Cm-p5, a synthetic antimicrobial peptide shows anti-Trypanosoma cruzi activity

Ana C. Mengarda; Fidel E. Morales-Vicente; Ernesto M. Martell-Huguet; Anselmo J. Otero-Gonzalez; Ariel M. Silber

doi:10.1371/journal.pntd.0013975

Abstract

Chagas disease is a neglected disease caused by Trypanosoma cruzi, which affects 6–7 million people worldwide. More than one hundred years after its description, the performance of available drugs for treating the T. cruzi infection remains largely unsatisfactory. Antimicrobial peptides (AMPs) are new alternatives that may have potential as trypanocides. Herein, we assessed Cm-p5, a synthetic peptide with previously shown antimicrobial activity, and 10 derivatives. After screening assays using epimastigote forms of the parasite to test their potential as proliferation inhibitors, Cm-p5 was selected. Cm-p5 showed an EC₅₀ against T. cruzi of 16.9 ± 1.2 μM and a cytotoxicity towards CHO-K₁ mammalian cells (CC₅₀) of 124.8 ± 0.1 µM. After further investigation, it was evidenced that part of the epimastigote population underwent necrosis-like cell death, while those that remained alive showed a cell-cycle arrest at the phases G2_M and S_G2. When infected cells were treated, the peptide diminished the release of the infective trypomastigote form, with an EC₅₀ of 25.2 ± 1.4 µM. Furthermore, Cm-p5 inhibited the number of intracellular amastigotes as well as the number of infected cells by 64.3 and 75%, respectively. Taken together, these numbers resulted in a reduction of the infection index by 91.1%. Additionally, we showed that Cm-p5 trypanocidal activity against intracellular amastigotes was attributable to cell membrane damage and cell cycle partial arrest, as described for epimastigotes. Our data suggest that Cm-p5 may be a promising template to design new peptides for the treatment of Chagas disease.

Author summary

Trypanosoma cruzi, a hemoflagellate parasite that belongs to the group of kinetoplastids, causes Chagas disease. It is estimated that 7 million people are infected and therefore more than 75 million people are at risk of acquiring Chagas disease. Only two drugs are available for its treatment, nifurtimox and benznidazole, which have limitations regarding efficacy and tolerance. Therefore, there is an urgent need for new molecules with anti-T. cruzi potential. In this work, we assessed Cm-p5 and 10 derivatives and their potential as a trypanocidal drug. Cm-p5 is a synthetic peptide with antimicrobial activity, which showed a potent effect on the intracellular forms of the parasite (EC₅₀ of 25.2 ± 1.4 µM) and inhibits the intracellular cycle of T. cruzi. This peptide could be a promising alternative to the development of new peptides to improve the chemotherapy of Chagas disease.

Citation: Mengarda AC, Morales-Vicente FE, Martell-Huguet EM, Otero-Gonzalez AJ, Silber AM (2026) Cm-p5, a synthetic antimicrobial peptide shows anti-Trypanosoma cruzi activity. PLoS Negl Trop Dis 20(3): e0013975. https://doi.org/10.1371/journal.pntd.0013975

Editor: Abhay R. Satoskar, Ohio State University, UNITED STATES OF AMERICA

Received: June 17, 2025; Accepted: January 27, 2026; Published: March 5, 2026

Copyright: © 2026 Mengarda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This study was financed, in part, by the São Paulo Research Foundation (FAPESP), Brazil, Process Number 2021/12938-0 (awarded to A.M.S.), and Conselho Nacional de Pesquisas Científicas e Tecnológicas (CNPq) grant 307487/2021-0 (awarded to A.M.S.). A.C.M. is a FAPESP post-doctoral fellow (FAPESP Process 2022/16258-6). A.J.O.G. received a FAPESP fellowship during this research (FAPESP Process 2023/08572-5 awarded to A.M.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

American trypanosomiasis, also known as Chagas disease, is a protozoan disease caused by Trypanosoma cruzi affecting 6–7 million people worldwide, mostly in the Americas [1]. Only two drugs are available for its treatment, nifurtimox and benznidazole, which have limitations regarding efficacy and tolerance [2]. Moreover, the World Health Organization (WHO) published in 2021 a new roadmap proposing the control and elimination of diseases associated with poverty by 2030, including Chagas disease [3]. Within this agenda, the finding of new molecules with trypanocidal effect is urgent.

In recent decades, host defense peptides (HDPs) and their synthetic derivatives have gained prominence in biomedicine as a novel alternative to combat pathogenic microorganisms, especially those resistant to conventional drugs [4]. HDPs, which include antimicrobial peptides, are a group of evolutionarily conserved peptides with a broad spectrum of actions including antimicrobial, immunomodulatory, antioxidant, antiparasitic, and anticancer activity [5]. Defense peptides are essential components of the innate immune system in most living organisms, especially in invertebrates, which lack the responses of adaptive immunity and produce a large number of HDPs as a primary immune mechanism [6]. Microorganisms, mammalian cells, marine, fluvial and terrestrial molluscs, insects, and reptiles are sources of HDPs that act against pathogenic microorganisms and human parasites. These HDPs have been widely explored as promising therapeutic alternatives to conventional antibacterial, antifungal, antiviral and anti-parasite agents, aiming to treat infections caused by these pathogens. In this sense, more than 20 AMPs have been reported with activity against T. cruzi [7].

The synthetic peptide Cm-p5 was bioinformatically designed from the natural peptide Cm-p1, originally isolated from the marine mollusc Cenchritis muricatus. [8]. This cationic peptide has shown a fungicidal activity at low concentrations through interaction with specific lipids of the cytoplasmic membrane, especially phosphatidyl serine [8].

In the present work we obtained a collection of peptides derived from Cm-p5 (Fig 1), and assessed their anti-T. cruzi activity in vitro. We showed that the lead Cm-p5 was the peptide with the best anti-T. cruzi activity in the collection, exhibiting antiproliferative activity in epimastigotes (EC₅₀ = 16.9 ± 1.2 µM) and a relevant trypanocidal effect on the proliferative (amastigote) and infective (trypomastigote) forms occurring during the mammalian host-cell infection. Our results suggest its potential for further developments of a new treatment against Chagas disease.

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Fig 1. Chemical structures of Cm-p5 and their 10 derivatives.

https://doi.org/10.1371/journal.pntd.0013975.g001

Materials and methods

Reagents

All chemicals, reagents and solvents, were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise mentioned. Probes, such as Fluo-4 AM and Annexin V-FITC were purchased from Invitrogen (Eugene, Oregon, USA). Culture media and fetal calf serum (FCS) were purchased from Cultilab (Campinas, SP, Brazil).

Peptide synthesis

Cm-p5 and derivatives were synthetized according to Vicente and collaborators [9]. Briefly, solid phase peptide synthesis was carried out using Fmoc/t-Bu chemistry on rink amide resin based on polystyrene or Polyethylene glycol (PEG) (ChemMatrix). The resin was washed twice with DMF (complete nomenclature) (2 mL, 1 min), twice with DCM (complete nomenclature) (2 mL, 1 min), twice with methanol (MeOH) (2 mL, 1 min), and twice again with DCM and DMF as mentioned earlier. Fluorenylmethyloxycarbonyl (Fmoc) removal was achieved with 20% piperidine in DMF (2 × 10 min), and the subsequent amino acids were added using the following coupling condition: Fmoc-Aa-OH/DIC/Oxyma (4 equivalents of each) in DMF after negative ninhydrin test (approximately 30 min). After the last coupling reaction, Fmoc was eliminated, and peptide-resin was four-times washed subsequently with DMF (1 min), MeOH (1 min), and ethyl ether (Et2O) (1 min). Acetylation and lipidation were achieved using the following condition: Acetic anhydride/N,N-Diisopropylethylamine (AC2O/DIEA) (8 equiv) in DMF for 10 min. The peptide was obtained with more than 95% of purity as ascertained by analytical Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The molecular mass determined experimentally by Electrospray Ionization Mass Spectrometry (ESI-MS) corresponded with the theoretically calculated monoisotopic mass for each peptide. When cyclization was performed, it was made by the dissolution of the crude mixture of peptides (160 mg) in H2O 0.1% Trifluoroacetic Acid/Acetonitrile/isopropylalcohol (0.1% TFA/CH3CN/i-PrOH), 1:1:1) at 8.517 µg mL−1 ammonia (ac) (NH3 (ac)) (25%) was added until pH 8.0–9.0, and the reaction was stirred for 4–12 h. At pH 7.0 the H2O/CH3CN/i-PrOH mixture was removed. Cyclization of the Parallel Dimer (Dimer 1) using S-acetamidomethyl-L-cysteine (Cys(Acm)) peptides (150 mg) containing Cys(Acm) and free Cysteine (Cys) in the first or the second Cys residue were submitted to dimerization at high concentrations in 30 mL of DMF/H2O (1:1) for 72 h (analytical RP-HPLC) in which the formation of white suspension was observed. Subsequently, H2O (0.1% TFA)/MeOH (1:1) (VT = 250 mL) was added. Finally, 1.5 equiv. of hydrochloric acid (HCl) (37%) (pH = 3.8) and 5 equiv. of Iodine (I2) (MeOH) by the S-acetamidomethyl (Acm) group were supplemented, and stirring was maintained over 3 h or until no dimeric initial material remaining by RP-HPLC. Iodine was removed by adding 158.120 µg mL−1 aqueous sodium thiosulfate (Na₂S₂O₃) dropwise until the mixture became colourless or by treatment with activated charcoal and centrifugation. At a pH of 7.0 the H2O/MeOH mixture was removed by vacuum rotatory evaporator and residue was lyophilized.

