Molecular hybridization strategy for tuning bioactive peptide function.

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1-128 µmol L-1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 µmol L-1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

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.

Molecular hybridization strategy for tuning bioactive peptide function

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1–128 μmol L−1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 μmol L−1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

The BR2 peptide associated with 2-aminoethyl dihydrogen phosphate is a formulation with antiproliferative potential for a triple-negative breast cancer model.

Triple-negative breast cancer is the most aggressive subtype of breast cancer, with worse clinical evolution and tumor-free survival, leading to the need to develop new effective therapies for its control. The present study evaluated the action of tumor-penetrating peptide BR2 associated with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on triple-negative breast tumor cells. Cell viability was evaluated by the MTT colorimetric method, mitochondrial electrical potential, and proteins involved in cell proliferation and death control were evaluated by flow cytometry and structural and morphological analysis by confocal microscopy. The results obtained showed that the peptide BR2 and the association 2-AEH2P + BR2 promoted significant cytotoxicity in tumor lines, compared to 2-AEH2P alone. In addition, the association 2-AEH2P + BR2 promoted tumor cells arrest in the G0/G1 phases. Interestingly, both treatments modulated the expression of markers CD44, CD34, CD24, cyclin D1, and Bcl-2, increased p21, Bax, and released cytochrome c. The association proved to be more effective, providing modulation of proteins involved in cell death and senescence, more pronounced cytotoxicity for tumor cells compared to normal cells, and the reduction of markers related to aggressiveness profile, progression, and tumor metastasis.

The BR2 peptide associated with 2-aminoethyl dihydrogen phosphate is a formulation with antiproliferative potential for a triple-negative breast cancer model

Triple-negative breast cancer is the most aggressive subtype of breast cancer, with worse clinical evolution and tumor-free survival, leading to the need to develop new effective therapies for its control. The present study evaluated the action of tumor-penetrating peptide BR2 associated with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on triple-negative breast tumor cells. Cell viability was evaluated by the MTT colorimetric method, mitochondrial electrical potential, and proteins involved in cell proliferation and death control were evaluated by flow cytometry and structural and morphological analysis by confocal microscopy. The results obtained showed that the peptide BR2 and the association 2-AEH2P + BR2 promoted significant cytotoxicity in tumor lines, compared to 2-AEH2P alone. In addition, the association 2-AEH2P + BR2 promoted tumor cells arrest in the G0/G1 phases. Interestingly, both treatments modulated the expression of markers CD44, CD34, CD24, cyclin D1, and Bcl-2, increased p21, Bax, and released cytochrome c. The association proved to be more effective, providing modulation of proteins involved in cell death and senescence, more pronounced cytotoxicity for tumor cells compared to normal cells, and the reduction of markers related to aggressiveness profile, progression, and tumor metastasis.

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.

Net charge tuning modulates the antiplasmodial and anticancer properties of peptides derived from scorpion venom.

VmCT1, a linear helical antimicrobial peptide isolated from the venom of the scorpion Vaejovis mexicanus, displays broad spectrum antimicrobial activity against bacteria, fungi, and protozoa. Analogs derived from this peptide containing single Arg-substitutions have been shown to increase antimicrobial and antiparasitic activities against Trypanossoma cruzi. Here, we tested these analogs against malaria, an infectious disease caused by Plasmodium protozoa, and assessed their antitumoral properties. Specifically, we tested VmCT1 synthetic variants [Arg]3 -VmCT1-NH2 , [Arg]7 -VmCT1-NH2 , and [Arg]11 -VmCT1-NH2 , against Plasmodium gallinaceum sporozoites and MCF-7 mammary cancer cells. Our screen identified peptides [Arg]3 -VmCT1-NH2 and [Arg]7 -VmCT1-NH2 as potent antiplasmodial agents (IC50 of 0.57 and 0.51 µmol L-1 , respectively), whereas [Arg]11 -VmCT1-NH2 did not show activity against P. gallinaceum sporozoites. Interestingly, all peptides presented activity against MCF-7 and displayed lower cytotoxicity toward healthy cells. We demonstrate that increasing the net positive charge of VmCT1, through arginine substitutions, modulates the biological properties of this peptide family yielding novel antiplasmodial and antitumoral molecules.

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.

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens.

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 µmol L-1 against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 µmol L-1, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.

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.

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 µmol L-1 against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 µmol L-1, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.

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.

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 µmol L-1 against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 µmol L-1, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.

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.

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.

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

Linear cationic a-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 a-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.