Antiproliferative Modulation and Pro-Apoptotic Effect of BR2 Tumor-Penetrating Peptide Formulation 2-Aminoethyl Dihydrogen Phosphate in Triple-Negative Breast Cancer.

Breast cancer is the most common cancer in women, the so-called "Triple-Negative Breast Cancer" (TNBC) subtype remaining the most challenging to treat, with low tumor-free survival and poor clinical evolution. Therefore, there is a clear medical need for innovative and more efficient treatment options for TNBC. The aim of the present study was to evaluate the potential therapeutic interest of the association of the tumor-penetrating BR2 peptide with monophosphoester 2-aminoethyl dihydrogen phosphate (2-AEH2P), a monophosphoester involved in cell membrane turnover, in TNBC. For that purpose, viability, migration, proliferative capacity, and gene expression analysis of proteins involved in the control of proliferation and apoptosis were evaluated upon treatment of an array of TNBC cells with the BR2 peptide and 2-AEH2P, either separately or combined. Our data showed that, while possessing limited single-agent activity, the 2-AEH2P+BR2 association promoted significant cytotoxicity in TNBC cells but not in normal cells, with reduced proliferative potential and inhibition of cell migration. Mechanically, the 2-AEH2P+BR2 combination promoted an increase in cells expressing p53 caspase 3 and caspase 8, a reduction in cells expressing tumor progression and metastasis markers such as VEGF and PCNA, as well as a reduction in mitochondrial electrical potential. Our results indicate that the combination of the BR2 peptide with 2-AEH2P+BR2 may represent a promising therapeutic strategy in TNBC with potential use in clinical settings.

Effect from dinoponeratoxin M-PONTXDq3a arginine and lysine substituted analogues against Staphylococcus aureus strains.

The growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections is associated with increased mortality rates, which has generated interest in the development of antimicrobial peptides (AMP), such as those found in the giant ant Dinoponera quadríceps. In order to improve the net positive charge and the antibacterial activity of the AMP, amino acids with positive side chain single substituted analogues have been proposed, mainly arginine or lysine. The present work aims to study the antimicrobial activity of the analogues of M-PONTX-Dq3a, a 23 amino acid AMP identified in the D. quadriceps venom. M-PONTX-Dq3a[1-15], a fragment containing the 15 central amino acids, and eight derivatives of single arginine or lysine substituted analogues were proposed. The antimicrobial activity of peptides was evaluated against Staphylococcus aureus ATCC 6538 P (MSSA) and ATCC 33591 (MRSA) strains, followed by minimum inhibitory concentration (MIC), minimum lethal concentration (MLC), and minimum biofilm inhibitory concentration (MBIC) measurement. The membrane permeability was then assessed via crystal violet assay and flow cytometry analysis. The effect of exposure time on microbial viability (Time-Kill) was evaluated. Finally, ultrastructural alterations were evaluated through scanning electron microscopy (SEM). Both arginine-substituted peptides [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15], showed lowest MIC and MLC values (each 0.78 µM). In the biofilm formation assays, the peptide [Arg]3M-PONTX-Dq3a [1-15] showed MBIC of 3.12 µM against the two tested strains. Both peptides were able to alter the membrane permeability approximately by 80%. The treatment with MIC was able to eliminate bacteria after 2 h of contact on the other hand, treatment with half of the MIC, the population of both bacterial strains remained constant for up to 12 h, indicating a possible bacteriostatic effect. The SEM results showed that the treatment with the lowest concentration (0.78 µM) of both peptides caused disruption of the cell membrane, destabilization of the intercellular interaction and the CLM of [Arg]4M-PONTX-Dq3a [1-15] resulted in the complete eradication of the bacteria. Thus, this study describes two AMPs active against MSSA and MRSA and also inhibits the biofilm formation of these stains. This study finds [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15] as alternative substances to treat resistant and/or biofilm-forming strains.