Cells and parasite cultures

T. cruzi epimastigotes (CL strain, clone Brener) were maintained in exponential proliferation by subculturing the parasites every 48 h in Brain Heart Infusion (BHI) medium at 28 °C [10]. The Chinese Hamster Ovary cell line CHO-K₁ was cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 0.15% (w/v) sodium bicarbonate (NaHCO₃), 100 units mL⁻¹ penicillin and 100 µg mL⁻¹ at 37 °C in a humidified atmosphere containing 5% CO₂. Trypomastigotes were obtained by infection in CHO-K₁ cells with previously obtained and stocked trypomastigotes, as previously described [11]. For the first obtainment of culture-derived trypomastigotes, metacyclic trypomastigotes were obtained from axenically cultured epimastigotes following a previously described [12] method with small modifications. Briefly, late exponentially proliferating epimastigotes were harvested and washed with PBS and then transferred to Triatomine Artificial Medium (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM potassium phosphate buffer, pH 6) at a density of 5 × 10⁷ parasites mL^-1 and maintained for two hours at 28 °C. The parasites were subsequently transferred to TAU 3AAG media (TAU + 10 mM glucose, 2 mM aspartic acid, 50 mM glutamic acid, 10 mM Pro) and maintained for 8 days in a CO₂ incubator at 28 °C until differentiation. The metacyclic trypomastigotes were transferred to bottles containing previously cultured CHO-K₁ [13]). As CL has been described as a thermosensitive strain [14], infected cells were maintained for 24 h at 37 °C in RPMI supplemented with 10% FBS, and then at 33 °C in RPMI supplemented with 2% FBS in order to control the proliferation of mammalian cells [15]. Five days after infection and onward, CHO-K₁-derived trypomastigotes could be collected form the supernatants and stocked for new infections.

In vitro inhibition of proliferation assays on epimastigotes

The cell density of exponentially proliferating epimastigotes (approximately 5 × 10⁷ parasites mL^-1) was adjusted to 5 × 10⁶ parasites mL^-1. The parasites were then transferred into 96-well plates (200 μL/well) and, firstly incubated with 10 µM of AMPs during the whole culture time (10 days). Then, the AMP that cause an inhibitory effect on the proliferation curve was chosen to test at different concentrations (1.56-50 µM) and the half-maximal effective concentrations (EC₅₀) was calculated. Epimastigotes proliferation was measured as previously reported, by reading the optical density (OD) at 620 nm every 24 h during 10 days. The OD values were converted to cell density values (parasites mL^-1) by using a calibration curve obtained by measuring the OD values at 620 nm of parasite suspensions at different known densities [16]. EC₅₀ were determined from cell density data obtained at the 5th day of proliferation, which corresponded to the mid exponential proliferation phase. Data were analysed by a non-linear regression to a sigmoidal dose-response curve using GraphPad Prism v.8. Dimethyl Sulfoxide (DMSO)-dissolved benznidazole (BNZ; final concentration of 20 μM) and untreated parasites grown in the presence of the same volume of DMSO used for the BNZ treatment were used as positive and negative controls, respectively. Combined treatment of Rotenone at 60 µM and Antimycin at 0.5 µM was used as a death control [16]. The peptides were assessed in quadruplicate in each experiment, and the results correspond to three independent experiments.

The effect of the Cm-p5 on mammalian cell viability

The viability of CHO-K₁ cells was assessed measuring oxidoreductases activity as a proxy, by analyzing the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [17]. Briefly, CHO-K₁ cells (1.0 × 10⁵ per well) in 200 μL of RPMI medium supplemented with FBS (10%) were seeded in 96-well plates cells and after a 24 h adhesion period at 37 °C in 5% CO₂ were incubated for 48 h in the presence of Cm-p5 at 12.5-500 µM. Next, the peptide solution was removed and MTT was added to each well, and the cells were incubated in the presence of 100 µL at 1 mg/mL MTT for 3 h at 37 °C in the absence of light. After this incubation time, the MTT excess was removed and DMSO was added to dissolve the crystals that had formed. The absorbance was read on Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 595 nm. The half-maximal cytotoxicity concentrations (CC₅₀) values were determined by fitting a sigmoidal dose-response curve to the data using GraphPad Prism v.8. Each assay was developed in triplicate and the results correspond to the mean of three independent experiments.

Analysis of phosphatidylserine exposure and membrane permeabilization in epimastigotes

Epimastigotes (5 × 10⁶ parasites mL^-1) were incubated (or not, negative control) for five days in the presence or absence of 16.9 μM and 33.8 µM of Cm-p5 (concentrations corresponding to 1 and 2 times the EC₅₀. To determine the exposure of phosphatidyl serine and membrane permeabilization, the cells (5.0 × 10⁷ parasites mL^-1) were labelled with propidium iodide (PI) and Annexin-V FITC (Molecular Probes) according to the manufacturer’s instructions. As positive controls for plasma membrane permeabilization and extracellular exposure of phosphatidylserine (PS), the parasites were treated with 150 μM digitonin for 30 min. The cells were analysed by flow cytometry on a BD Accuri C6 Plus, each condition was run in three biological independent replicas with 10,000 events collected and analyzed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v07, Carrboro, North Carolina, USA) [18].

DNA content and cell cycle analysis in epimastigotes

Epimastigotes (5 × 10⁶ parasites mL^-1) were incubated (or not, negative control) in the presence of 16.9 μM and 33.8 µM of Cm-p5 (concentrations corresponding to 1 and 2 times the EC₅₀) for five days. Then, the cells (5.0 × 10⁷ parasites mL^-1) were collected by centrifugation (2,700 × g for 5 min), washed in PBS and fixed in 70% ethanol for 4 h. The parasites were washed twice in PBS and incubated with 10 μg/mL RNase A (Thermo Fisher Scientific, Massachusetts, USA) for 30 min at 37 °C. To measure the DNA content, the cells were stained with 10 μg/mL PI (Molecular Probes/Invitrogen) and analyzed by flow cytometry on a BD Accuri C6 Plus, with 10,000 events collected from three biological independent experiments [19]. Histograms (number of counts by FL2 area), scatter plots (side scatter [FSC] area by forward scatter [SSC] area) and gates for each cell cycle phase were analysed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v10). Cell cycle data were fitted by a model included in the FlowJo software (v10).

