Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.

Two Tachykinin-Related Peptides with Antimicrobial Activity Isolated from Triatoma infestans Hemolymph.

Antimicrobial peptides and proteins (AMPs) are molecules that can interact with microbial cells and lead to membrane disruption or intracellular molecule interactions and death. Several molecules with antimicrobial effects also present other biological activities. One such protein group representing the duplicity of activities is the tachykinin family. Tachykinins (TKs) form a family of neuropeptides in vertebrates with a consensus C-terminal region (F-X-G-Y-R-NH2). Invertebrate TKs and TK-related peptides (TKRPs) are subfamilies found in invertebrates that present high homology with TKs and have similar biological effects. Several of these molecules have already been described but reports of TKRP in Hemiptera species are limited. By analyzing the Triatoma infestans hemolymph by reversed-phase high-performance liquid chromatography, biological assays, and mass spectrometry, two antimicrobial molecules were isolated and identified as TKRPs, which we named as TRP1-TINF and TRP2-TINF (tachykinin-related peptides I and II from T. infestans). TRP1-TINF is a random secondary structure peptide with 9 amino acid residues. It is susceptible to aminopeptidases degradation and is active mainly against Micrococcus luteus (32 µM). TRP2-TINF is a 10-amino acid peptide with a 310 helix secondary structure and is susceptible to carboxypeptidases degradation. It has major antimicrobial activity against both Pseudomonas aeruginosa and Escherichia coli (45 µM). Neither molecule is toxic to human erythrocytes and both present minor toxicity toward Vero cells at a concentration of 1000 µM. As the first description of TKRPs with antimicrobial activity in T. infestans, this work contributes to the wider comprehension of the insects' physiology and describes pharmacological relevant molecules.

The interaction of sodium trimetaphosphate with collagen I induces conformational change and mineralization that prevents collagenase proteolytic attack.

OBJECTIVES: This study evaluated the cell viability and expression of different major genes involved in mineralization in odontoblast-like cells exposed to sodium trimetaphosphate (STMP). It was also investigated the influence of STMP on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization. METHODS: Immortalized rat odontoblast MDPC-23 cells were cultured. Cell viability was assessed by trypan blue staining, and the changes in gene expression balance induced by STMP were assessed by quantitative reverse transcription (qRT) PCR assays. Crystalline particle formation was monitored by light-scattering detectors to estimate pH variation and the radial size of the crystalline particles as a function of reaction time (pH 7.4, 25°C) in the presence of STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under atomic force microscopy (AFM) to measure the particle size in the presence of STMP. A three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams after immersion in STMP solution. The binding mechanism of STMP to collagen fibrils and potential stabilization mechanism was assessed with circular dichroism spectrometry (CD). The data were analyzed statistically (α=0.05). RESULTS: STMP had no significant influence on the cell viability and gene expression of the MDPC-23 cells. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM-treated crystal size. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen. SIGNIFICANCE: The increased resistance of collagen against the proteolytic activity of collagenases appears to be related to the conformational change induced by STMP binding in collagen I and the STMP capacity for promoting biomimetic mineralization in type I collagen fibrils.

IsCT-based analogs intending better biological activity.

IsCT1-NH2 is a cationic antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis that has a tendency to form an α-helical structure and shows potent antimicrobial activity and also inopportunely shows hemolytic effects. In this study, five IsCT1 (ILGKIWEGIKSLF)-based analogs with amino acid modifications at positions 1, 3, 5, or 8 and one analog with three simultaneous substitutions at the 1, 5, and 8 positions were designed. The net charge of each analog was between +2 and +3. The peptides obtained were characterized by mass spectrometry and analyzed by circular dichroism for their structure in different media. Studies of antimicrobial activity, hemolytic activity, and stability against proteases were also carried out. Peptides with a substitution at position 3 or 5 ([L]3 -IsCT1-NH2 , [K]3 -IsCT1-NH2 , or [F]5 -IsCT1-NH2 ) showed no significant change in an activity relative to IsCT1-NH2 . The addition of a proline residue at position 8 ([P]8 -IsCT1-NH2 ) reduced the hemolytic activity as well as the antimicrobial activity (MIC ranging 3.13-50 µmol L-1 ), and the addition of a tryptophan residue at position 1 ([W]1 -IsCT1-NH2 ) increased the hemolytic activity (MHC = 1.56 µmol L-1 ) without an improvement in antimicrobial activity. The analog [A]1 [F]5 [K]8 -IsCT1-NH2 , which carries three simultaneous modifications, presented increasing or equivalent values in antimicrobial activity (MIC approximately 0.38 and 12.5 µmol L-1 ) with a reduction in hemolytic activity. In addition, this analog presented the best resistance against proteases. This kind of strategy can find functional hotspots in peptide molecules in an attempt to generate novel potent peptide antibiotics.