Antiproliferative mdulation and pro-apoptotic effect of BR2 tumor-penetrating peptide formulation 2-aminoethyl dihydrogen phosphate in triple-negative breast cancer

Breast cancer is the most common cancer in women, the so-called “Triple-Negative Breast Cancer” (TNBC) subtype remaining the most challenging to treat, with low tumor-free survival and poor clinical evolution. Therefore, there is a clear medical need for innovative and more efficient treatment options for TNBC. The aim of the present study was to evaluate the potential therapeutic interest of the association of the tumor-penetrating BR2 peptide with monophosphoester 2-aminoethyl dihydrogen phosphate (2-AEH2P), a monophosphoester involved in cell membrane turnover, in TNBC. For that purpose, viability, migration, proliferative capacity, and gene expression analysis of proteins involved in the control of proliferation and apoptosis were evaluated upon treatment of an array of TNBC cells with the BR2 peptide and 2-AEH2P, either separately or combined. Our data showed that, while possessing limited single-agent activity, the 2-AEH2P+BR2 association promoted significant cytotoxicity in TNBC cells but not in normal cells, with reduced proliferative potential and inhibition of cell migration. Mechanically, the 2-AEH2P+BR2 combination promoted an increase in cells expressing p53 caspase 3 and caspase 8, a reduction in cells expressing tumor progression and metastasis markers such as VEGF and PCNA, as well as a reduction in mitochondrial electrical potential. Our results indicate that the combination of the BR2 peptide with 2-AEH2P+BR2 may represent a promising therapeutic strategy in TNBC with potential use in clinical settings.

Synthetic Peptide Derived from Scorpion Venom Displays Minimal Toxicity and Anti-infective Activity in an Animal Model.

Multidrug-resistant bacteria represent a global health problem increasingly leading to infections that are untreatable with our existing antibiotic arsenal. Therefore, it is critical to identify novel effective antimicrobials. Venoms represent an underexplored source of potential antibiotic molecules. Here, we engineered a peptide (IsCT1-NH2) derived from the venom of the scorpion Opisthacanthus madagascariensis, whose application as an antimicrobial had been traditionally hindered by its high toxicity. Through peptide design and the knowledge obtained in preliminary studies with single and double-substituted analogs, we engineered IsCT1 derivatives with multiple amino acid substitutions to assess the impact of net charge on antimicrobial activity and toxicity. We demonstrate that increased net charge (from +3 to +6) significantly reduced toxicity toward human erythrocytes. Our lead synthetic peptide, [A]1[K]3[F]5[K]8-IsCT1-NH2 (net charge of +4), exhibited increased antimicrobial activity against Gram-negative and Gram-positive bacteria in vitro and enhanced anti-infective activity in a mouse model. Mechanism of action studies revealed that the increased antimicrobial activity of our lead molecule was due, at least in part, to its enhanced ability to permeabilize the outer membrane and depolarize the cytoplasmic membrane. In summary, we describe a simple method based on net charge tuning to turn highly toxic venom-derived peptides into viable therapeutics.

Arg-substituted VmCT1 analogs reveals promising candidate for the development of new antichagasic agent.

VmCT1 is an antimicrobial peptide (AMP) isolated from the venom of the scorpion Vaejovis mexicanus with antimicrobial, anticancer and antimalarial activities, which the rational design with Arg-substitution has yielded AMPs with higher antimicrobial activity than VmCT1. Chagas is a neglected tropical disease, becoming the development of new antichagasic agents is urgent. Thus, we aimed to evaluate the antichagasic effect of VmCT1 and three Arg-substituted analogues, as well their action mechanism. Peptides were tested against the epimastigote, trypomastigote, amastigote forms of Trypanossoma cruzi Y strain and against LLC-MK2 mammalian cells. The mechanism of action of these peptides was evaluated by means of flow cytometry and scanning electron microscopy. VmCT1 presented activity against all three forms of T. cruzi, with EC50 against trypomastigote forms of 1.37 µmol L-1 and selectivity index (SI) of 58. [Arg]3-VmCT1, [Arg]7-VmCT1 and [Arg]11-VmCT1 also showed trypanocidal effect, but [Arg]11-VmCT1 had the best effect, being able to decrease the EC50 against trypomastigote forms to 0.8 µmol L-1 and increase SI to 175. Necrosis was cell death pathway of VmCT1, as well [Arg]7-VmCT1 and [Arg]11-VmCT1, such as observed by membrane damage in flow cytometry analyses and scanning-electron-microscopy. In conclusion, [Arg]11-VmCT1 revealed promising as a candidate for new antichagasic therapeutics.

Wasp venom peptide as a new antichagasic agent.

Chagas disease is caused by Trypanosoma cruzi and affects approximately 10 million people a year worldwide. The only two treatment options, benznidazole and nifurtimox, have low efficacy and high toxicity towards human cells. Mastoporan peptide (MP) a small cationic AMP from the venom of the wasp Polybia paulista has been reported as a potent trypanocidal agent. Thus, we evaluated the antichagasic effect of another AMP from the venom of the same wasp Polybia paulista, polybia-CP (ILGTILGLLSKL-NH2), and investigated its mechanism of action against different stages of the trypanosomal cells life cycle. Polybia-CP was tested against the epimastigote, trypomastigote and amastigote forms of the T. cruzi Y strain (benznidazole-resistant strain) and inhibited the development of these forms. We also assessed the selectivity of the AMP against mammalian cells by exposing LLC-MK2 cells to polybia-CP, the peptide presented a high selectivity index (>106). The mechanism of action of polybia-CP on trypanosomal cells was investigated by flow cytometry, scanning electron microscopy (SEM) and enzymatic assays with T. cruzi GAPDH (tcGAPDH), enzyme that catalyzes the sixth step of glycolysis. Polybia-CP induced phosphatidylserine exposure, it also increased the formation of reactive species of oxigen (ROS) and reduced the transmembrane mitochondrial potential. Polybia-CP also led to cell shrinkage, evidencing apoptotic cell death. We did not observe the inhibition of tcGAPDH or autophagy induction. Altogether, polybia-CP has shown the features of a promising template for the development of new antichagasic agents.

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.

The effect of lysine substitutions in the biological activities of the scorpion venom peptide VmCT1.

Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1-6.3 µmol L-1) than VmCT1 (0.8-50 µmol L-1) and higher activity against mammary cancer cells MCF-7 (6.3-12.5 µmol L-1) than VmCT1 (12.5 µmol L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8 µmol L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.

The effect of lysine substitutions in the biological activities of the scorpion venom peptide VmCT1

Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1–6.3?µmol?L-1) than VmCT1 (0.8–50?µmol?L-1) and higher activity against mammary cancer cells MCF-7 (6.3–12.5?µmol?L-1) than VmCT1 (12.5?µmol?L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8?µmol?L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.

The effect of lysine substitutions in the biological activities of the scorpion venom peptide VmCT1

Antimicrobial peptides (AMPs) are biologically active molecules with a broad-spectrum activity against a myriad of microorganisms. Aside from their antimicrobial functions, AMPs present physicochemical and structural properties that allow them to exert activity against other kind of cells, such as cancer cells. VmCT1 is a potent cationic amphipathic AMP from the venom of the scorpion Vaejovis mexicanus. In this study, we designed lysine-substituted VmCT1 analogs for verifying the influence of changes in the net positive charge on biological activities. The increase in the net positive charge caused by lysine substitutions in the hydrophilic portion, led to higher antimicrobial activity values (0.1–6.3?µmol?L-1) than VmCT1 (0.8–50?µmol?L-1) and higher activity against mammary cancer cells MCF-7 (6.3–12.5?µmol?L-1) than VmCT1 (12.5?µmol?L-1). Contrarily, when lysine-substitutions were made at the hydrophobic portion of the helical projection, the activity values decreased. However, the lysine-substitution at the center of the hydrophobic face led to the generation of an analog with antiplasmodial activity at the same concentration presented by VmCT1 (0.8?µmol?L-1). In this study, we demonstrated that it is possible to modulate biological activities and cytotoxicity of VmCT1 peptides by increasing their net positive charge using lysine residues, thus creating alternatives for standard-of-care therapeutics against different types of microorganisms and MCF-7 human breast cancer cells.