Effect of Cm-p5 on trypomastigote invasion

CHO-K₁ cells (1.0 × 10⁴ per well) were maintained in 96-well plates in RPMI medium supplemented with 10% FBS and maintained at 37 °C. After 24 h, the cells were infected with trypomastigote forms (5.0 × 10⁵ per well) and, firstly incubated with 50 µM of Cm-p5. As Cm-p5 cause an inhibitory effect on the invasion, different concentrations (3.12-50 µM) were tested, and the EC₅₀ was calculated. The treatment was conducted for four hours. DMSO-dissolved BNZ (final concentration of 5 μM) was used as a positive control and parasites in the presence of the same volume of DMSO used for the BNZ treatment were used as negative controls. After this period, free parasites and the Cm-p5 were removed. The infected cells were washed twice with PBS. The RPMI medium was replaced, and the plates were incubated at 33 °C [14]. Trypomastigotes were collected from the extracellular medium on the 5^th day of incubation and counted in a Neubauer chamber. The EC₅₀ values were determined by fitting a sigmoidal dose-response curve to the data using GraphPad Prism v.8. Each assay was developed in triplicate and the results correspond to the mean of three independent experiments.

Effect of Cm-p5 on trypomastigote cellular egress

CHO-K₁ cells (1.0 × 10⁴ per well) were maintained in 96-well plates in RPMI medium supplemented with 10% FBS and maintained at 37° C. After 24 h, the cells were infected with trypomastigote forms (5.0 × 10⁵ per well) for four hours. After this period, free parasites were removed. The infected cells were washed twice with PBS, the RPMI medium was replaced, and the cells were kept in culture firstly incubated with 50 µM of Cm-p5 for 5 days. Then, if the peptide caused an inhibitory effect on cellular egress, different concentrations (3.12-50 µM) was tested and the EC₅₀ was calculated. Negative and positive controls were used as the same as for invasion analysis. The plates were then incubated at 33 °C [14]. Trypomastigotes were collected from the extracellular medium on the 5^th day of incubation and counted in a Neubauer chamber. The EC₅₀ values were determined by fitting a sigmoidal dose-response curve to the data using GraphPad Prism v.8 and the selectivity index (SI) for trypomastigotes egress was calculated. Each assay was developed in triplicate and the results correspond to the mean of three independent experiments.

Effect of Cm-p5 on amastigote replication

CHO-K₁ cells (1.0 × 10⁴ per well) were maintained in 96-well plates in RPMI medium supplemented with 10% FBS and maintained at 37 °C. After 24 h, the cells were infected with trypomastigote forms (5.0 × 10⁵ per well) for 4 h. After this period, free parasites were removed by washing the plates twice with PBS, and cells were incubated with 25.2 μM of Cm-p5 (concentrations corresponding to 1 time the EC₅₀ for trypomastigotes egress) for 48 h. After this period, the CHO-K₁ cells and parasites were fixed with 4% paraformaldehyde and stained with Hoechst 33342. Images were taken by fluorescence microscopy. Negative and positive controls were used as the same as for invasion and cellular egress analysis. Cells, parasites, and infected cells were counted manually. The infection index (percentage of infected cells × the number of parasites per cell) was calculated. Each assay was developed in triplicate and the results correspond to the mean of three independent experiments.

Analysis of phosphatidylserine exposure, membrane permeabilization and cell cycle in amastigotes

CHO-K₁ cells were infected as described above, and treated with 25.2 μM of Cm-p5 (the concentration corresponding to the EC₅₀ for trypomastigotes egress) dissolved in DMSA, or only DMSO as a control, for 48 h. The infected host cells were washed with PBS and lysed using 0.05% SDS [20]. The lysates of the cells were monitored by microscopy and stopped by the addition of 10% FBS in PBS and further washing with PBS. The parasites were separated from cellular debris by two steps of centrifugation: the first of 10 min of centrifugation at 115 × g and 4 °C, the supernatant was recovered and submitted to centrifugation for 10 min at 2,900 × g and 4 °C. The parasites were recovered from the pellets by resuspension in PBS. The purity of intracellular forms was microscopically evaluated, and the yield was determined by counting them in a Neubauer chamber [20].

To determine the exposure of phosphatidylserine and membrane permeabilization, amastigotes (5.0 × 10⁷ parasites mL^-1) were labelled with propidium iodide (PI) and Annexin-V FITC (Molecular Probes) according to the manufacturer’s instructions. As positive controls for plasma membrane permeabilization and extracellular exposure of phosphatidylserine (PS), the parasites were treated with 150 μM digitonin for 30 min. The cells were analysed by flow cytometry on a BD Accuri C6 Plus, each condition was run in three biological independent replicas with 10,000 events collected and analyzed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v07, Carrboro, North Carolina, USA) [18].

To analyze the possible cell cycle alterations in intracellular amastigotes obtained from cells treated with Cm-p5, the parasites (5 × 10⁶ amastigotes mL^-1). To measure the DNA content, the cells were stained with 10 μg/mL PI (Molecular Probes/Invitrogen) and analyzed by flow cytometry on a BD Accuri C6 Plus, with 10,000 events collected from three biological independent experiments [19]. Histograms (number of counts by FL2 area), scatter plots (side scatter [FSC] area by forward scatter [SSC] area) and gates for each cell cycle phase were analysed using BD CSampler Plus Software (v 1.0.27.1) and FlowJo software (v10). Cell cycle data were fitted by a model included in the FlowJo software (v10).

Statistical analysis

Curve adjustments, regressions, and statistical analyses were performed with the GraphPad Prism v.8 analysis tools (San Diego, CA, USA). All assays were performed at least in biological triplicates. p < 0.05 were considered statistically significant. One-way ANOVA followed by the Tukey post-test was used for multi-comparison statistical analysis. The t test was used to analyze differences between pairs of groups.

Results

Cm-p5 affects the growth of T. cruzi epimastigotes

To make an initial selection of active peptides against T. cruzi among the Cm-p5 and 10 derivatives, the parasites were initially incubated in the presence of each peptide at a concentration of 10 μM (Fig 2A). For this, the cell density was followed for 10 days. Cm-p5 was the only peptide of the collection that significantly inhibited the increase of cell density at 10 µM, showing an inhibition of 46.4% (calculated at 5^th proliferation day, which corresponds to the mid-exponential proliferation phase) when compared to untreated cultures. Subsequently, different concentrations of Cm-p5 (ranging between 1.56 and 50 µM) were tested in order to calculate the EC₅₀ (S15 Fig). Cm-p5 exhibited a dose-dependent inhibition of cell proliferation with EC₅₀ values of 16.9 ± 1.2 μM (Fig 2B).

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Fig 2. Effect of Cm-p5 on the proliferation of the epimastigotes of T. cruzi and on CHO-K₁ A) Proliferation curves of epimastigotes of T. cruzi in the presence of 10 μM for each peptide.

Positive control for the inhibition of proliferation corresponds to curves obtained in the presence of 20 μM BNZ, and negative controls correspond to curves obtained in the presence of the solvent (DMSO 1%). Rotenone at 60 µM and Antimycin at 0.5 µM was used as a death control. Each experiment was developed in quadruplicates in three independent experiments and values correspond to Mean ± SD. B) Dose-response curve. Values for proliferation were obtained from the proliferation curves at day 5^th day. Proliferation of the epimastigotes without treatment (Control) was used as a reference of 0% inhibition. C) Viability of CHO-K₁ cells treated with different concentrations of Cm-p5 (range 12.5 µM to 500 µM). Viability was assessed by MTT assay. Results correspond to Mean ± SD of three independent experiments for each condition. Each curve in each independent experiment was made in quadruplicate. Original data in S1 Table.

https://doi.org/10.1371/journal.pntd.0013975.g002

Cytotoxicity of Cm-p5 on mammalian cells

To further assess Cm-p5 as potential anti-T. cruzi agent, its toxicity against CHO-K₁, used here as a model of a mammalian host cell, was tested. CHO-K₁ cells were cultured in the presence or not (control) of different concentrations of Cm-p5 ranging between 0 and 500 μM, for 24 h. As a proxy for cell viability, a classical MTT assay was used. The CC₅₀ value obtained was 124.8 ± 0.1 µM (Fig 2C).

