Optimizing Bothropstoxin-I-Derived Peptides: Exploring the Antibacterial Potential of p-BthW.

Antimicrobial peptides are an emerging class of antibiotics that present a series of advantageous characteristics such as wide structural variety, broad spectrum of activity, and low propensity to select for resistance. They are found in all classes of life as defense molecules. A group of peptides derived from the protein Bothropstoxin-I has been previously studied as an alternative treatment against multi-drug-resistant bacteria. The peptide p-BthTX-I (sequence: KKYRYHLKPFCKK) and its homodimer, linked by disulfide oxidation through the residues of Cys11 and the serum degradation product [sequence: (KKYRYHLKPFC)2], were evaluated and showed similar antimicrobial activity. In this study, we synthesized an analogue of p-BthTX-I that uses the strategy of Fmoc-Lys(Fmoc)-OH in the C-terminal region for dimerization and tryptophan for all aromatic amino acids to provide better membrane interactions. This analogue, named p-BthW, displayed potent antibacterial activity at lower concentrations and maintained the same hemolytic levels as the original molecule. Our assessment revealed that p-BthW has a quick in vitro bactericidal action and prolonged post-antibiotic effect, comparable to the action of polymyxin B. The mode of action of p-BthW seems to rely not only on membrane depolarization but also on necrosis-like effects, especially in Gram-negative bacteria. Overall, the remarkable results regarding the propensity to develop resistance reaffirmed the great potential of the developed molecule.

Antibiofilm Activity and Biocompatibility of Temporin-SHa: A Promising Antimicrobial Peptide for Control of Fluconazole-Resistant Candida albicans.

The aim of the study was to investigate the effect of antimicrobial peptides (AMPs) Hylin-a1, KR-12-a5, and Temporin-SHa in Candida albicans as well as the biocompatibility of keratinocytes spontaneously immortalized (NOK-si) and human gingival fibroblasts (FGH) cells. Initially, the susceptible (CaS-ATCC 90028) and fluconazole-resistant (CaR-ATCC 96901) C. albicans strains were grown to evaluate the effect of each AMP in planktonic culture, biofilm, and biocompatibility on oral cells. Among the AMPs evaluated, temporin-SHa showed the most promising results. After 24 h of Temporin-SHa exposure, the survival curve results showed that CaS and CaR suspensions reduced 72% and 70% of cell viability compared to the control group. The minimum inhibitory/fungicide concentrations (MIC and MFC) showed that Temporin-SHa was able to reduce ≥50% at ≥256 µg/mL for both strains. The inhibition of biofilm formation, efficacy against biofilm formation, and total biomass assays were performed until 48 h of biofilm maturation, and Temporin-SHa was able to reduce ≥50% of CaS and CaR growth. Furthermore, Temporin-SHa (512 µg/mL) was classified as non-cytotoxic and slightly cytotoxic for NOK-si and FGH, respectively. Temporin-SHa demonstrated an anti-biofilm effect against CaS and CaR and was biocompatible with NOK-si and FGH oral cells in monolayer.

The Synthetic Peptide GA-Hecate and Its Analogs Inhibit Multiple Steps of the Chikungunya Virus Infection Cycle In Vitro.

Chikungunya virus (CHIKV) belongs to the Alphavirus genus and is responsible for significant outbreaks worldwide. Currently, there is no approved antiviral therapy against CHIKV. Bioactive peptides have great potential for new drug development. Here, we evaluated the antiviral activity of the synthetic peptide GA-Hecate and its analogs PSSct1905 and PSSct1910 against CHIKV infection. Initial screening showed that all three peptides inhibited the CHIKV replication cycle in baby hamster kidney fibroblast cells (BHK-21) and human hepatocarcinoma epithelial cells (Huh-7). GA-Hecate and its analog PSSct1905 were the most active, demonstrating suppression of viral infection by more than 91%. The analog PSSct1905 exhibited a protective effect in cells against CHIKV infection. We also observed that the analogs PSSct1905 and PSSct1910 affected CHIKV entry into both cell lines, inhibiting viral attachment and internalization. Finally, all tested compounds presented antiviral activity on the post-entry steps of CHIKV infection in all cells evaluated. In conclusion, this study highlights the potential of the peptide GA-Hecate and its analogs as novel anti-CHIKV compounds targeting different stages of the viral replication cycle, warranting the development of GA-Hecate-based compounds with broad antiviral activity.