Anticancer activity of VmCT1 analogs against MCF-7 cells.

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe-Leu-Gly-Ala-Leu-Trp-Asn-Val-Ala-Lys-Ser-Val-Phe-NH2 ) is an α-helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF-7 mammary cancer cells. The results indicated that [Glu]7 -VmCT1-NH2 , [Lys]3 -VmCT1-NH2 , and [Lys]7 -VmCT1-NH2 analogs presented moderated helical tendency (0.23-0.61) and tendency of anticancer activity at 25 µmol/L in 24 hr of experiment; and [Trp]9 -VmCT1-NH2 analog that presented low helical tendency and moderated anticancer activity at 50 µmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.

Generation of Advanced Glycation End-Products (AGEs) by glycoxidation mediated by copper and ROS in a human serum albumin (HSA) model peptide: reaction mechanism and damage in motor neuron cells.

Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.

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.

Antimicrobial activity of leucine-substituted decoralin analogs with lower hemolytic activity.

Linear cationic α-helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine-substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α-helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]10 -Dec-NH2 analog showed similar activity against different microorganisms (c.a. 0.4-0.8 µmol L-1 ), helical structuration, and some hemolytic activity, [Leu]8 -Dec-NH2 analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 µmol L-1 ). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Novel designed VmCT1 analogs with increased antimicrobial activity.

Antimicrobial peptides are biologically active molecules produced by a wide range of organisms as an essential component of the innate immune response. They have recently attracted great interest, since they have antimicrobial activity against a broad spectrum of microorganisms. VmCT1 is a cationic peptide from the venom of Vaejovis mexicanus smithi scorpions, which presents antibacterial activity and tends to helical structures. Its analogs were synthesized, characterized and the conformational studies were performed by circular dichroism. The peptides were designed to verify if the single and double substitutions proposed at the hydrophilic and hydrophobic portions of the amphipathic structure would alter antimicrobial activity against Gram-negative and Gram-positive bacteria, yeast and filamentous fungus, besides the hemolytic activity in human erythrocytes. Total charge of the peptides were modified from +2 to +3 by the introduction of a Lysine residue in the hydrophilic face of the amphiphilic helical structure leading to enhanced antimicrobial activity. [K]11-VmCT1-NH2 presented the lower MIC value against the microorganisms (from 0.39 to 6.25 µmol L-1), however it showed higher hemolytic activity. The other Lysine-substituted analogs presented also lower MIC values ranging from 0.39 to 25 µmol L-1 for the microorganisms assessed. The circular dichroism spectra analyses suggest that the Lysine-substituted analogs tend to adopt helical structures in trifluoroethanol solution and vesicles (fH: 0.43-1), however they were coiled in water. Alanine substitution by a Glutamic acid residue in the hydrophilic face promotes the increase of polar angle in [E]4-VmCT1-NH2 analog, which was important to led lower hemolytic activity (MHC value = 25 µmol L-1). [W]9-VmCT1-NH2 and [E]4[W]9-VmCT1-NH2 were designed to favors hydrophobic interactions by the introduction of Tryptophan residue. [W]9-VmCT1-NH2 presented MIC values lower or similar than the model molecule in the most of microorganisms tested. These results provided information about the structure-activity relationship and showed the influence of physicochemical parameters on antimicrobial and hemolytic activity.