Cm-p5 leads to necrosis-like cell death and triggers a cell cycle arrest in epimastigotes

Cm-p5-treated epimastigotes were assessed for plasma membrane integrity and for the exposure of PS in its external leaflet, a signal of programmed cell-death. As shown in Fig 3, epimastigotes treated with Cm-p5 1 × EC₅₀ and 2 × EC₅₀ presented 20.2% and 22.3% of the population labelled with PI, respectively (Fig 3B-3C) when compared to control (Fig 3A). Cells treated with digitonin (control inducing membrane permeabilization) presented 84.9% PI labelling (Fig 3D). Statistical tests indicated significant values in the treated parasites when compared to untreated parasites (Fig 3E). These data indicate that epimastigotes treated with Cm-p5 presented cell death through the loss of plasma membrane integrity.

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Fig 3. Cell death analysis of epimastigotes treated with Cm-p5.

Cells were treated or not (control) with Cm-p5 and simultaneously assessed for the extracellular exposure of phosphatidylserine (by using Annexin V) and plasma membrane integrity (by using PI labelling) followed by flow cytometry. Cells were treated or not with Cm-p5 for five days. A) Non-treated parasites (control); B) parasites treated with 1 × EC₅₀ Cm-p5; C) parasites treated with 2 × EC₅₀ Cm-p5 and; D) parasites treated with 150 µM of digitonin. E) Quantitative analysis of three independent experiments. Each experiment was run in three biological independent replicates with 10,000 events being collected and analysed. Quadrants correspond to: Q1 (dead cells), Q2 (late stages of programmed cell-death or necrotic), Q3 (early stages of programmed cell-death or PS exposing cells) and Q4 (normal and healthy cells). Original data in S2 Table.

https://doi.org/10.1371/journal.pntd.0013975.g003

Considering that Cm-p5 is capable of reducing the proliferation of epimastigotes, it was also investigated whether the peptide is capable of causing a cell cycle arrest. To verify this, the parasites were treated with 16.9 μM and 33.8 µM of Cm-p5 (corresponding to 1× and 2 × EC₅₀, respectively), or left untreated (control) for five days. The cells were then labelled with PI and analyzed by flow cytometry. Cm-p5 at a concentration corresponding to 1 × EC₅₀ caused an accumulation of 13.8% in G2_M and 11.6% in S_G2, while Cm-p5 at a concentration equivalent to 2 × EC₅₀ caused an accumulation of 12.9% in G2_M and 14.1% in S_G2. According to Elias et al. [21], the complete cell cycle of T. cruzi epimastigotes during cellular replication lasts approximately 24 hours. Based on the previously presented findings from cell cycle assays, we calculated the delay in cell cycle progression of epimastigotes treated with Cm-p5 at 1× and 2 × EC₅₀. In untreated parasites, in our experimental setup, the total cell cycle duration was 21.9 hours, with the G2_M and S_G2 phases lasting 3.1 and 1.8 hours, respectively. In contrast, treatment with Cm-p5 at 1 × EC₅₀ extended the duration of these phases to 4.6 hours (G2_M) and 3.8 hours (S_G2). Similarly, treatment at 2 × EC₅₀ resulted in phase durations of 4.5 hours (G2_M) and 4.9 hours (S_G2). These results are consistent with the cell cycle analysis, which showed that Cm-p5 treatment leads to accumulation of parasites in the G2_M and S_G2 phases (Fig 4).

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Fig 4. Effect of Cm-p5 on the T. cruzi epimastigotes cell cycle.

Cells were treated or not (control) with 1 and 2 times EC₅₀ Cm-p5 for five days. Then, the cells were washed, treated with RNase A, and stained with PI, and their DNA content was analysed by cytometry. In total, 10,000 events were analysed for each sample. Histograms obtained for the labelled cells in each experimental condition: A) Control (black line) and 1 × EC₅₀ (orange line) treatments resulted in a cell cycle accumulation of 13.8% in G2_M phase and 11.6% in S_G2 phase. B) Control (black line) and 2 × EC₅₀ (green line) treatments led to a cell cycle accumulation of 12.9% in G2_M phase and 14.1% in S_G2 phase. C) Quantification of the labeled cells at each phase of the cell cycle revealed statistically significant values for the G2_M and S_G2 phases. Results correspond to three independent biological replicas and the values were plotted as the Mean ± SD and compared to the control. Data correspond to the three independent biological experiments. Original data in S2 Table.

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Taken together, the results obtained in both experiments presented above demonstrate that 45.6% of epimastigotes treated with Cm-p5 1 × EC₅₀ were affected, and 49.3% of epimastigotes treated with Cm-p5 2 × EC₅₀ were affected. These data show that Cm-p5 inhibited epimastigote proliferation by a combination of cell death due to loss of plasma membrane integrity and a partial cell cycle arrest in the G2_M and S_G2 phases.

Effect of Cm-p5 on the host-cell invasion and egress of trypomastigotes

The effect of Cm-p5 was assessed on the invasion of the mammalian host-cell and further egress of trypomastigotes, the infective forms of T. cruzi. To this end, CHO-K₁ cells were incubated with trypomastigote forms in the presence of the peptide at a concentration of 50 µM (less than half the CC₅₀ value). BNZ at 5 µM was used as a positive control. The treatment was conducted for 4 h, and then the trypomastigotes that did not adhere or were not internalized into the host-cells were removed together with the treatment. The parasites that invaded the mammalian cells proceeded with the infection cycle. The trypomastigotes produced by the infected cells were collected from the extracellular medium on the 5^th day of incubation onward, and counted in a Neubauer chamber. Cm-p5 treatment during the host-cell invasion did not show a significant effect on the number of trypomastigotes produced when compared to untreated controls while BNZ caused a 25.7% of reduction on the number of trypomastigotes (Fig 5A). To assess the effect of Cm-p5 on the intracellular infection cycle, CHO-K₁ cells were treated during the infection. For this purpose, the cells were incubated with trypomastigotes for 4 h, and then the non-adhered or internalized parasites were removed. The infected cells were then incubated with Cm-p5 at 50 µM (less than half the CC₅₀ value) or 5 µM BNZ as a positive control. The treatment was conducted for until the 5th. day post-infection, and the trypomastigotes produced by the infected cells were collected from the extracellular medium on the 5^th day post-infection onward, and counted in a Neubauer chamber. When the infected cells were treated with Cm-p5 a 96% diminution on trypomastigote production was observed, when compared to untreated controls. Importantly, BNZ caused a 98% reduction on the number of egressed trypomastigotes (Fig 5B). Based on this results, Cm-p5 was tested at different concentrations (6.25-25 µM), showing a decrease of 41.8% on trypomastigote egress at a 25 µM concentration (Fig 5C). These results indicate that Cm-p5 interferes with the infection process, and the EC₅₀ for trypomastigotes egress under these conditions was determined as 25.2 ± 1.4 µM, presenting a SI of 4.9 (Table 1).

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Table 1. Cell viability and trypanocidal activity of AMPs.

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Fig 5. Effect of Cm-p5 on invasion and bursting of trypomastigotes.

Effect on the infectivity (A) and after invasion (B) of trypomastigotes treated with 50 µM of Cm-p5. Effect of treatment after invasion of parasites in CHO-K₁ cells (3.12–50 µM) (C). In all experiments, the bursting of trypomastigotes on the 5th day post-infection was evaluated by counting parasites in a Neubauer chamber. Original data in S3 Table.

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Cm-p5 inhibits the intracellular cycle of T. cruzi

In order to prove that the Cm-p5 is able to affect the intracellular infection cycle, and thus, to decrease the amastigotes proliferation, the infected cells were treated with 25.2 µM of Cm-p5 (the concentration corresponding to the gEC₅₀ for trypomastigote egress) for 2 days after infection. It is worth remarking that 2 days after infection, the predominant intracellular stage of the parasite is the amastigote [15]. BNZ at 5 µM was used as a positive control. After fixing and staining the infected cells the effect of Cm-p5 on the total number of cells, the number of infected cells, and the number of amastigotes per infected cell, were quantified. Cm-p5 significantly diminished the infected cells by 75% (Fig 6A) and the number of parasites per cell by 64.3% (Fig 6B) which reduced the infection index by 91.1% (Fig 6C). BNZ (5 µM) caused a reduction of 59% in the infected cells, 81.4% in the number of parasites per cell and 92.4% in the infection index. All together, these results indicate that Cm-p5 interferes the parasites proliferation and/or differentiation during the intracellular infection.

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Fig 6. Effect of Cm-p5 on the mammalian cell infection by T. cruzi.

