Antonio Miranda

Universidade Federal de São Paulo, San Paulo, São Paulo, Brazil

Are you Antonio Miranda?

Claim your profile

Publications (78)179.31 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. 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. IC 50 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. 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 β-turn conformation in different solutions, except peptide 3. Besides the biological assays IC 50 , the haemolysis assays and contractile response activities were applied for peptides 5 and 6, which did not present expressive results. 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
    Full-text · Article · Nov 2015 · Malaria Journal
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gomesin (Gm) is an antimicrobial peptide first isolated from the hemolymph of a Brazilian spider. Its powerful antimicrobial activity is, however, accompanied by hemolysis. As an alternative to this issue, a linear analogue (named GmL) lacking the disulfide bonds was designed. Here, CD spectroscopy, a fluorescence-based leakage assay, isothermal titration calorimetry (ITC) and light scattering are used to study the interaction of both Gm and GmL with large unilamellar vesicles (LUVs) composed of POPC (palmitoyl oleoyl phosphatidylcholine) with 25 and 50 mol% POPG (palmitoyl oleoyl phosphatidylglycerol). The activities of Gm and GmL in respect to their binding affinity/enthalpy, ability to permeabilize membranes and to induce vesicle aggregation are correlated with peptide secondary structure. Whereas Gm displays a quite stable β-hairpin motif irrespective of the environment, GmL assumes a random conformation in aqueous solution and in the presence of 25 mol% POPG but adopts a β-like structure in the presence of 50 mol% POPG. Gm exhibited high lytic activity against both surface charge densities. Instead, the activity of GmL was found to be negligible in the presence of 25 mol% POPG LUVs, but comparable to that of the native peptide against 50 mol% POPG as a consequence of peptide structuring. We conclude that the activity of Gm and its linear analogue is intimately related to the formation of a β-turn motif, in which the hydrophobic residues form a hydrophobic face able to insert into the membrane and disrupt it. Copyright © 2015 Elsevier B.V. All rights reserved.
    No preview · Article · Jul 2015 · Biochimica et Biophysica Acta
  • [Show abstract] [Hide abstract]
    ABSTRACT: To find effective new candidate antimalarial drugs, bradykinin and its analogs were synthesized and tested for effectiveness against Plasmodium gallinaceum sporozoites and Plasmodium falciparum on erythrocytes. Among them, bradykinin and its P2 analog presented high activity against Plasmodium gallinaceum, but they degrade in plasma. On the other hand, RI-BbKI did not degrade and reached high activity. No analog was active against Plasmodium falciparum. Copyright © 2015. Published by Elsevier Ltd.
    No preview · Article · May 2015 · Bioorganic & medicinal chemistry letters
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
    No preview · Article · Apr 2015 · Journal of Peptide Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Several studies have shown the important actions of cytokine leptin that regulates food intake and energy expenditure. Additionally, the ability to modulate hematopoiesis has also been demonstrated. Previous reports have shown that some synthetic sequences of leptin molecules can activate leptin receptor. Herein, decapeptides encompassing amino acids from positions 98 to 122 of the leptin molecule were constructed to evaluate their effects on hematopoiesis. Among them, the synthetic peptide Lep110-119 -NH2 (LEP F) was the only peptide that possessed the ability to increase the percentage of hematopoietic stem cells (HSC). Moreover, LEP F also produced an increase of granulocyte/macrophage colony-forming units and activated leptin receptor. Furthermore, LEP F also improves the grafting of HSC in bone marrow, but did not accelerate the recovery of bone marrow after ablation with 5-fluorouracil. These results show that LEP F is a positive modulator of the in vivo expansion of HSC and could be useful in bone marrow transplantation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Mar 2015 · Journal of Cellular Biochemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Experimental Parasitology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malaria is an infectious disease for which effective treatment and prevention strategies remain limited. Our group recently reported that angiotensin II (AII) presents antiplasmodial activity and inhibits the development of Plasmodium gallinaceum in Aedes aegypti. However, details concerning role of each amino acid residue in the antiplasmodial activity of the peptide and information about the minimal structure responsible for this activity remain unknown. In this work, we investigated the effects of specific deletions (i.e., mono-, di-, tri- and tetra-deletions) of AII amino acids on the antiplasmodial activity of this molecule. The peptides were synthesized on solid phase method using the t-Boc strategy, purified using high performance liquid chromatography and characterized using mass spectrometry. The lytic activity of the peptides was assessed in vitro using mature sporozoites extracted from the salivary glands of infected Aedes aegypti mosquitoes. The results demonstrate that all of the deletions reduced antiplasmodial activity compared to native AII and that active analogs tend to adopt β-turn conformations; however, the deletion of bulky hydrophobic residues causes greater reductions of bioactivity than the deletion of hydrophilic residues. Corroborating previous studies, we observed that analog extremities are susceptible to changes and can be carefully modified without compromising the activity of this compound. This research contributes to our understanding of the role of each AII amino acid residue in activity against Plasmodium gallinaceum and identifies two short analogs with similar antiplasmodial activity to AII. These analogs may be candidates for additional antimalarial assays because they are inexpensive and easy to synthesize.
    Full-text · Article · Dec 2014 · International Journal of Peptide Research and Therapeutics
  • Source