Novel designed VmCT1 analogs with increased antimicrobial activity

Antimicrobial peptides are biologically active molecules produced by a wide range of organisms as an essential component of the innate immune response. They have recently attracted great interest, since they have antimicrobial activity against a broad spectrum of microorganisms. VmCell is a cationic peptide from the venom of Vaejovis mexicanus smithi scorpions, which presents antibacterial activity and tends to helical structures. Its analogs were synthesized, characterized and the conformational studies were performed by circular dichroism. The peptides were designed to verify if the single and double substitutions proposed at the hydrophilic and hydrophobic portions of the amphipathic structure would alter antimicrobial activity against Gram-negative and Gram-positive bacteria, yeast and filamentous fungus, besides the hemolytic activity in human erythrocytes. Total charge of the peptides were modified from +2 to +3 by the introduction of a Lysine residue in the hydrophilic face of the amphiphilic helical structure leading to enhanced antimicrobial activity. [K](11)-Vmal-NH2 presented the lower MIC value against the microorganisms (from 0.39 to 6.25 mol L-1), however it showed higher hemolytic activity. The other Lysine-substituted analogs presented also lower MIC values ranging from 0.39 to 25 mu mol L-1 for the microorganisms assessed. The circular dichroism spectra analyses suggest that the Lysine substituted analogs tend to adopt helical structures in trifluoroethanol solution and vesicles (f(H): 0.43 1), however they were coiled in water. Alanine substitution by a Glutamic acid residue in the hydrophilic face promotes the increase of polar angle in [E](4)-VmCT1-NH2 analog, which was important to led lower hemolytic activity (MHC value = 25 !Imo! L-1). [W-19-VmCT1-NH2 and [E](4)[W](9)-VmC11-NH2 were designed to favors hydrophobic interactions by the introduction of Tryptophan residue. W-19-VmalNH(2) presented MIC values lower or similar than the model molecule in the most of microorganisms tested. These results provided information about the structure -activity relationship and showed the influence of physicochemical parameters on antimicrobial and hemolytic activity.

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.

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.

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.

Angiotensin II restricted analogs with biological activity in the erythrocytic cycle of Plasmodium falciparum.

The anti-plasmodial activity of conformationally restricted analogs of angiotensin II against Plasmodium gallinaceum has been described. To observe activity against another Plasmodium species, invasion of red blood cells by Plasmodium falciparum was analyzed. Analogs restricted with lactam or disulfide bridges were synthesized to determine their effects and constraints in the peptide-parasite interaction. The analogs were synthesized using tert-butoxycarbonyl and fluoromethoxycarbonyl solid phase methods, purified by liquid chromatography, and characterized by mass spectrometry. Results indicated that the lactam bridge restricted analogs 1 (Glu-Asp-Arg-Orn-Val-Tyr-Ile-His-Pro-Phe) and 3 (Asp-Glu-Arg-Val-Orn-Tyr-Ile-His-Pro-Phe) showed activity toward inhibition of ring formation stage of P. falciparum erythrocytic cycle, preventing invasion in about 40% of the erythrocytes. The disulfide-bridged analog 10 (Cys-Asp-Arg-Cys-Val-Tyr-Ile-His-Pro-Phe) was less effective yet significant, showing a 25% decrease in infection of new erythrocytes. In all cases, the peptides presented no pressor activity, and hydrophobic interactions between the aromatic and alkyl amino acid side chains were preserved, a factor proven important in efficacy against P. gallinaceum. In contrast, hydrophilic interactions between the Asp(1) carboxyl and Arg(2) guanidyl groups proved not to be as important as they were in the case of P. gallinaceum, while interactions between the Arg(2) guanidyl and Tyr(4) hydroxyl groups were not important in either case. The ß-turn conformation was predominant in all of the active peptides, proving importance in anti-plasmodial activity. This approach provides insight for understanding the importance of each amino acid residue on the native angiotensin II structure and a new direction for the design of potential chemotherapeutic agents.