The effect of Cm-p5 after infection on CHO-K₁ cells with trypomastigotes forms. A) The effect of Cm-p5 on the successful establishment of the infection was measured as a percent of infected cells at 2 days post-infection. B) The number of intracellular amastigotes per infected cell was recorded, and C) the infection index (percentage of infected cells × the number of parasites per infected cell) was computed for parasites treated or not (control) with 25.2 µM of Cm-p5. Results correspond to three independent biological replicas and the values were plotted as the Mean ± SD and compared to control. Original data in S3 Table.

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Cm-p5 leads to necrosis-like cell death and triggers a cell cycle arrest in amastigotes

Cm-p5-treated amastigotes were assessed for plasma membrane integrity and for the exposure of PS in its external leaflet, a signal of programmed cell-death. Amastigotes treated with Cm-p5 1 × EC₅₀ presented 47.7% of the population labelled with PI (Fig 7B) when compared to control (Fig 7A). Cells treated with digitonin (control inducing membrane permeabilization) presented 98.5% PI labelling (Fig 7C). Statistical tests indicated significant values in the treated parasites when compared to untreated parasites (Fig 7D). These data indicate that amastigotes treated with Cm-p5 presented cell death through the loss of plasma membrane integrity.

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Fig 7. Cell death analysis of amastigotes treated with Cm-p5.

Cells were treated or not (control) with Cm-p5 and simultaneously assessed for the extracellular exposure of phosphatidylserine (by using Annexin V) and plasma membrane integrity (by using PI labelling) followed by flow cytometry. Cells were treated or not with Cm-p5 for 48 hours. A) Non-treated parasites (control); B) parasites treated with 1 × EC₅₀ Cm-p5 on egress of trypomastigotes; C) parasites treated with 150 µM of digitonin. D) Quantitative analysis of three independent experiments. Each experiment was run in three biological independent replicates with 10,000 events being collected and analysed. Quadrants correspond to: Q1 (dead cells), Q2 (late stages of programmed cell-death or necrotic), Q3 (early stages of programmed cell-death or PS exposing cells) and Q4 (normal and healthy cells). Original data in S4 Table.

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Considering that Cm-p5 is capable of reducing the replication of amastigotes, it was also investigated whether the peptide is capable of causing a cell cycle arrest. To verify this, the parasites were treated with 25.2 μM of Cm-p5 (corresponding to 1 × EC₅₀), or left untreated (control) for 48 hours. The cells were then labelled with PI and analyzed by flow cytometry. Cm-p5 caused an 18.7% reduction in the Sub_G1 phase and an increase in the remaining phases of the cell cycle of 27.1% in G1_S, 27% in G2_M, and 11.2% in S_G2 (Fig 8). These data reveal that Cm-p5 treatment decreased the number of amastigotes in the Sub_G1 phase compared to untreated cells, indicating an accumulation in the G1_S, G2_M, and S_G2 phases.

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Fig 8. Effect of Cm-p5 on the T. cruzi amastigotes cell cycle.

Cells were treated or not (control) with 1 × EC₅₀ Cm-p5 for 48 hoours. Then, the cells were washed, treated with RNase A, and stained with PI, and their DNA content was analysed by cytometry. In total, 10,000 events were analysed for each sample. Histograms obtained for the labelled cells in each experimental condition: A) Control (black line) and 1 × EC₅₀ (red line) treatments resulted in a decrease of 18.7% in Sub_G1 phase and an increase in the other phases of the cell cycle. B) Quantification of the labeled cells at each phase of the cell cycle revealed statistically significant values for the Sub_G1 phase. Results correspond to three independent biological replicas and the values were plotted as the Mean ± SD and compared to the control. Data correspond to the three independent biological experiments. Original data in S1 Table.

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Taken together, the results obtained in both experiments presented above demonstrate that 66.4% of amastigotes treated with Cm-p5 1 × EC₅₀ were affected. These data demonstrate that Cm-p5 inhibits amastigote replication through a combination of cell death due to loss of plasma membrane integrity, and partial cell cycle arrest in the G1_S, G2_M, and S_G2 phases.

Discussion

Due to their extraordinary structural diversity and physicochemical properties, peptides have been pointed out as sources of a myriad of biological functions, from immunomodulation to anti-microbials [22]. Based on the urgent need of new drugs for Chagas disease, we assessed here Cm-p5, a synthetic peptide with known antimicrobial activity, and 10 of its derivatives for an anti-T. cruzi activity. After an initial screening, Cm-p5 was identified as the only peptide in the collection that significantly inhibited the parasite proliferation, by a combination of cell death through the loss of plasma membrane integrity, and a partial cell-cycle arrest at the G2_M and S_G2 phases. When assessed against the infective forms of the parasite the peptide showed an EC₅₀ of 25.2 ± 1.4 µM for trypomastigote egress, with a SI of 4.9, which when assessed against the infected cells, Cm-p5 reduced the infection index by 91.1%. Notably, Cm-p5 showed no activity against trypomastigotes. For a therapeutic to be effective against both acute and chronic infection, future peptides based on the Cm-p5 scaffold will need to overcome this hurdle to target these forms present in the patient´s blood, responsible for the prominent acute parasitemia and the chronic tissue dissemination and insect infection. We investigated as well the mechanism underlying Cm-p5 inhibition in intracellular forms and found that, similar to epimastigotes, amastigote replication is impaired by a dual effect: induction of cell death through loss of plasma membrane integrity and partial cell cycle arrest at the G1_S, G2_M, and S_G2 phases. It is worth remarking that benznidazole performed better than Cm-p5. However, in a landscape in which new drugs are urgently necessary for treating Chagas disease, our data allow to conceive a new source of chemical entities to be optimized for this purpose.

Previous works already showed the effect of different AMPs on T. cruzi infection. Fieck et al. reported LC₁₀₀ values of 199 µM for Apidaecin, 30 µM for Melittin, 80 µM for Cecropin A, 33 µM for Magainin II, and 10 µM for Moricin against epimastigotes form [23]. Pinto and colleagues evaluated four antimicrobial peptides (AMPs)—Dermaseptins 1 and 4 and Phylloseptins 7 and 8—from Phyllomedusa nordestina against T. cruzi [24]. Similar to Cm-p5, these AMPs demonstrated high activity against the trypomastigote form. Specifically, Dermaseptin 4 exhibited an IC₅₀ value of 0.25 µM, while Dermaseptin 1 showed an IC₅₀ of 0.68 µM. Both Phylloseptins 7 and 8 exhibited selective anti-T. cruzi activity, with EC₅₀ values of 0.34 µM and 0.46 µM, respectively, and a selectivity index (SI) of 100. Additionally, Dermaseptins 1 and 4, along with Phylloseptins 7 and 8, induced time-dependent plasma membrane permeabilization in T. cruzi, ultimately leading to cell death. In another study, Díaz-Garrido et al. evaluated the effect of rDef1.3, a defensin isoform described in Triatoma (Meccus) pallidipennis, on T. cruzi [25]. The EC₅₀ value for epimastigotes was 84 µM, and the observed cytotoxic effect was associated with membrane damage.

When analyzing the trypanocidal effects of AMPs in relation to necrosis, other peptides such as Batroxycidin (BatxC) and Crotalicidin (Ctn), isolated from the venom glands of Bothrops atrox and Crotalus durissus terrificus, respectively, were found to induce the death of all developmental stages of T. cruzi strain Y. This occurred through the formation of pores in the plasma membrane, the promotion of reactive oxygen species (ROS) production, the loss of mitochondrial membrane potential, and ultimately cell death by necrosis [26,27]. Another study investigated the effect of the peptide Tachyplesin (Tach), isolated from the crab Tachypleus tridentatus, on T. cruzi [28]. This AMP demonstrated a promising trypanocidal effect at micromolar concentrations, with low cytotoxicity against mammalian cell lines. Although the exact mechanism of action in T. cruzi remains unclear, it has been reported that this peptide forms transient pores in membranes and subsequently translocates across them after pore disintegration, triggering necrotic cell death.

Cm-p5 has demonstrated antimicrobial effects against Candida albicans, Candida parapsilosis, and Candida auris [29], in addition, this peptide has been shown to induce cell death and ROS production in the melanoma cell line A375 [8]. Isothermal titration calorimetry (ITC) studies have shown that Cm-p5 has high affinity for PS, with a binding constant (K) of approximately 4.22 × 10⁵ M ⁻ ¹ significantly higher than its affinity for other phospholipids such as phosphatidylcholine and phosphatidylethanolamine. This specific interaction suggests that Cm-p5 may preferentially associate with regions of the plasma membrane rich in PS, especially during cell death processes in which PS is externalized [8]. Our study in T. cruzi have shown that Cm-p5’s effect is primarily associated with necrosis, and the PS labelling by Annexin-V only occurs upon the loss of plasma membrane integrity, evidenced by the propidium iodide labelling. Differently from Cm-p5, other peptides such as Pep5, have shown that they can partially induce cell death via programmed cell-death combined with necrosis [30]. Pep5-cpp, for instance, activates caspases 3/7 and 9, promotes phosphorylation of proteins related to the MAPK pathway, and inhibits the beta-5 subunit of the proteasome. In addition, Pep5-cpp induced signalling pathways typically associated with programmed cell death by increasing intracellular ROS production and calcium levels [31]. Although specific studies on Cm-p5 remain limited, it is plausible that similar mechanisms are involved in its action.

In summary, despite the already demonstrated ability of Cm-p5 to specifically interact with PS in Candida spp. [28,29] and showing moderate antibacterial and antiviral activity [30], this seems not to be the case when assessed against T. cruzi. Instead, Cm-p5 has an activity driven by two concomitant effects: cell-death induction due to plasma membrane damage, and a partial cell-cycle arrest at the G2_M and S_G2 phases in the surviving cells. Considering the results of the present study, particularly the limited effect of Cm-p5 on both the infectious and proliferative forms of the parasite in mammalian hosts, Cm-p5 demonstrates promising profile as a template to be optimized through a new round of derivatives. Therefore, we propose that this peptide could serve as a valuable candidate for the development of new therapeutic agents to improve chemotherapy for Chagas disease.

Conclusions

Our work contributes to the understanding of the anti-trypanosomatid activity of Cm-p5, which demonstrated an in vitro antiparasitic effects against the infective forms of the parasite during the infection of mammalian host cells. In vitro analyses, such as flow cytometry, revealed that the inhibition of parasite proliferation involves a necrosis-like cell death and induces cell cycle arrest in the G2_M and S_G2 phases, ultimately impairing the proliferation of T. cruzi epimastigotes. Furthermore, the anti-trypanosomatid activity of Cm-p5 appears to be linked to its ability to inhibit cellular egress events, reducing the release of trypomastigotes. Together with the limited effects of Cm-p5, its selectivity index (SI) does not appear to be sufficiently high to consider it as a strong candidate molecule for the treatment of Chagas disease, since ideally, a good candidate should have a considerably lower EC₅₀ and a SI > 10 [32]. Nevertheless, our findings provide valuable insights that may contribute to the development of new peptides to improve the chemotherapy of Chagas disease.

We are grateful to Dr. Ludger Standker (Core Facility of Functional Peptidomics, University of Ulm) for the support with the synthesis and quality of the Cm-p5 peptide, and to the DAAD-German Ministry for Foreign Affairs via the program Global Health and Pandemic Prevention Centers (project 57592717-GLACIER).

Supporting information

S1 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 1.

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S2 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 2.

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S3 Fig. RP-HPLC (left panel) and ESI-MS (right panel) of peptide 3.

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S4 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 4.

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S5 Fig. RP-HPLC (left panel) after 6h of cyclization and ESI-HRMS (right panel) of cyclic peptide 4.

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S6 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 5.

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S7 Fig. RP-HPLC (left upper panel) and ESI-HRMS (right upper panel and lower panel) of cyclization of peptide 5 at 0,5 mM after 24 h.

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S8 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 6.

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S9 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 7.

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S10 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 8.

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S11 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of cyclization of CysCysCm-p5 peptide at 0,03 mM after 24 h.

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S12 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of cyclization of peptide 9 at 1 mM after 24 h.

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S13 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 10.

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S14 Fig. RP-HPLC (left panel) and ESI-MS (right panel) of the antiparallel dimer of CysCysCm-p5, peptide 11.

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S15 Fig. Effect of Cm-p5 on the proliferation of the epimastigotes of T. cruzi.

Proliferation curves of epimastigotes of T. cruzi in the presence of different concentrations of Cm-p5. Positive control for the inhibition of proliferation corresponds to curves obtained in the presence of 20 μM BNZ, and negative controls correspond to curves obtained in the presence of the solvent (DMSO 1%). Rotenone at 60 µM and Antimycin at 0.5 µM was used as a death control. Each experiment was developed in quadruplicates in three independent experiments and values correspond to Mean ± SD. Original data in S1 Table.

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S1 Table. Original data used for Figs 2 and S15.

Optical density values (620 nm) corresponding to calibration and proliferation curves performed using epimastigote forms.

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S2 Table. Original data used for Figs 3 and 4.

Percentage values of epimastigotes labelled with propidium iodide (PI) and annexin calculated using FlowJo software (v07, Carrboro, North Carolina, USA).

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S3 Table. Original author used for Figs 5 and 6.

Values corresponding to the parasite count from mammalian cells (CHO-K₁).

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S4 Table. Original data used for Figs 7 and 8.

Percentage values of amastigotes labelled with propidium iodide (PI) and annexin calculated using FlowJo software (v07, Carrboro, North Carolina, USA).

