Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity.

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100­alanyl­hexadecyl­1­amine (BP100-Ala-NH-C16H33) and cyclo(1­4)­d­Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1­4)­cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1­4)­cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Biochemical characterisation of a Kunitz-type inhibitor from Tamarindus indica L. seeds and its efficacy in reducing plasma leptin in an experimental model of obesity.

A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M + 14H]+ = 19,594,690 Da and [M + 13H]+ = 19,578,266 Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the same sequence. pTTI presented resistance to temperature and pH variations; IC50 was 2.7 × 10-10 mol.L-1 and Ki was 2.9 × 10-11 mol.L-1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and one near pH 8. pTTI action on leptin decrease was confirmed. We conclude that pTTI is a Kunitz trypsin inhibitor with possible biotechnological health-related application.

Evaluation of in vitro biological properties of Senna occidentalis (L) Link / Avaliação in vitro de propriedades biológicas de Senna occidentalis (L) Link

Senna species have been widely used by American, African and Indian ethic groups mainly in the treatment of feebleness, constipation, liver disorders and skin infections. Senna occidentalis (L.) Link is a perennial shrub native to South America and indigenous to tropical regions throughout the world. Current study evaluated the antimicrobial activity of aqueous and hydroalcoholic extracts from S. occidentalis prepared from different parts of the plant. Antimicrobial activity was assessed against standard pharmaceutical microorganisms by spectrophotometry and microdilution technique. Escherichia coli was sensitive only to compounds extracted from seeds which may be proteinaceous. A broader antimicrobial spectrum was demonstrated by the hydroalcoholic extract of seeds, mostly against Pseudomonas aeruginosa. The in vitro toxicity using mouse fibroblasts indicated that the extract might be a biocompatible ingredient for topical formulations, while the hydroalcoholic extract of aerial parts demonstrated to be potentially cytotoxic.

Espécies de Senna são amplamente utilizadas por tribos americanas, africanas e indianas, principalmente para tratar a fraqueza, a constipação, as desordens do fígado e também em preparações tópicas para infecções de pele. A Senna occidentalis (L.) Link é um arbusto perene nativo da América do Sul encontrado em regiões tropicais. Este trabalho avaliou a atividade antimicrobiana de extratos aquosos e hidroalcoólicos de diferentes partes da planta. A atividade antimicrobiana foi estabelecida frente aos microrganismos padrões farmacêuticos por espectrofotometria e técnica de microdiluição. A Escherichia coli apresentou sensibilidade apenas a componentes extraídos das sementes, os quais podem ser de natureza proteica. O espectro mais amplo de atividade antimicrobiana foi obtido com o extrato hidroalcoólico das sementes, principalmente contra Pseudomonas aeruginosa. A toxicidade in vitro utilizando fibroblastos de camundongo indicou que este extrato pode ser um ingrediente biocompatível para formulações de uso tópico. Já o extrato hidroalcoólico de partes aéreas demonstrou ser potencialmente citotóxico.

Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.

Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

Effects of a novel pathogenesis-related class 10 (PR-10) protein from Crotalaria pallida Roots with papain inhibitory activity against root-knot nematode Meloidogyne incognita.

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M(r) of 15 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K(i) value of 1.8 x 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC)-CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.

Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.

A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.

Isolation and structural characterization of microcystin-LR and three minor oligopeptides simultaneously produced by Radiocystis feernandoi (Chroococcales, Cyanobacteriae): a Brazilian toxic cyanobacterium.

Several blooms of cyanobacteria naturally occurring in freshwater reservoirs have been associated to numerous fatalities and cases of livestock and human poisoning. Microcystins (Mcs) are the most frequently found cyclic heptapeptide toxins in the cyanobacterial extracts. In previous work, Radiocystis fernandoi (strain SPC 714) lyophilized extracts were found to be hepatotoxic to mice with LD100 of about 60 mg kg(-1) and Mc LR was suggested as responsible for that toxicity. Here, we describe the isolation of four oligopeptides from R. fernandoi methanol extract by reversed-phase high performance liquid chromatography (RP-HPLC). The major component, which eluted with 65% acetonitrile from acetonitrile/water gradient, was identified as Mc-LR and its structure was confirmed by the presence of molecular related ion species [M+H]+ at m/z 996.3, ([M+H-Adda])+ at m/z 861.5, [Arg-Adda-Glu+H]+ at m/z 599.8, and [PhCH2CH(OMe)]+ at m/z 135.1 in the ESI spectra. Two components corresponding to small signals eluted from C18 column, respectively, with 44 and 45% acetonitrile had their structures proposed as isomers of aeruginosin derivatives showing molecular ions at m/z 651.7 and a [CHOI]+ immonium at m/z 140.1. Finally, the structure of the third minor and most hydrophobic component (68% acetonitrile elution) isolated from R. fernandoi extract seemed to correspond to a cyclic cyanopeptolin like micropeptin K139, a trypsin inhibitor firstly isolated from Microcystis aeruginosa, showing similar ions fragmentation pattern and [M+H]+ at m/z 987.6 in its ESI spectra.

Introduction of a chemical constraint in a short peptide derived from human acidic fibroblast growth factor elicits mitogenic structural determinants.

Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.

Peptídeos de conformação restrita relacionados ao sítio 2 do fator de crescimento de fibroblastos ácido humano (hFGF-1): estudo sobre estrutura e atividade / Study of structure-activity relationship of synthetic peptides derived from human acidic Fibroblast Growth Factor Site 2 primary sequence

Na busca por agonistas, antagonistas e inibidores de natureza peptídica do fator de crescimento de fibroblastos ácido humano (hFGF-1), iniciamos o presente trabalho fazendo uma análise conformacional teórica do peptídeo Ac-WFVGLKKNGSSKRGPRT-N`H IND. 2ï (107-123[hFGF-1]). Em trabalho anterior, este composto havia se mostrado um agonista da atividade mitogênica da proteína capaz de inibir a ligação de `ANTPOT. 125Iï-hFGF-1 aos seus receptores celulares e de se ligar à resina heparina-Sepharose (Oyama et al. 1996). Os cálculos das propriedades dinâmicas deste peptídeo (I; Tabela 1) demonstraram que ele não adotava nenhuma conformação preferencial, o que poderia justificar a baixa atividade apresentada pelo mesmo (`10 POT. 4ï vêzes menor do que a da proteína nativa)...

Induction of mitogenesis by synthetic peptides related to the structure of human fibroblast growth factor-1

The synthetic peptide Ac-WFVGLKKNGSSKRGPRT-NH2,related to the human FGF-1 sequence, was shown to be mitogenic upon Balb/c 3T3 fibroblasts in culture (ED50=10-20 muM) and to compete with the growth factor for cellular binding (ID50 = 30-50 muM). The results described suggest that the mitogenic activity of the peptide is dependent on the presence of the residues 122-127 (WFVGLK) in its structure. Also, its affinity for the cellular receptors seems to be dependent on the presence of residues that are important for FGF-heparin binding such as K127, K133 and R137.