Purification and Biological Properties of Raniseptins-3 and -6, Two Antimicrobial Peptides from Boana raniceps (Cope, 1862) Skin Secretion.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

Ts17, a Tityus serrulatus ß-toxin structurally related to α-scorpion toxins.

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Nav1.1-Nav1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na+-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Nav1.2 and Nav1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Nav1.5. Circular dichroism spectra showed that Ts17 presents mainly ß-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug. Key contribution Purification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, ß-toxin classification.

Crystal structure and physicochemical characterization of a phytocystatin from Humulus lupulus: Insights into its domain-swapped dimer.

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Figainin 1, a Novel Amphibian Skin Peptide with Antimicrobial and Antiproliferative Properties.

Amphibian skin secretions are abundant in bioactive compounds, especially antimicrobial peptides. These molecules are generally cationic and rich in hydrophobic amino acids, have an amphipathic structure and adopt an α-helical conformation when in contact with microorganisms membranes. In this work, we purified and characterized Figainin 1, a novel antimicrobial and antiproliferative peptide from the cutaneous secretion of the frog Boana raniceps. Figainin 1 is a cationic peptide with eighteen amino acid residues-rich in leucine and isoleucine, with an amidated C-terminus-and adopts an α-helical conformation in the presence of trifluoroethanol (TFE). It displayed activity against Gram-negative and especially Gram-positive bacteria, with MIC values ranging from 2 to 16 µM, and showed an IC50 value of 15.9 µM against epimastigote forms of T. cruzi; however, Figanin 1 did not show activity against Candida species. This peptide also showed cytolytic effects against human erythrocytes with an HC50 of 10 µM, in addition to antiproliferative activity against cancer cells and murine fibroblasts, with IC50 values ranging from 10.5 to 13.7 µM. Despite its adverse effects on noncancerous cells, Figainin 1 exhibits interesting properties for the development of new anticancer agents and anti-infective drugs against pathogenic microorganisms.

Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 µg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

Optimizing liposomes for delivery of Bowman-Birk protease inhibitors - Platforms for multiple biomedical applications.

One of the major challenges in the administration of therapeutic proteins involves delivery limitations. Liposomes are well-known drug delivery systems (DDS) that have been used to overcome this drawback; nevertheless, low protein entrapment efficiency (EE) still limits their wide biomedical application on a commercial scale. In the present work, different methods for protein entrapment into liposomes were tested in order to obtain tailored DDS platforms for multiple biomedical applications. The protein used as model was the Black-eyed pea Trypsin and Chymotrypsin Inhibitor (BTCI), a member of the Bowman-Birk protease inhibitor family (BBIs), which has been largely explored for its potential application in many biomedical therapies. We optimized reverse-phase evaporation (REV) and freeze/thaw (F/T) entrapment methods, using a cationic lipid matrix to entrap expressive amounts of BTCI (∼100 µM) in stable liposomes without affecting its protease inhibition activity. The influence of various parameters (e.g. entrapment method, liposome composition, buffer type) on particle size, charge, polydispersity, and EE of liposomes was investigated to provide an insight on how to control such parameters in view of obtaining a high entrapment yield. In addition, BTCI liposome platforms obtained herein showed to be versatile vesicles, allowing surface modification with moieties/polymers of interest (e.g. PEG, transferrin). The aforementioned results are relevant to focusing on the entrapment of other promising BBIs or protein agents sharing similar structural features. These findings encourage future studies to investigate the advantages of using the liposome platforms presented herein to broaden the use of this type of DDS for BBI biomedical applications.

Dendropsophin 1, a novel antimicrobial peptide from the skin secretion of the endemic Colombian frog Dendropsophus columbianus

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher -helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.

Dendropsophin 1, a novel antimicrobial peptide from the skin secretion of the endemic Colombian frog Dendropsophus columbianus

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher -helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.