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References

1. World Health Organization. Chagas disease (also known as American trypanosomiasis). https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis). 2024.
- View Article
- Google Scholar
2. Pérez-Molina JA, Crespillo-Andújar C, Bosch-Nicolau P, Molina I. Trypanocidal treatment of Chagas disease. Enferm Infecc Microbiol Clin (Engl Ed). 2020;:S0213-005X(20)30193-2. pmid:32527494
- View Article
- PubMed/NCBI
- Google Scholar
3. World Health Organization. Ending the neglect to attain the Sustainable Development Goals: A road map for neglected tropical diseases 2021–2030. 2021. https://www.who.int/publications/i/item/9789240010352
- View Article
- Google Scholar
4. Hancock REW, Haney EF, Gill EE. The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol. 2016;16(5):321–34. pmid:27087664
- View Article
- PubMed/NCBI
- Google Scholar
5. Kumar P, Kizhakkedathu JN, Straus SK. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules. 2018;8(1):4. pmid:29351202
- View Article
- PubMed/NCBI
- Google Scholar
6. Alba A, López-Abarrategui C, Otero-González AJ. Host defense peptides: an alternative as antiinfective and immunomodulatory therapeutics. Biopolymers. 2012;98(4):251–67. pmid:23193590
- View Article
- PubMed/NCBI
- Google Scholar
7. Hagemann CL, Macedo AJ, Tasca T. Therapeutic potential of antimicrobial peptides against pathogenic protozoa. Parasitol Res. 2024;123(2).
- View Article
- Google Scholar
8. López-Abarrategui C, McBeth C, Mandal SM, Sun ZJ, Heffron G, Alba-Menéndez A, et al. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). FASEB J. 2015;29(8):3315–25. pmid:25921828
- View Article
- PubMed/NCBI
- Google Scholar
9. Vicente FEM, González-Garcia M, Diaz Pico E, Moreno-Castillo E, Garay HE, Rosi PE, et al. Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity. ACS Omega. 2019;4(21):19081–95. pmid:31763531
- View Article
- PubMed/NCBI
- Google Scholar
10. Brener Z, Chiari E. Aspects of early growth of different Trypanosoma cruzi strains in culture medium. J Parasitol. 1965;51(6):922–6. pmid:5848818
- View Article
- PubMed/NCBI
- Google Scholar
11. Girard RMBM, Crispim M, Stolić I, Damasceno FS, Santos da Silva M, Pral EMF, et al. An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells. Antimicrob Agents Chemother. 2016;60(10):5867–77. pmid:27431229
- View Article
- PubMed/NCBI
- Google Scholar
12. Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol Biochem Parasitol. 1985;16(3):315–27. pmid:3903496
- View Article
- PubMed/NCBI
- Google Scholar
13. de Freitas Nascimento J, Barisón MJ, Torres Montanaro G, Marchese L, Oliveira Souza RO, Silva LS, et al. Knocking out histidine ammonia-lyase by using CRISPR-Cas9 abolishes histidine role in the bioenergetics and the life cycle of Trypanosoma cruzi. Microb Cell. 2025;12:157–72. pmid:40584587
- View Article
- PubMed/NCBI
- Google Scholar
14. Brener Z, Golgher R, Bertelli MS, Teixeira JA. Strain-dependent thermosensitivity influencing intracellular differentiation of Trypanosoma cruzi in cell culture. J Protozool. 1976;23(1):147–50. pmid:775065
- View Article
- PubMed/NCBI
- Google Scholar
15. Tonelli RR, Silber AM, Almeida-de-Faria M, Hirata IY, Colli W, Alves MJM. L-proline is essential for the intracellular differentiation of Trypanosoma cruzi. Cell Microbiol. 2004;6(8):733–41. pmid:15236640
- View Article
- PubMed/NCBI
- Google Scholar
16. Magdaleno A, Ahn I-Y, Paes LS, Silber AM. Actions of a proline analogue, L-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi. PLoS One. 2009;4(2):e4534. pmid:19229347
- View Article
- PubMed/NCBI
- Google Scholar
17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. pmid:6606682
- View Article
- PubMed/NCBI
- Google Scholar
18. Damasceno FS, Barisón MJ, Pral EMF, Paes LS, Silber AM. Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi. PLoS Negl Trop Dis. 2014;8(2):e2717. pmid:24587468
- View Article
- PubMed/NCBI
- Google Scholar
19. Cuevas-Hernández RI, Girard RMBM, Krstulović L, Bajić M, Silber AM. An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection. PLoS Negl Trop Dis. 2021;15(11):e0009994. pmid:34843481
- View Article
- PubMed/NCBI
- Google Scholar
20. Damasceno FS, Barisón MJ, Crispim M, Souza ROO, Marchese L, Silber AM. L-Glutamine uptake is developmentally regulated and is involved in metacyclogenesis in Trypanosoma cruzi. Mol Biochem Parasitol. 2018;224:17–25. pmid:30030130
- View Article
- PubMed/NCBI
- Google Scholar
21. Elias MC, da Cunha JPC, de Faria FP, Mortara RA, Freymüller E, Schenkman S. Morphological events during the Trypanosoma cruzi cell cycle. Protist. 2007;158(2):147–57. pmid:17185034
- View Article
- PubMed/NCBI
- Google Scholar
22. Zhang R, Xu L, Dong C. Antimicrobial Peptides: An Overview of their Structure, Function and Mechanism of Action. Protein Pept Lett. 2022;29(8):641–50. pmid:35702771
- View Article
- PubMed/NCBI
- Google Scholar
23. Fieck A, Hurwitz I, Kang AS, Durvasula R. Trypanosoma cruzi: synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts. Exp Parasitol. 2010;125(4):342–7. pmid:20206169
- View Article
- PubMed/NCBI
- Google Scholar
24. Pinto EG, Pimenta DC, Antoniazzi MM, Jared C, Tempone AG. Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi. Exp Parasitol. 2013;135(4):655–60. pmid:24113627
- View Article
- PubMed/NCBI
- Google Scholar
25. Díaz-Garrido P, Cárdenas-Guerra RE, Martínez I, Poggio S, Rodríguez-Hernández K, Rivera-Santiago L, et al. Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis. Insect Biochem Mol Biol. 2021;139:103673. pmid:34700021
- View Article
- PubMed/NCBI
- Google Scholar
26. Mello CP, Lima DB, Menezes RRPPBd, Bandeira ICJ, Tessarolo LD, Sampaio TL, et al. Evaluation of the antichagasic activity of batroxicidin, a cathelicidin-related antimicrobial peptide found in Bothrops atrox venom gland. Toxicon. 2017;130:56–62. pmid:28246023
- View Article
- PubMed/NCBI
- Google Scholar
27. Bandeira ICJ, Bandeira-Lima D, Mello CP, Pereira TP, De Menezes RRPPB, Sampaio TL, et al. Antichagasic effect of crotalicidin, a cathelicidin-like vipericidin, found in Crotalus durissus terrificus rattlesnake’s venom gland. Parasitology. 2018;145(8):1059–64. pmid:29208061
- View Article
- PubMed/NCBI
- Google Scholar
28. Monteiro ML, Lima DB, Menezes RRPPBd, Sampaio TL, Silva BP, Serra Nunes JV, et al. Antichagasic effect of hemocyanin derived from antimicrobial peptides of penaeus monodon shrimp. Exp Parasitol. 2020;215:107930. pmid:32464221
- View Article
- PubMed/NCBI
- Google Scholar
29. Amann V, Kissmann A-K, Mildenberger V, Krebs I, Perez-Erviti JA, Martell-Huguet EM, et al. Cm-p5 Peptide Dimers Inhibit Biofilms of Candida albicans Clinical Isolates, C. parapsilosis and Fluconazole-Resistant Mutants of C. auris. Int J Mol Sci. 2023;24(12):9788. pmid:37372935
- View Article
- PubMed/NCBI
- Google Scholar
30. de Araujo CB, Russo LC, Castro LM, Forti FL, do Monte ER, Rioli V, et al. A novel intracellular peptide derived from g1/s cyclin d2 induces cell death. J Biol Chem. 2014;289(24):16711–26. pmid:24764300
- View Article
- PubMed/NCBI
- Google Scholar
31. de Araujo CB, de Lima LP, Calderano SG, Silva Damasceno F, Silber AM, Elias MC. Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity. Antimicrob Agents Chemother. 2019;63(5):e01806-18. pmid:30833431
- View Article
- PubMed/NCBI
- Google Scholar
32. Don R, Ioset J-R. Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections. Parasitology. 2014;141(1):140–6. pmid:23985066
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. World Health Organization. Chagas disease (also known as American trypanosomiasis). https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis). 2024.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Pérez-Molina JA, Crespillo-Andújar C, Bosch-Nicolau P, Molina I. Trypanocidal treatment of Chagas disease. Enferm Infecc Microbiol Clin (Engl Ed). 2020;:S0213-005X(20)30193-2. pmid:32527494
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. World Health Organization. Ending the neglect to attain the Sustainable Development Goals: A road map for neglected tropical diseases 2021–2030. 2021. https://www.who.int/publications/i/item/9789240010352
View Article
Google Scholar

[9] View Article

[10] Google Scholar

[ref4] 4. Hancock REW, Haney EF, Gill EE. The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol. 2016;16(5):321–34. pmid:27087664
View Article
PubMed/NCBI
Google Scholar

[12] View Article

[13] PubMed/NCBI

[14] Google Scholar

[ref5] 5. Kumar P, Kizhakkedathu JN, Straus SK. Antimicrobial Peptides: Diversity, Mechanism of Action and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules. 2018;8(1):4. pmid:29351202
View Article
PubMed/NCBI
Google Scholar

[16] View Article

[17] PubMed/NCBI

[18] Google Scholar

[ref6] 6. Alba A, López-Abarrategui C, Otero-González AJ. Host defense peptides: an alternative as antiinfective and immunomodulatory therapeutics. Biopolymers. 2012;98(4):251–67. pmid:23193590
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref7] 7. Hagemann CL, Macedo AJ, Tasca T. Therapeutic potential of antimicrobial peptides against pathogenic protozoa. Parasitol Res. 2024;123(2).
View Article
Google Scholar

