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.

Nanobiotechnology with Therapeutically Relevant Macromolecules from Animal Venoms: Venoms, Toxins, and Antimicrobial Peptides.

Some diseases of uncontrolled proliferation such as cancer, as well as infectious diseases, are the main cause of death in the world, and their causative agents have rapidly developed resistance to the various existing treatments, making them even more dangerous. Thereby, the discovery of new therapeutic agents is a challenge promoted by the World Health Organization (WHO). Biomacromolecules, isolated or synthesized from a natural template, have therapeutic properties which have not yet been fully studied, and represent an unexplored potential in the search for new drugs. These substances, starting from conglomerates of proteins and other substances such as animal venoms, or from minor substances such as bioactive peptides, help fight diseases or counteract harmful effects. The high effectiveness of these biomacromolecules makes them promising substances for obtaining new drugs; however, their low bioavailability or stability in biological systems is a challenge to be overcome in the coming years with the help of nanotechnology. The objective of this review article is to describe the relationship between the structure and function of biomacromolecules of animal origin that have applications already described using nanotechnology and targeted delivery.

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.

Baltetin: a new C-type lectin-like isolated from Bothrops alternatus snake venom which act as a platelet aggregation inhibiting.

C-type lectin-like proteins found in snake venom, known as snaclecs, have important effects on hemostasis through targeting membrane receptors, coagulation factors and other hemostatic proteins. Here, we present the isolation and functional characterization of a snaclec isolated from Bothrops alternatus venom, designated as Baltetin. We purified the protein in three chromatographic steps (anion-exchange, affinity and reversed-phase chromatography). Baltetin is a dimeric snaclec that is approximately 15 and 25 kDa under reducing and non-reducing conditions, respectively, as estimated by SDS-PAGE. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and Edman degradation sequencing revealed that Baltetin is a heterodimer. The first 40 amino acid residues of the N-terminal region of Baltetin subunits share a high degree of sequence identity with other snaclecs. Baltetin had a specific, dose-dependent inhibitory effect on epinephrine-induced platelet aggregation in human platelet-rich plasma, inhibiting up to 69% of platelet aggregation. Analysis of the infrared spectra suggested that the interaction between Baltetin and platelets can be attributed to the formation of hydrogen bonds between the PO32- groups in the protein and PO2- groups in the platelet membrane. This interaction may lead to membrane lipid peroxidation, which prevents epinephrine from binding to its receptor. The present work suggests that Baltetin, a new C-type lectin-like protein isolated from B. alternatus venom, is the first snaclec to inhibit epinephrine-induced platelet aggregation. This could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

Understanding the mechanism of action of peptide (p-BthTX-I)2 derived from C-terminal region of phospholipase A2 (PLA2)-like bothropstoxin-I on Gram-positive and Gram-negative bacteria.

Based on the antimicrobial activity of bothropstoxin-I (BthTX-I) and on the premise that a C-terminal peptide of Lys49 myotoxin can reproduce the antimicrobial activity of the parent protein, we aimed to study the mechanism of action of a peptide derived from the C-terminal region of the myotoxin BthTX-I [(p-BthTX-I)2, sequence: KKYRYHLKPFCKK, disulfide-linked dimer] against Gram-positive and Gram-negative bacteria. Fluorescence quenching technique showed that the carboxyfluorescein labeled-peptide [CF-(p-BthTX-I)2] when incubated with E. coli displayed a superior penetration activity than when incubated with S. aureus. Cell death induced by the peptide (p-BthTX-I)2 showed a loss of membrane integrity in E. coli and S. aureus; however, the mechanisms of cell death were different, characterized by the presence of necrosis-like and apoptosis-like deaths, respectively. Scanning electron microscopy studies in E. coli and S. aureus showed morphological changes in the cells, with superficial deformities, appearance of wrinkles and bubbles, and formation of vesicles. Our results demonstrate that the mechanism of action of the peptide (p-BthTX-I)2 is different in Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. Knowledge of the mechanism of action of these peptides is important, since they are promising prototypes for new antimicrobial drugs.

In vitro tracking of phospholipase A2 from snake venom conjugated with magic-sized quantum dots.

Phospholipases A2 represent a family of enzymes with important application in medicine. However, direct tracking is difficult due to the absence of a stable, effective and specific marker for these enzymes. Magic-sized quantum dots (MSQDs) are inorganic semiconducting nanocrystals with unique physical properties. They have the ability to conjugate to proteins, making them excellent markers for biological systems. In this work, we labelled phospholipase A2 from Bothrops alternatus snake venom with Cadmium selenide (CdSe)/cadmium sulphate (CdS) MSQDs-a biocompatible and luminescent probe-. Bioconjugation was confirmed using infrared spectra and fluorescence microscopy, which demonstrated that the CdSe/CdS MSQDs interact with phospholipase A2 without interfering with its activity. This probe may be an important tool for the elucidation of many biological mechanisms, because it allows the pathway of phospholipase A2 to be tracked from its entry through the plasma membrane until its incorporation into the nucleus of myoblasts.

Antimicrobial peptide-loaded liquid crystalline precursor bioadhesive system for the prevention of dental caries.

BACKGROUND: Anticaries agents must interfere with the adhesion of Streptococcus mutans and its proliferation in dental biofilm, without causing host toxicity and bacterial resistance. Natural substances, including cationic antimicrobial peptides (CAMPs) and their fragments, such as ß-defensin-3 peptide fragment (D1-23), have been widely studied. However, the chemical and physical stability of CAMPs may be compromised by external factors, such as temperature and pH, reducing the period of antimicrobial activity. METHODS: To overcome the aforementioned disadvantage, this study developed and character-ized a drug delivery system and evaluated the cytotoxicity and effect against S. mutans biofilm of a D1-23-loaded bioadhesive liquid crystalline system (LCS). LCS was composed of oleic acid, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol, Carbopol® 974P and Carbopol® 971P. LCS was analyzed by polarized light microscopy (PLM), rheology (viscoelasticity and flow properties) and in vitro bioadhesion. The viability of epithelial cells was evaluated. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans were determined for D1-23 for further evaluation of the effect against S. mutans biofilm after 4 and 24 h of exposure to treatments. RESULTS: PLM, rheology, and in vitro bioadhesion tests showed that both viscosity and bioadhesion of LCS increased after it was diluted with artificial saliva. D1-23-loaded LCS system presented better activity against S. mutans biofilm after 24 h when compared to 4 h of treatment, showing a cumulative effect. Neither LCS nor D1-23-loaded LCS presented toxicity on human epithelial cells. CONCLUSION: D1-23-loaded LCS is a promising drug delivery system for the prevention of dental caries.

Antimicrobial Photodynamic therapy enhanced by the peptide aurein 1.2.

In the past few years, the World Health Organization has been warning that the post-antibiotic era is an increasingly real threat. The rising and disseminated resistance to antibiotics made mandatory the search for new drugs and/or alternative therapies that are able to eliminate resistant microorganisms and impair the development of new forms of resistance. In this context, antimicrobial photodynamic therapy (aPDT) and helical cationic antimicrobial peptides (AMP) are highlighted for the treatment of localized infections. This study aimed to combine the AMP aurein 1.2 to aPDT using Enterococcus faecalis as a model strain. Our results demonstrate that the combination of aPDT with aurein 1.2 proved to be a feasible alternative capable of completely eliminating E. faecalis employing low concentrations of both PS and AMP, in comparison with the individual therapies. Aurein 1.2 is capable of enhancing the aPDT activity whenever mediated by methylene blue or chlorin-e6, but not by curcumin, revealing a PS-dependent mechanism. The combined treatment was also effective against different strains; noteworthy, it completely eliminated a vancomycin-resistant strain of Enterococcus faecium. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections as an alternative to antibiotics.

LmrBPP9: A synthetic bradykinin-potentiating peptide from Lachesis muta rhombeata venom that inhibits the angiotensin-converting enzyme activity in vitro and reduces the blood pressure of hypertensive rats.

Bradykinin-potentiating peptides (BPPs) are an important group of toxins present in Lachesis muta rhombeata venom. They act directly at renin-angiotensin-aldosterone system, through the inhibition of angiotensin-converting enzyme (ACE). This action may contribute to the hypotensive shock observed during the envenoming by this species. Thus, the main goal of this study was the solid-phase synthesis of a BPP found in L. m. rhombeata venom and its in vitro and in vivo characterization in relation to ACE inhibition and hypotensive activity, respectively. The LmrBPP9 peptide was synthesized using an automated solid-phase peptide synthesizer and purified by reversed-phase fast protein liquid chromatography (FPLC). The in vitro IC50 of the synthetic peptide is 4.25 ±â€¯0.10 µM, showing a great capacity of ACE inhibition. The in vivo studies showed that LmrBPP9 induces blood pressure reduction, both in normotensive and hypertensive rats, being more pronounced in the last ones. These results agree with the in vitro results, showing that the synthetic peptide LmrBPP9 is a potential molecule to the development of a new antihypertensive drug.

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens.

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3-1CV and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 µg ml-1, respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.

Identification and characterisation of elongation factor Tu, a novel protein involved in Paracoccidioides brasiliensis-host interaction.

Paracoccidioides spp., which are temperature-dependent dimorphic fungi, are responsible for the most prevalent human systemic mycosis in Latin America, the paracoccidioidomycosis. The aim of this study was to characterise the involvement of elongation factor Tu (EF-Tu) in Paracoccidioides brasiliensis-host interaction. Adhesive properties were examined using recombinant PbEF-Tu proteins and the respective polyclonal anti-rPbEF-Tu antibody. Immunogold analysis demonstrated the surface location of EF-Tu in P. brasiliensis. Moreover, PbEF-Tu was found to bind to fibronectin and plasminogen by enzyme-linked immunosorbent assay, and it was determined that the binding to plasminogen is at least partly dependent on lysine residues and ionic interactions. To verify the participation of EF-Tu in the interaction of P. brasiliensis with pneumocytes, we blocked the respective protein with an anti-rPbEF-Tu antibody and evaluated the consequences on the interaction index by flow cytometry. During the interaction, we observed a decrease of 2- and 3-fold at 8 and 24 h, respectively, suggesting the contribution of EF-Tu in fungal adhesion/invasion.

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom.

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bß chain and BpirSP41 on both Aα and Bß chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of ∼3.5 µg versus 20 µg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu(2+) ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs.