Guilherme D. Brand

Phylloseptins: a novel class of anti-bacterial and anti-protozoan peptides from the Phyllomedusa genus.

Six novel peptides called phylloseptins (PS-1, -2, -3, -4, -5, and -6) showing anti-bacterial (PS-1) and anti-protozoan (PS-4 and -5) activities were isolated from the skin secretion of the Brazilian tree-frogs, Phyllomedusa hypochondrialis and Phyllomedusa oreades. Phylloseptins have a primary structure consisting of 19-21 amino acid residues (1.7-2.1 kDa). They have common structural features, such as a highly conserved N-terminal region and C-terminal amidation. Phylloseptin-1 (FLSLIPHAINAVSAIAKHN-NH2) demonstrated a strong effect against gram-positive and gram-negative bacteria (MICs ranging from 3 to 7.9 microM), without showing significant hemolytic activity (<0.6% at the MIC range) towards mammalian cells. Atomic force microscopy experiments indicated that the bacteriolytic properties of these peptides might be related to their disruptive action on the cell membrane, characterized by a number of bubble-like formations, preceding every cell lysis. PS-4 and PS-5 showed anti-protozoan activity with IC50 at about 5 microM for Trypanosoma cruzi.

Dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta. Anti-Trypanosoma cruzi activity without cytotoxicity to mammalian cells.

Amphibian skin secretions are known as a rich source of biologically active molecules, most of which are alkaloids, biogenic amines, and peptides. Dermaseptins are a class of antimicrobial peptides present in tree frogs of thePhyllomedusa genus. They are cationic molecules of 28–34 residues that permeabilize the membrane of Gram-positive and Gram-negative bacteria, yeasts, and filamentous fungi, showing little or no hemolytic activity. This work reports the isolation, molecular mass analysis, primary structure determination, biological activities, and potential therapeutic applications of an antimicrobial peptide found in the skin secretion of Phyllomedusa oreades, which is a newly described amphibian species endemic of the Brazilian savanna. DS 01 is a 29-residue-long peptide with a molecular mass of 2793.39 Da showing antibacterial properties against Gram-positive and Gram-negative bacteria in the range of 3–25 μm. Anti-protozoan activity was investigated using T. cruzi in its trypomatigote and epimastigote forms cultivated in both cell culture and blood media. Within 2 h after incubation with DS 01 at a final concentration of ∼6 μm, no protozoan cells were detected. Two synthetic dermaseptins, described previously by our group and named dermadistinctins K and L (DD K and DD L), also had their anti-Trypanosoma cruzi activity investigated and demonstrated similar properties. Toxicity of DS 01 to mouse erythrocytes and white blood cells was evaluated by means of atomic force microscopy and flow cytometry. No morphological alterations were observed at a lytic concentration of DS 01, suggesting its therapeutic value especially as an anti-T. cruzi agent to prevent infections during blood transfusion.

Bradykinin-related peptides from Phyllomedusa hypochondrialis

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.

The Skin Secretion of the Amphibian Phyllomedusa nordestina: A Source of Antimicrobial and Antiprotozoal Peptides

Antimicrobial peptides (AMPs) from the dermaseptin and phylloseptin families were isolated from the skin secretion of Phyllomedusa nordestina, a recently described amphibian species from Northeastern Brazil. One dermaseptin and three phylloseptins were chosen for solid phase peptide synthesis. The antiprotozoal and antimicrobial activities of the synthetic peptides were determined, as well as their cytotoxicity in mouse peritoneal cells. AMPs are being considered as frameworks for the development of novel drugs inspired by their mechanism of action.

The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance

DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies.

The Antifungal Plant Defensin HsAFP1 Is a Phosphatidic Acid-Interacting Peptide Inducing Membrane Permeabilization

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

Towards an experimental classification system for membrane active peptides

Mature proteins can act as potential sources of encrypted bioactive peptides that, once released from their parent proteins, might interact with diverse biomolecular targets. In recent work we introduced a systematic methodology to uncover encrypted intragenic antimicrobial peptides (IAPs) within large protein sequence libraries. Given that such peptides may interact with membranes in different ways, resulting in distinct observable outcomes, it is desirable to develop a predictive methodology to categorize membrane active peptides and establish a link to their physicochemical properties. Building upon previous work, we explored the interaction of a range of IAPs with model membranes probed by differential scanning calorimetry (DSC) and circular dichroism (CD) techniques. The biophysical data were submitted to multivariate statistical methods and resulting peptide clusters were correlated to peptide structure and to their antimicrobial activity. A re-evaluation of the physicochemical properties of the peptides was conducted based on peptide cluster memberships. Our data indicate that membranolytic peptides produce characteristic thermal transition (DSC) profiles in model vesicles and that this can be used to categorize novel molecules with unknown biological activity. Incremental expansion of the model presented here might result in a unified experimental framework for the prediction of novel classes of membrane active peptides.

Synthesis and structure–activity relationships of novel arylpiperazines as potent antagonists of α1-adrenoceptor

Arylpiperazines 2-11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional experiments in isolated rat vas deferens (α1A), spleen (α1B), and aorta (α1D). Modifications at the 1,3-benzodioxole and phenyl phamacophoric units resulted in the identification of a number of potent compounds (moderately selective with respect to the α1b-AR), in binding experiments. Notably, compound 7 (LDT451) showed a subnanomolar pKi of 9.41 towards α1a-AR. An encouragingly lower α1B-potency was a general trend for all the series of compounds, which showed α1A/D over α1B selectivity in functional assays. If adequately optimized, such peculiar selectivity could have relevance for a potential LUTS/BPH therapeutic application.

Intragenic antimicrobial peptides from Theobroma cacao.

Background It is well known that many bioactive peptides (intragenic) are encrypted in source proteins and that they can exert their function once released by proteolytic cleavage; e.g. hypotensive, opioids and antimicrobial peptides. However, other bioactive peptides may be “stuck” on a polipeptide chain with no cleavage sites for its release. These “non-obvious” intragenic peptides are also of interest in the search for new biologically active peptides, mainly antimicrobial peptides, in an alternative way for new drug discovery and for the control of different phytopathogens, mainly fungi, that can cause several losses to different crops of interest; e.g. rice, soybean, common bean, cocoa. In Brazil, Theobroma cacao production can be decimate by the basidiomycete Moniliophtora perniciosa, the causative agent of cocoa witch’s broom disease. In this report we present preliminary results of the search, synthesis and activity of intragenic antimicrobial peptides (IAPs) selected from Theobroma cacao genome.