    Full-text · Dataset · Oct 2014
  • Source

    Full-text · Dataset · Sep 2014
  • Source

    Full-text · Dataset · Sep 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malaria is caused by the protozoa Plasmodium and is responsible for approximately one million deaths annually. The antimalarial effects of angiotensin II and its analogs against Plasmodium gallinaceum and falciparum have recently been reported. Here, 12 angiotensin II restricted analogs that contain i − (i + 2), i − (i + 3) and i − (i + 4) lactam bridges were synthesized to analyze their effect on antiplasmodial activity. To accomplish this, peptides containing two amino acid residues (aspartic or glutamic acids and lysine or ornithine), were synthesized by the t-Boc solid phase method, purified by liquid chromatography, and characterized by mass spectrometry, and conformational studies were performed by circular dichroism. The results indicate that some of the analogs had anti-plasmodium activity similar to angiotensin II (88 % activity). Among those, eight compounds exhibited high activity (>70 %), measured by fluorescence microscopy. The analogs with smaller lactam rings and an aspartic acid residue as the bridgehead element had lower levels of lytic activity. The results obtained with the new restricted analogs showed that the insertion position (near the N-terminus), the ring size, and the number of residues between the rings are as important as the components of lactam bridge, regardless of their chirality. The circular dichroism studies suggest that the active analogs, and native angiotensin II, adopt a β-fold conformation in different solutions. In conclusion, this approach provides insight for understanding the effects of restricting the ring size and position on the bioactivity of angiotensin II and provides a new direction for the design of potential chemotherapeutic agents.
    Full-text · Article · Sep 2014 · International Journal of Peptide Research and Therapeutics
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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 Ile5 or the His6 residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala6]-AII (79%), and [Ala5]-AII (75%). Analogs [Ala3]-AII, [Ala1]-AII, and AII-NH2 showed antiplasmodial activity around 65%, whereas the activity of the [Ala8]-AII, [Ala7]-AII, [Ala4]-AII, and [Ala2]-AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β-fold conformation in different solutions. All AII analogs, except [Ala4]-AII and [Ala8]-AII, show contractile responses and interact with the AT1 receptor, [Ala5]-AII and [Ala6]-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. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
    Full-text · Article · Aug 2014 · Journal of Peptide Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. In this study, we analyzed three Gm analogs, [Trp1]-Gm, [Trp7]-Gm, and [Trp9]-Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. The incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (~8 mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0–15 mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3-6.0 mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent-peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally-restricted in the presence of SDS, probably because this residue is located at the N-terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
    Full-text · Article · Jun 2014 · Journal of Peptide Science
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Malaria is an infectious disease responsible for approximately one million deaths annually. The antimalarial effects of angiotensin II and its analogs against Plasmodium gallinaceum and P. falciparum have recently been reported. To evaluate anti-plasmodial activity, we synthesized five angiotensin II restricted analogs that containing disulfide bridges. To accomplish this, peptides containing two inserted amino acid residues (cysteine), were synthesized by the Fmoc solid phase method, purified by liquid chromatography, and characterized by mass spectrometry. Conformational studies were performed by circular dichroism. The results indicated that two of the analogs had higher anti-plasmodium activity (92% and 98% activity) than angiotensin II (88% activity), measured by fluorescence microscopy. Results showed that the insertion position must be selected, to preserve the hydrophobic interactions between the nonpolar residues, as this affects anti-plasmodial activity. The circular dichroism studies suggested that the active analogs as well as the native angiotensin II adopt a β-turn conformation in different solutions. This approach provided insight for understanding the effects of restricting the ring size and position on the bioactivity of angiotensin II and provides a new direction for the design of potential chemotherapeutic agents. This article is protected by copyright. All rights reserved.
    Full-text · Article · May 2014 · Chemical Biology & Drug Design
  • Source
    Bruno Mattei · Antonio Miranda · Katia Regina Perez · Karin A Riske
    [Show abstract] [Hide abstract]
    ABSTRACT: Antimicrobial peptides are part of the innate immune system of animals and plants. Their lytic activity against microorganisms generally depends on their ability to disrupt and permeabilize membranes. Here we study the structure-activity relationship of the antimicrobial peptide gomesin (Gm), from the spider Acanthoscurria gomesiana, with large unilamellar vesicles (LUVs) composed of 3:7 palmitoyloleoyl phosphatidylglycerol : palmitoyloleoyl phosphatidylcholine. Several synthetic analogues of Gm were designed to alter the hydrophobicity/charge of the molecule, whereby selected amino acid residues were replaced by alanine. Isothermal titration calorimetry (ITC) was used to assess the thermodynamic parameters of peptide binding to LUVs and light scattering measurements were made to evaluated peptide-induced vesicle aggregation. The ability of the peptides to permeabilize vesicles was quantified through the leakage of an entrapped fluorescent probe. The activity of peptides could be quantified in terms of the leakage extent induced and their affinity to the membrane, which was largely dictated by the exothermic enthalpy change. The results show that analogues more hydrophobic than Gm display higher activity, whereas peptides more hydrophilic than Gm have their activity almost abolished. Vesicle aggregation, on the other hand, largely increases with peptide charge. We conclude that interaction of Gm with membranes depends on an interplay between surface electrostatic interactions, which drive anchoring to the membrane surface and vesicle aggregation, and insertion of the hydrophobic portion into the membrane core, responsible for causing membrane rupture/permeabilization.
    Full-text · Article · Mar 2014 · Langmuir
  • Source

    Full-text · Dataset · Feb 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.
    Full-text · Article · Nov 2013 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study we have isolated and identified strains of lactic acid bacteria (LAB) that are potential bacteriocin producers from rocket salad samples acquired in Sao Paulo, SP, Brazil. Using the PCR-ARDRA method, Lactococcus lactis subsp. lactis MK02R was identified and characterized as the only bacteriocinogenic among 12 strains initially isolated. The analysis of the antimicrobial peptide produced by MK02R revealed the presence of nisin variant, a well-known lantibiotic, and one of the most studied and important antimicrobial peptides known today. The bacteriocin MK02R showed heat stability for 1 h at 60 °C and 100 °C and it was inactivated by treatment with proteolytic enzymes. The antimicrobial activity did not change with pH adjustment between 2.0 and 9.0 and the production was stimulated by the addition of 0.5 and 1.0% cysteine in MRS broth at 37 °C. Bacteriocin MK02R inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Lactobacillus delbrueckii, Lb. sakei subsp. sakei, Listeria innocua and Listeria monocytogenes from different serological groups. Low levels of adsorption of bacteriocin MK02R were detected in the cell surface of the producer cells suggesting that bacteriocin MK02R remains bound to the outer surface and that it is released when the pH of the environment increases. The chromatographic studies and the genetic tests using primers nisF and nisR related to specific genes of nisin production confirmed that the antimicrobial MK02R is a natural variant of nisin. The partial sequencing of the reconstructed protein showed that the peptide has an amino acid change in the sequence of the leader peptide compared with nisin A, Z, Q, U and F, but the structure of the mature is homologous to that of nisin F.
    Full-text · Article · Oct 2013 · Food Control
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Leptin is a cytokine that regulates food intake, energy expenditure and hematopoiesis. Based on the tridimensional structure of the human leptin molecule, six fragments have been synthesized, (Ac-Lep23-47-NH2, [LEP1]; Ac-Lep48-71-NH2, [LEP2]; Ac-Lep72-88-NH2, [LEP3]; Ac-Lep92-115-NH2, [LEP4], Ac-[Ser(117)]-Lep116-140-NH2, [LEP5] and Ac-Lep141-164-NH2, [LEP6]), and their effects on hematopoiesis were evaluated. The mice were treated with 1mg/kg LEP5 for 3 days. The mature and primitive hematopoietic populations were quantified. We observed that the mature populations from the bone marrow and spleen were not affected by LEP5. However, the peptide caused at least a two-fold increase in the number of hematopoietic stem cells, the most primitive population of the bone marrow. Additionally, the number of granulocyte/macrophage colony-forming units produced by bone marrow cells in methylcellulose also increased by 40% after treatment with LEP5, and the leptin receptor was activated. These results show that the leptin fragment LEP5 is a positive modulator of the in vivo expansion of hematopoietic stem cells.
    Full-text · Article · Sep 2013 · Peptides
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti-plasmodium activity. The peptides were synthesized by a conventional solid-phase method on Merrifield's resin using the t-Boc strategy, purified by RP-HPLC and characterized by liquid chromatography/ESI (+) MS (LC-ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti-plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least-square analysis, assessing the position-wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C-terminus, as well as that of hydrophobic amino acids in the N-terminus, suggests that the mechanism underlying the anti-malarial activity of these peptides is attributed to its amphiphilic character. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
    Full-text · Article · Sep 2013 · Journal of Peptide Science