Development of New Leishmanicidal Compounds via Bioconjugation of Antimicrobial Peptides and Antileishmanial Guanidines.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity.

Antimicrobial and Antibiofilm Activity of Synthetic Peptide [W7]KR12-KAEK Against Enterococcus faecalis Strains.

The emergence of infections caused by microorganisms in the oral cavity and increasing concerns regarding the use of antibiotics have resulted in the development of novel antimicrobial molecules, such as antimicrobial synthetic peptides. The purpose of this study was to evaluate the antimicrobial and antibiofilm activities of the native peptide KR-12 and its derivative, the synthetic peptide [W7]KR12-KAEK, against planktonic and biofilms Enterococcus faecalis strains. The methods used to evaluate the antimicrobial activity in planktonic cultures include minimum inhibitory concentration and minimum bactericidal concentration assays. The effects of [W7]KR12-KAEK on biofilm formation and mature biofilms were evaluated by quantifying biomass (crystal violet staining) and counting colony-forming units. Structural assessments of the biofilms and cellular morphological changes were performed using scanning electron microscopy. Peptide [W7]KR12-KAEK showed potential antimicrobial activity against planktonic cells. Interestingly, the native peptide KR-12 showed no antimicrobial activity. Moreover, it inhibited biofilm formation and disrupted the mature biofilms of E. faecalis strains. These results suggest that [W7]KR12-KAEK may be a potential molecule for the development of auxiliary antimicrobial therapies against oral infections.

Resveratrol-Loaded Attalea funifera Oil Organogel Nanoparticles: A Potential Nanocarrier against A375 Human Melanoma Cells.

This study aimed to evaluate Attalea funifera seed oil with or without resveratrol entrapped in organogel nanoparticles in vitro against A375 human melanoma tumor cells. Organogel nanoparticles with seed oil (SON) or with resveratrol entrapped in the seed oil (RSON) formed functional organogel nanoparticles that showed a particle size <100 nm, polydispersity index <0.3, negative zeta potential, and maintenance of electrical conductivity. The resveratrol entrapment efficiency in RSON was 99 ± 1%. The seed oil and SON showed no cytotoxicity against human non-tumor cells or tumor cells. Resveratrol at 50 µg/mL was cytotoxic for non-tumor cells, and was cytotoxic for tumor cells at 25 µg/mL. Resveratrol entrapped in RSON showed a decrease in cytotoxicity against non-tumor cells and cytotoxic against tumor cells at 50 µg/mL. Thus, SON is a potential new platform for the delivery of resveratrol with selective cytotoxic activity in the treatment of melanoma.

Antiviral Evaluation of New Synthetic Bioconjugates Based on GA-Hecate: A New Class of Antivirals Targeting Different Steps of Zika Virus Replication.

Re-emerging arboviruses represent a serious health problem due to their rapid vector-mediated spread, mainly in urban tropical areas. The 2013-2015 Zika virus (ZIKV) outbreak in South and Central America has been associated with cases of microcephaly in newborns and Guillain-Barret syndrome. We previously showed that the conjugate gallic acid-Hecate (GA-FALALKALKKALKKLKKALKKAL-CONH2)-is an efficient inhibitor of the hepatitis C virus. Here, we show that the Hecate peptide is degraded in human blood serum into three major metabolites. These metabolites conjugated with gallic acid were synthesized and their effect on ZIKV replication in cultured cells was evaluated. The GA-metabolite 5 (GA-FALALKALKKALKKL-COOH) was the most efficient in inhibiting two ZIKV strains of African and Asian lineage at the stage of both virus entry (virucidal and protective) and replication (post-entry). We also demonstrate that GA-metabolite 5 does not affect cell growth after 7 days of continuous treatment. Thus, this study identifies a new synthetic antiviral compound targeting different steps of ZIKV replication in vitro and with the potential for broad reactivity against other flaviviruses. Our work highlights a promising strategy for the development of new antivirals based on peptide metabolism and bioconjugation.

K-aurein: A notable aurein 1.2-derived peptide that modulates Candida albicans filamentation and reduces biofilm biomass.

Candida albicans is considered one of the most important opportunistic fungi due to the large arsenal of virulence factors that help throughout the progress of the infection. In this sense, antimicrobial peptides (AMPs) appear as an alternative, with great antifungal action. Among these, aurein 1.2 has been widely explored, becoming the basis for the discovery of new AMPs, such as K-aurein (K-au). Thus, this study evaluated the anti-C. albicans potential of K-au against virulence factors, planktonic growth, and biofilm formation of clinical isolates. Firstly, K-au antifungal activity was determined by the microdilution method and time-kill curve. The inhibition of hydrolytic enzyme secretion (proteinase, phospholipase, and hemolysin) and germ tube formation was tested. Then, the antibiofilm potential of K-au was verified through biomass quantification and scanning electron microscopy (SEM). All tests were compared with the classical antifungal drug, amphotericin B (AmB). The outcomes showed fungicidal action of K-au at 62.50 µg mL-1 for all isolates, with a time of action around 150-180 min, determined by the time-kill curve. K-au-treated cells decreased by around 40% of the germinative tube compared to the control. Additionally, K-au inhibited the biofilm formation by more than 90% compared to AmB and the control group. SEM images show apparent cellular disaggregation without the formation of filamentous structures. Therefore, the findings suggest a promising anti-C. albicans effect of K-au due to its fungicidal activity against planktonic cells, or its ability to inhibit important virulence factors like germ tube and biofilm formation. Thus, this peptide could be explored as a useful compound against C. albicans-related infection.

Effects of Conjugation of Ferrocene and Gallic Acid On desCys11/Lys12/Lys13-(p-BthTX-I)2K Peptide: Structure, Permeabilization and Antibacterial Activity.

BACKGROUND: Antimicrobial resistance is an emerging global health challenge that has led researchers to study alternatives to conventional antibiotics. A promising alternative is antimicrobial peptides (AMPs), produced as the first line of defense by almost all living organisms. To improve its biological activity, the conjugation of AMPs is a promising approach. OBJECTIVE: In this study, we evaluated the N-terminal conjugation of p-Bt (a peptide derived from Bothrops Jararacuçu`s venom) with ferrocene (Fc) and gallic acid (GA). Acetylated and linear versions of p-Bt were also synthesized to evaluate the importance of N-terminal charge and dimeric structure. METHODS: The compounds were obtained using solid-phase peptide synthesis. Circular dichroism, vesicle permeabilization, antimicrobial activity, and cytotoxicity studies were conducted. RESULTS: No increase in antibacterial activity against Escherichia coli was observed by adding either Fc or GA to p-Bt. However, Fc-p-Bt and GA-p-Bt exhibited improved activity against Staphylococcus aureus. No cytotoxicity upon fibroblast was observed for GA-p-Bt. On the other hand, conjugation with Fc increased cytotoxicity. This toxicity may be related to the membrane permeabilization capacity of this bioconjugate, which showed the highest carboxyfluorescein leakage in vesicle permeabilization experiments. CONCLUSION: Considering these observations, our findings highlight the importance of adding bioactive organic compounds in the N-terminal position as a tool to modulate the activity of AMPs.

The Dimeric Peptide (KKYRYHLKPF)2K Shows Broad-Spectrum Antiviral Activity by Inhibiting Different Steps of Chikungunya and Zika Virus Infection.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are important disease-causing agents worldwide. Currently, there are no antiviral drugs or vaccines approved to treat these viruses. However, peptides have shown great potential for new drug development. A recent study described (p-BthTX-I)2K [(KKYRYHLKPF)2K], a peptide derived from the Bothropstoxin-I toxin in the venom of the Bothrops jararacussu snake, showed antiviral activity against SARS-CoV-2. In this study, we assessed the activity of this peptide against CHIKV and ZIKV and its antiviral action in the different stages of the viral replication cycle in vitro. We observed that (p-BthTX-I)2K impaired CHIKV infection by interfering with the early steps of the viral replication cycle, reducing CHIKV entry into BHK-21 cells specifically by reducing both the attachment and internalization steps. (p-BthTX-I)2K also inhibited the ZIKV replicative cycle in Vero cells. The peptide protected the cells against ZIKV infection and decreased the levels of the viral RNA and the NS3 protein of this virus at viral post-entry steps. In conclusion, this study highlights the potential of the (p-BthTX-I)2K peptide to be a novel broad-spectrum antiviral candidate that targets different steps of the replication cycle of both CHIKV and ZIKV.

Effects of Dimerization, Dendrimerization, and Chirality in p-BthTX-I Peptide Analogs on the Antibacterial Activity and Enzymatic Inhibition of the SARS-CoV-2 PLpro Protein.

Recent studies have shown that the peptide [des-Cys11,Lys12,Lys13-(p-BthTX-I)2K] (p-Bth) is a p-BthTX-I analog that shows enhanced antimicrobial activity, stability and hemolytic activity, and is easy to obtain compared to the wild-type sequence. This molecule also inhibits SARS-CoV-2 viral infection in Vero cells, acting on SARS-CoV-2 PLpro enzymatic activity. Thus, the present study aimed to assess the effects of structural modifications to p-Bth, such as dimerization, dendrimerization and chirality, on the antibacterial activity and inhibitory properties of PLpro. The results showed that the dimerization or dendrimerization of p-Bth was essential for antibacterial activity, as the monomeric structure led to a total loss of, or significant reduction in, bacterial activities. The dimers and tetramers obtained using branched lysine proved to be prominent compounds with antibacterial activity against Gram-positive and Gram-negative bacteria. In addition, hemolysis rates were below 10% at the corresponding concentrations. Conversely, the inhibitory activity of the PLpro of SARS-CoV-2 was similar in the monomeric, dimeric and tetrameric forms of p-Bth. Our findings indicate the importance of the dimerization and dendrimerization of this important class of antimicrobial peptides, which shows great potential for antimicrobial and antiviral drug-discovery campaigns.

Plant Defense Elicitation by the Hydrophobin Cerato-Ulmin and Correlation with Its Structural Features.

Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time.

BACKGROUND: (p-BthTX-I)2 K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required. OBJECTIVES: To determine whether (p-BthTX-I)2 K affects the hemostatic system. METHODS: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I)2 K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg- 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I)2 K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline administration. RESULTS: (p-BthTX-I)2 K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I)2 K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I)2 K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I)2 K did not prolong the bleeding time in the mouse model of arterial thrombosis. CONCLUSION: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I)2 K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.

Novel Selective and Low-Toxic Inhibitor of LmCPB2.8ΔCTE (CPB) One Important Cysteine Protease for Leishmania Virulence.

Leishmaniasis is a highly prevalent, yet neglected disease caused by protozoan parasites of the genus Leishmania. In the search for newer, safer, and more effective antileishmanial compounds, we herein present a study of the mode of action in addition to a detailed structural and biological characterization of LQOF-G6 [N-benzoyl-N'-benzyl-N″-(4-tertbutylphenyl)guanidine]. X-ray crystallography and extensive NMR experiments revealed that LQOF-G6 nearly exclusively adopts the Z conformation stabilized by an intramolecular hydrogen bond. The investigated guanidine showed selective inhibitory activity on Leishmania major cysteine protease LmCPB2.8ΔCTE (CPB) with ~73% inhibition and an IC50-CPB of 6.0 µM. This compound did not show any activity against the mammalian homologues cathepsin L and B. LQOF-G6 has been found to be nontoxic toward both organs and several cell lines, and no signs of hepatotoxicity or nephrotoxicity were observed from the analysis of biochemical clinical plasma markers in the treated mice. Docking simulations and experimental NMR measurements showed a clear contribution of the conformational parameters to the strength of the binding in the active site of the enzyme, and thus fit the differences in the inhibition values of LQOF-G6 compared to the other guanidines. Furthermore, the resulting data render LQOF-G6 suitable for further development as an antileishmanial drug.

Efficacy of the combination of P5 peptide and photodynamic therapy mediated by bixin and chlorin-e6 against Cutibacterium acnes biofilm.

In this study, the action of antimicrobial peptide (AMP) P5 and antimicrobial photodynamic therapy (aPDT) mediated by bixin and chlorin-e6 (Ce6) on Cutibacterium acnes (C. acnes) in planktonic phase and biofilm were evaluated both as monotherapies and combined therapies. Microbial viability after treatments were quantified by colony-forming units per milliliter of the sample (CFU/mL) and have demonstrated that all treatments employed exerted bactericidal activity, reducing the microbial load by more than 3 log10 CFU/mL, also demonstrating for the first time in the literature the antimicrobial photodynamic effect of bixin that occurs mostly through type I mechanism which was proved by the quantification of superoxide anion production. Bacterial biofilm was completely eliminated only after its exposure to aPDT mediated by this PS, however, Ce6 proved to be a more efficient PS, considering that most of the photodynamic effect of bixin- aPDT was exerted by excitation of the endogenous C porphyrins of C. acnes with blue light. The combination of P5 with Ce6-aPDT showed a synergistic effect on the bacterial biofilm with a reduction in microbial load by more than 10 log10 CFU/mL, in which the ability of P5 to permeabilize the polymeric extracellular matrix of the biofilm explains the obtained results, with greater internalization of the PS as shown by the Confocal Laser Scanning Microscopy. One-way ANOVA (Analysis of Variance) with Tukey's post-test and two-way ANOVA with Bonferroni's post-test were used to compare the values of continuous variables between the control group and the treatment groups.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Use of Photodynamic Therapy Associated with Antimicrobial Peptides for Bacterial Control: A Systematic Review and Meta-Analysis.

Considering the challenges related to antimicrobial resistance, other strategies for controlling infections have been suggested, such as antimicrobial photodynamic therapy (aPDT) and antimicrobial peptides (AMP). This study aims to perform a systematic review and meta-analysis to obtain evidence on the antimicrobial effectiveness of aPDT associated with AMP and establish in vitro knowledge on this topic for further study designs. The PubMed, Scopus, Web of Science, Science Direct, Scielo, and Cochrane Library databases were searched. Two independent and calibrated researchers (Kappa = 0.88) performed all the systematic steps according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The odds ratio (OR) was used as the effect measure. The Peto method was used to perform the meta-analysis due to the sparse data. Twenty studies were included in the present review. The result was significant (OR = 0.14/p = 0.0235/I-squared = 0%), showing better outcomes of aPDT associated with peptides than those of aPDT alone for controlling the microbial load. Only 20% of the studies included evaluated this approach in a biofilm culture. Combined treatment with aPDT and AMP highly increased the ability of microbial reduction of Gram-positive and Gram-negative bacteria. However, additional blind studies are required to evaluate the efficacy of this therapy on microbial biofilms.

ZIKV B-cell epitopes for immunodiagnostic tests.

The use of serological tests is valuable to diagnose Zika virus (ZIKV) infection and carry out epidemiological surveillance. However, ZIKV serological tests may result in false positives due to cross-reactivity between antibodies against other Flavivirus, especially dengue virus that worldwide disseminated. We used three online tools to predict amino acid sequences of B-cell epitopes. We selected and synthetized two epitopes that showed appropriate features in the molecular dynamic simulation and demonstrated to be suitable for serological assays.

Insights on the inhibition properties of Jatromollistatin (a cyclic heptapeptide) against Crotalus adamanteus metalloendopeptidase using molecular docking analysis.

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites.

Cytocompatibility and Synergy of EGCG and Cationic Peptides Against Bacteria Related to Endodontic Infections, in Planktonic and Biofilm Conditions.

This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of epigallocatechin-3-gallate (EGCG) associated with peptide LL-37 and its analogue KR-12-a5 against oral pathogens. The effect of the compounds on metabolism of fibroblasts was evaluated by methyltetrazolium assays. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on single- and dual-species biofilms and E. faecalis biofilms in dentinal tubules and analyzed by bacterial counts and confocal microscopy. Data were statistically analyzed considering p < 0.05. EGCG and peptide combinations were not toxic to fibroblasts. KR-12-a5 showed synergistic or addictive effects with EGCG and LL-37 against all bacteria tested. However, EGCG associated with KR-12-a5 demonstrated the highest bactericidal activity on all bacteria tested, at lower concentrations. In single-species biofilms, EGCG + KR-12-a5 eliminated S. mutans and A. israelii and reduced E. faecalis and F. nucleatum counts around 5 log CFU/mL. EGCG + KR-12-a5 reduced E. faecalis (-3.93 log CFU/mL) and eliminated S. mutans in dual-species biofilms. No growth of E. faecalis and significant reduction in A. israelii (-6.24 log CFU/mL) and F. nucleatum (-4.62 log CFU/mL) counts were detected in dual-species biofilms. The combination of EGCG and KR-12-a5 led to 88% of E. faecalis dead cells inside dentin tubules. The association of EGCG and KR-12-a5 was cytocompatible and promoted synergistic effect against biofilms of bacteria associated with endodontic infections.