaling pathways. Dot blotting, enzyme-linked immunosorbent assay, and pull-down experiments confirmed GPVI as the target of Cath-HG. Molecular docking and amino acid residue truncations/mutations identified crucial sites of Cath-HG. These findings suggest that GPVI represents a promising therapeutic target for sepsis, and Cath-HG may serve as a potential treatment for sepsis-related thrombocytopenia and thrombotic events. Additionally, identifying Cath-HG as a GPVI inhibitor provides insights for developing novel antithrombotic therapies targeting platelet activation mediated by GPVI.

The in vitro anticancer effects of FS48 from salivary glands of Xenopsylla cheopis on NCI-H460 cells via its blockage of voltage-gated K+ channels.

Voltage-gated K+ (Kv) channels play a role in the cellular processes of various cancer cells, including lung cancer cells. We previously identified and reported a salivary protein from the Xenopsylla cheopis, FS48, which exhibited inhibitory activity against Kv1.1-1.3 channels when assayed in HEK 293T cells. However, whether FS48 has an inhibitory effect on cancer cells expressing Kv channels is unclear. The present study aims to reveal the effects of FS48 on the Kv channels and the NCI-H460 human lung cancer cells through patch clamp, MTT, wound healing, transwell, gelatinase zymography, qRT-PCR and WB assays. The results demonstrated that FS48 can be effective in suppressing the Kv currents, migration, and invasion of NCI-H460 cells in a dose-dependent manner, despite the failure to inhibit the proliferation. Moreover, the expression of Kv1.1 and Kv1.3 mRNA and protein were found to be significantly reduced. Finally, FS48 decreases the mRNA level of MMP-9 while increasing TIMP-1 mRNA level. The present study highlights for the first time that blood-sucking arthropod saliva-derived protein can inhibit the physiological activities of tumour cells via the Kv channels. Furthermore, FS48 can be taken as a hit compound against the tumour cells expressing Kv channels.

Novel Amphibian Bowman-Birk-Like Inhibitor with Antioxidant and Anticoagulant Effects Ameliorates Pancreatitis Symptoms in Mice.

Acute pancreatitis (AP) is a serious inflammatory disorder and still lacks effective therapy globally. In this study, a novel Ranacyclin peptide, Ranacin, was identified from the skin of Pelophylax nigromaculatus frog. Ranacin adopted a compact ß-hairpin conformation with a disulfide bond (Cys5-Cys15). Ranacin was also demonstrated effectively to inhibit trypsin and have anticoagulant and antioxidant activities in vitro. Furthermore, the severity of pancreatitis was significantly alleviated in l-Arg-induced AP mice after treatment with Ranacin. In addition, structure-activity studies of Ranacin analogues confirmed that the sequences outside the trypsin inhibitory loop (TIL), especially at the C-terminal side, might be closely associated with the efficacy of its trypsin inhibitory activity. In conclusion, our data suggest that Ranacin can improve pancreatic injury in mice with severe AP through its multi-activity. Therefore, Ranacin is considered a potential drug candidate in AP therapy.

The Protective Effects of Cath-MH With Anti-Propionibacterium Acnes and Anti-Inflammation Functions on Acne Vulgaris.

Acne vulgaris is a common adolescent skin condition which is mainly caused by Propionibacterium acnes overcolonization and subsequent inflammation. Our previous studies have demonstrated that Cath-MH, an antimicrobial peptide from the skin of the frog Microhyla heymonsivogt, possesses potential antimicrobial, LPS-binding, and anti-septicemic properties. However, its protective effects and potential mechanisms against acne vulgaris are still unclear. In the present study, its anti-P. acnes effects were measured by two-fold broth dilution method, agglutination assay, scanning electron microscopy and confocal laser scanning microscopy experiments. Its treatment potential for acne vulgaris was further evaluated in mice ear inoculated by P. acnes. In addition, the binding ability between Cath-MH and LTA was measured by the Circular Dichroism and antibacterial assay. Moreover, the anti-inflammatory efficiency of Cath-MH was evaluated in LTA- and LPS-induced RAW 264.7 macrophage cells. Cath-MH was found to kill P. acnes with a MIC value of about 1.56 µM by membrane disruption mechanism. It also exhibited agglutination activity against P. acnes. Cath-MH was able to bind LTA as well as LPS, inhibit LTA/LPS-stimulated TLR2/4 expression, and subsequently decreased the inflammatory response in RAW 264.7 cells. As expected, Cath-MH alleviated the formation of edema and the infiltration of inflammatory cells in acne mouse model with concurrent suppression of P. acnes growth and inflammatory cytokines expression in vivo. The potent P. acnes inhibition activity combined with powerful anti-inflammatory effect of Cath-MH indicates its potential as a novel therapeutic option for acne vulgaris.

FM-CATH, A Novel Cathelicidin From Fejervarya Multistriata, Shows Therapeutic Potential for Treatment of CLP-Induced Sepsis.

Sepsis is an exacerbated inflammatory reaction induced by severe infection. As important defensive molecules in innate immunity, several AMPs are reported to prevent septic shock. In this study, we characterized a novel cathelicidin, FM-CATH, from the frog skin of F. multistriata. FM-CATH was found to adopt an amphipathic α-helix structural in membrane-mimetic environments and possess favorable antimicrobial effects against bacteria and fungus. In addition, it triggered the agglutination of bacteria. It could also strongly bind to LPS and LTA. Additionally, FM-CATH affected the enzymatic activities of thrombin, plasmin, ß-tryptase, and tPA, leading to coagulation inhibition in vitro and in vivo. Finally, we observed that FM-CATH improved survival rate and inhibited pathological alteration, bacterial count, serum biochemistry, and pro-inflammatory cytokine expression in the cecal ligation and puncture-induced sepsis mice. Taken together, these findings suggest that FM-CATH might be served as a promising agent for the treatment of sepsis.

Brevinin-2GHk, a Peptide Derived from the Skin of Fejervarya limnocharis, Inhibits Zika Virus Infection by Disrupting Viral Integrity.

Several years have passed since the Zika virus (ZIKV) pandemic reoccurred in 2015-2016. However, there is still a lack of proved protective vaccines or effective drugs against ZIKV. The peptide brevinin-2GHk (BR2GK), pertaining to the brevinin-2 family of antimicrobial peptides, has been reported to exhibit only weak antibacterial activity, and its antiviral effects have not been investigated. Thus, we analyzed the effect of BR2GK on ZIKV infection. BR2GK showed significant inhibitory activity in the early and middle stages of ZIKV infection, with negligible cytotoxicity. Furthermore, BR2GK was suggested to bind with ZIKV E protein and disrupt the integrity of the envelope, thus directly inactivating ZIKV. In addition, BR2GK can also penetrate the cell membrane, which may contribute to inhibition of the middle stage of ZIKV infection. BR2GK blocked ZIKV E protein expression with an IC50 of 3.408 ± 0.738 µΜ. In summary, BR2GK was found to be a multi-functional candidate and a potential lead compound for further development of anti-ZIKV drugs.

Tablysin-15 inhibits osteoclastogenesis and LPS-induced bone loss via attenuating the integrin αvß3 pathway.

Excessive osteoclast leads to the imbalance in bone reconstruction and results in osteolytic diseases, such as osteoporosis and rheumatic arthritis. Integrin αvß3 abundantly expresses on osteoclast and plays a critical role in the formation and function of osteoclast, therefore, blockage of αvß3 has become an attractive therapeutic option for osteolytic diseases. In this study, we find that Tablysin-15, a RGD motif containing disintegrin, concentration-dependently suppresses RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption without affecting the cell viabilities. Tablysin-15 binds to integrin αvß3 and inhibits the activation of FAK-associated signaling pathways. Tablysin-15 also suppresses the activation of NF-кB, MAPK, and Akt-NFATc1 signaling pathways, which are crucial transcription factors during osteoclast differentiation. Moreover, Tablysin-15 decreases the osteoclastogenesis marker gene expression, including MMP-9, TRAP, CTSK, and c-Src. Finally, Tablysin-15 significantly inhibits LPS-induced bone loss in a mouse model. Taken together, our results indicate that Tablysin-15 significantly suppresses osteoclastogenesis in vitro and in vivo, thus it might be a excellent candidate for treating osteolytic-related diseases.