Angiotensin II-derived constrained peptides with antiplasmodial activity and suppressed vasoconstriction.

Angiotensin II (Ang II) is a natural mammalian hormone that has been described to exhibit antiplasmodial activity therefore constituting a promising alternative for the treatment of malaria. Despite its promise, the development of Ang II as an antimalarial is limited by its potent induction of vasoconstriction and its rapid degradation within minutes. Here, we used peptide design to perform targeted chemical modifications to Ang II to generate conformationally restricted (disulfide-crosslinked) peptide derivatives with suppressed vasoconstrictor activity and increased stability. Designed constrained peptides were synthesized chemically and then tested for antiplasmodial activity. Two lead constrained peptides were identified (i.e., peptides 1 and 2), each composed of 10 amino acid residues. These peptides exhibited very promising activity in both our Plasmodium gallinaceum (>80%) and Plasmodium falciparum (>40%) models, an activity that was equivalent to that of Ang II, and led to complete suppression of vasoconstriction. In addition, peptide 5 exhibited selective activity towards the pre-erythrocytic stage (98% of activity against P. gallinaceum), thus suggesting that it may be possible to design peptides that target specific stages of the malaria life cycle. The Ang II derived stable scaffolds presented here may provide the basis for development of a new generation of peptide-based drugs for the treatment of malaria.

Angiotensin II analogues with N-terminal lactam bridge cyclization: an overview on AT1 receptor activation and tachyphylaxis.

Angiotensin II (AngII) is the final active product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. To investigate the effect of N-terminal cyclization on AT1 activation and tachyphylaxis, we designed conformationally constrained analogues with an i-(i + 1) lactam bridge. All analogues presented the same binding coefficient and tachyphylactic index, but some of them such as Cyclo (0-1a) [Glu0 , endo-(Lys1a )]-AngII and Cyclo (0-1a) [Asp0 , endo-(Orn1a )]-AngII showed higher potency. The same tachyphylactic index presented by AngII and cyclic analogues was surprising. We expected a variation after the modification of AngII N-terminal region.

Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes.

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d-amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so-called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all-D-AII and all-D-VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro-AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a ß-turn conformation in different solutions. In the presence of membrane-mimetic micelles, AII had also a ß-turn conformation, while retro-AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro-AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro-AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation.

Identification and characterisation of serine protease inhibitors from Araucaria angustifolia seeds.

Araucaria angustifolia seeds are characterised by a relatively high content of starch and protein. This study aimed to verify the presence of α-amylase inhibitors in the seeds and to characterise a trypsin inhibitor found in the embryo tissues. Inhibitor purification was carried out by the saline extraction of proteins, acetone precipitation and affinity chromatography. Two protein bands of molecular weight estimated by SDS-PAGE at about 35 kDa were further examined by high-performance liquid chromatography coupled to a mass spectrometer and were shown to be 36.955 Da (AaTI-1) and 35.450 Da (AaTI-2). The sequence of the N-terminal region shows that AaTI-1 and AaTI-2 are structurally similar to plant inhibitors of the serpin family. A mixture of AaTI-1 and AaTI-2, identified as AaTI, shows selectivity for the inhibition of trypsin (Kiapp 85 nM) and plasmin (Kiapp 7.0 µM), but it does not interfere with the chymotrypsin, human plasma kallikrein, porcine kallikrein or other coagulation enzymes activity.