[24] View Article

[25] Google Scholar

[ref8] 8. López-Abarrategui C, McBeth C, Mandal SM, Sun ZJ, Heffron G, Alba-Menéndez A, et al. Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae). FASEB J. 2015;29(8):3315–25. pmid:25921828
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Vicente FEM, González-Garcia M, Diaz Pico E, Moreno-Castillo E, Garay HE, Rosi PE, et al. Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity. ACS Omega. 2019;4(21):19081–95. pmid:31763531
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Brener Z, Chiari E. Aspects of early growth of different Trypanosoma cruzi strains in culture medium. J Parasitol. 1965;51(6):922–6. pmid:5848818
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 11. Girard RMBM, Crispim M, Stolić I, Damasceno FS, Santos da Silva M, Pral EMF, et al. An Aromatic Diamidine That Targets Kinetoplast DNA, Impairs the Cell Cycle in Trypanosoma cruzi, and Diminishes Trypomastigote Release from Infected Mammalian Host Cells. Antimicrob Agents Chemother. 2016;60(10):5867–77. pmid:27431229
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S. In vitro differentiation of Trypanosoma cruzi under chemically defined conditions. Mol Biochem Parasitol. 1985;16(3):315–27. pmid:3903496
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. de Freitas Nascimento J, Barisón MJ, Torres Montanaro G, Marchese L, Oliveira Souza RO, Silva LS, et al. Knocking out histidine ammonia-lyase by using CRISPR-Cas9 abolishes histidine role in the bioenergetics and the life cycle of Trypanosoma cruzi. Microb Cell. 2025;12:157–72. pmid:40584587
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Brener Z, Golgher R, Bertelli MS, Teixeira JA. Strain-dependent thermosensitivity influencing intracellular differentiation of Trypanosoma cruzi in cell culture. J Protozool. 1976;23(1):147–50. pmid:775065
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Tonelli RR, Silber AM, Almeida-de-Faria M, Hirata IY, Colli W, Alves MJM. L-proline is essential for the intracellular differentiation of Trypanosoma cruzi. Cell Microbiol. 2004;6(8):733–41. pmid:15236640
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Magdaleno A, Ahn I-Y, Paes LS, Silber AM. Actions of a proline analogue, L-thiazolidine-4-carboxylic acid (T4C), on Trypanosoma cruzi. PLoS One. 2009;4(2):e4534. pmid:19229347
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63. pmid:6606682
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Damasceno FS, Barisón MJ, Pral EMF, Paes LS, Silber AM. Memantine, an antagonist of the NMDA glutamate receptor, affects cell proliferation, differentiation and the intracellular cycle and induces apoptosis in Trypanosoma cruzi. PLoS Negl Trop Dis. 2014;8(2):e2717. pmid:24587468
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref19] 19. Cuevas-Hernández RI, Girard RMBM, Krstulović L, Bajić M, Silber AM. An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection. PLoS Negl Trop Dis. 2021;15(11):e0009994. pmid:34843481
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref20] 20. Damasceno FS, Barisón MJ, Crispim M, Souza ROO, Marchese L, Silber AM. L-Glutamine uptake is developmentally regulated and is involved in metacyclogenesis in Trypanosoma cruzi. Mol Biochem Parasitol. 2018;224:17–25. pmid:30030130
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Elias MC, da Cunha JPC, de Faria FP, Mortara RA, Freymüller E, Schenkman S. Morphological events during the Trypanosoma cruzi cell cycle. Protist. 2007;158(2):147–57. pmid:17185034
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Zhang R, Xu L, Dong C. Antimicrobial Peptides: An Overview of their Structure, Function and Mechanism of Action. Protein Pept Lett. 2022;29(8):641–50. pmid:35702771
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Fieck A, Hurwitz I, Kang AS, Durvasula R. Trypanosoma cruzi: synergistic cytotoxicity of multiple amphipathic anti-microbial peptides to T. cruzi and potential bacterial hosts. Exp Parasitol. 2010;125(4):342–7. pmid:20206169
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. Pinto EG, Pimenta DC, Antoniazzi MM, Jared C, Tempone AG. Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi. Exp Parasitol. 2013;135(4):655–60. pmid:24113627
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Díaz-Garrido P, Cárdenas-Guerra RE, Martínez I, Poggio S, Rodríguez-Hernández K, Rivera-Santiago L, et al. Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis. Insect Biochem Mol Biol. 2021;139:103673. pmid:34700021
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Mello CP, Lima DB, Menezes RRPPBd, Bandeira ICJ, Tessarolo LD, Sampaio TL, et al. Evaluation of the antichagasic activity of batroxicidin, a cathelicidin-related antimicrobial peptide found in Bothrops atrox venom gland. Toxicon. 2017;130:56–62. pmid:28246023
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Bandeira ICJ, Bandeira-Lima D, Mello CP, Pereira TP, De Menezes RRPPB, Sampaio TL, et al. Antichagasic effect of crotalicidin, a cathelicidin-like vipericidin, found in Crotalus durissus terrificus rattlesnake’s venom gland. Parasitology. 2018;145(8):1059–64. pmid:29208061
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. Monteiro ML, Lima DB, Menezes RRPPBd, Sampaio TL, Silva BP, Serra Nunes JV, et al. Antichagasic effect of hemocyanin derived from antimicrobial peptides of penaeus monodon shrimp. Exp Parasitol. 2020;215:107930. pmid:32464221
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Amann V, Kissmann A-K, Mildenberger V, Krebs I, Perez-Erviti JA, Martell-Huguet EM, et al. Cm-p5 Peptide Dimers Inhibit Biofilms of Candida albicans Clinical Isolates, C. parapsilosis and Fluconazole-Resistant Mutants of C. auris. Int J Mol Sci. 2023;24(12):9788. pmid:37372935
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. de Araujo CB, Russo LC, Castro LM, Forti FL, do Monte ER, Rioli V, et al. A novel intracellular peptide derived from g1/s cyclin d2 induces cell death. J Biol Chem. 2014;289(24):16711–26. pmid:24764300
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. de Araujo CB, de Lima LP, Calderano SG, Silva Damasceno F, Silber AM, Elias MC. Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity. Antimicrob Agents Chemother. 2019;63(5):e01806-18. pmid:30833431
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref32] 32. Don R, Ioset J-R. Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections. Parasitology. 2014;141(1):140–6. pmid:23985066
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

Figures

Abstract

Author summary

Introduction

Materials and methods

Reagents

Peptide synthesis

Cells and parasite cultures

In vitro inhibition of proliferation assays on epimastigotes

The effect of the Cm-p5 on mammalian cell viability

Analysis of phosphatidylserine exposure and membrane permeabilization in epimastigotes

DNA content and cell cycle analysis in epimastigotes

Effect of Cm-p5 on trypomastigote invasion

Effect of Cm-p5 on trypomastigote cellular egress

Effect of Cm-p5 on amastigote replication

Analysis of phosphatidylserine exposure, membrane permeabilization and cell cycle in amastigotes

Statistical analysis

Results

Cm-p5 affects the growth of T. cruzi epimastigotes

Cytotoxicity of Cm-p5 on mammalian cells

Cm-p5 leads to necrosis-like cell death and triggers a cell cycle arrest in epimastigotes

Effect of Cm-p5 on the host-cell invasion and egress of trypomastigotes

Cm-p5 inhibits the intracellular cycle of T. cruzi

Cm-p5 leads to necrosis-like cell death and triggers a cell cycle arrest in amastigotes

Discussion

Conclusions

Supporting information

S1 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 1.

S2 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 2.

S3 Fig. RP-HPLC (left panel) and ESI-MS (right panel) of peptide 3.

S4 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 4.

S5 Fig. RP-HPLC (left panel) after 6h of cyclization and ESI-HRMS (right panel) of cyclic peptide 4.

S6 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 5.

S7 Fig. RP-HPLC (left upper panel) and ESI-HRMS (right upper panel and lower panel) of cyclization of peptide 5 at 0,5 mM after 24 h.

S8 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 6.

S9 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 7.

S10 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of the linear peptide 8.

S11 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of cyclization of CysCysCm-p5 peptide at 0,03 mM after 24 h.

S12 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of cyclization of peptide 9 at 1 mM after 24 h.

S13 Fig. RP-HPLC (left panel) and ESI-HRMS (right panel) of peptide 10.

S14 Fig. RP-HPLC (left panel) and ESI-MS (right panel) of the antiparallel dimer of CysCysCm-p5, peptide 11.

S15 Fig. Effect of Cm-p5 on the proliferation of the epimastigotes of T. cruzi.

S1 Table. Original data used for Figs 2 and S15.

S2 Table. Original data used for Figs 3 and 4.

S3 Table. Original author used for Figs 5 and 6.

S4 Table. Original data used for Figs 7 and 8.

References