New linear antiplasmodial peptides related to angiotensin II.

BACKGROUND: Antiplasmodial activities of angiotensin II and its analogues have been extensively investigated in Plasmodium gallinaceum and Plasmodium falciparum parasite species. Due to its vasoconstrictor property angiotensin II cannot be used as an anti-malarial drug. METHODS: This work presents the solid-phase syntheses and liquid chromatography and mass spectrometry characterization of ten linear peptides related to angiotensin II against mature P. gallinaceum sporozoites and erythrocyte invasion by P. falciparum. Conformational analyses were performed by circular dichroism. IC50 assays were performed to identify the ideal concentration used on the biological tests and haemolytical erythrocytic assays were made to verify the viability of the biological experiments. The contractile responses of the analogues were made to evaluate if they are promising candidates to be applied as antiplasmodial drugs. RESULTS: The results indicate two short-peptides constituted by hydrophobic residues (5 and 6) with antiplasmodial activity in these models, 89 and 94 % of biological activity against P. gallinaceum sporozoite, respectively, and around 50 % of activity against P. falciparum. Circular dichroism spectra suggested that all the peptides adopted ß-turn conformation in different solutions, except peptide 3. Besides the biological assays IC50, the haemolysis assays and contractile response activities were applied for peptides 5 and 6, which did not present expressive results. CONCLUSIONS: The hydrophobic portion and the arginine, tyrosine, proline, and phenylalanine, when present on peptide primary sequence, tend to increase the antiplasmodial activity. This class of peptides can be explored, as anti-malarial drugs, after in vivo model tests. Graphical abstract: The most active peptide presented 94 % activity on P. gallinaceum sporozoites and 53 % inhibited P. falciparum ring forms invasion.

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin-like activities.

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI51-62 , and a related analog (P2 ) in which a proline residue was inserted in order to mimic the N-terminal region of the bradykinin molecule. The related retro-inverso counterparts Ri-BbKI51-62 and Ri-P2 were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI51-62 nor its Ri-BbKI51-62 analog exhibit bradykinin-like activity, although the retro-inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P2 and Ri-P2 presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B-2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects.

Effects of the angiotensin II Ala-scan analogs in erythrocytic cycle of Plasmodium falciparum (in vitro) and Plasmodium gallinaceum (ex vivo).

The anti-plasmodium activity of angiotensin II and its analogs have been described in different plasmodium species. Here we synthesized angiotensin II Ala-scan analogs to verify peptide-parasite invasion preservation with residue replacements. The analogs were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (t-Boc) solid phase methods, purified by liquid chromatography and characterized by mass spectrometry. The results obtained in Plasmodium falciparum assays indicated that all analogs presented some influence in parasite invasion, except [Ala(4)]-Ang II (18% of anti-plasmodium activity) that was not statistically different from control. Although [Ala(8)]-Ang II presented a lower biological activity (20%), it was statistically different from control. The most relevant finding was that [Ala(5)]-Ang II preserved activity (45%) relative to Ang II (47%). In the results of Plasmodium gallinaceum assays all analogs were not statistically different from control, except [Ala(6)]-Ang II, which was able to reduce the parasitemia about 49%. This approach provides insight for understanding the importance of each amino acid on the native Ang II sequence and provides a new direction for the design of potential chemotherapeutic agents without pressor activity.

Antiplasmodial activity study of angiotensin II via Ala scan analogs.

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid-phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide-capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile(5) or the His(6) residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala(6) ]-AII (79%), and [Ala(5) ]-AII (75%). Analogs [Ala(3) ]-AII, [Ala(1) ]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala(8) ]-AII, [Ala(7) ]-AII, [Ala(4) ]-AII, and [Ala(2) ]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a ß-fold conformation in different solutions. All AII analogs, except [Ala(4) ]-AII and [Ala(8) ]-AII, show contractile responses and interact with the AT1 receptor, [Ala(5) ]-AII and [Ala(6) ]-AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds.