Batroxin I: A Novel Bradykinin-Potentiating Peptide with Cytotoxic Activity Isolated from Bothrops atrox Snake Venom

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.


Introduction
Cancer is one of the most impactful diseases in the world, which still has few prospects for safe and effective treatments that provide accessibility and quality of life for patients during the therapy. Data from the Global Cancer Observatory (Globocan) estimate an increase of 47% in the number of new cases in the next 20 years, corresponding to about 28.4 million new cases per year in 2040, which will mainly impact countries with low and medium human development rates (Sung et al. 2021). Not only the cancer incidence has alarming rates, but also the mortality rates. The Brazilian National Cancer Institute reports mortality rates of 17.19% for the disease, which reflects one death in every six cancer patients, with estimated growth rate of 0.2% per year (Brasil 2019).
Despite the high global incidence, scientific and technological advances, the classical antitumor treatments still rely mostly on surgery, hormone therapy, radiotherapy, chemotherapy, and photoradiation (Ferlay et al. 2015;Miller et al. 2019). Chemotherapy, the main therapeutic strategy used in neoplasia, is usually a combination of drugs that interferes in DNA synthesis, transcription, and translation processes; it is highly aggressive to patients due to its high toxicity potential in healthy cells. This fact determines the great academic and technological interest in the development of new that are more efficient and accessible to the patients and improve their quality of life during the treatment period (Gibbs 2000;Smith and Prasad 2021).
Snake venom compounds have been reported as potential agents to target specific molecular pathways in cancer cells (Koh et al. 2006;Vyas et al. 2013). Over the years, researchers have identified these molecules, studied their efficacy as diagnostic tools and therapeutic agents, and developed new synthetic molecules that mimetize their antitumor, analgesic, antimicrobial, neurotoxic, and anticoagulant properties (Péterfi et al. 2019). As the technological advances have enabled the development of comprehensive approaches and complementary techniques to isolate, characterize and explore the structure-function relationships, the venom components present in low amounts, such as peptides, have become of great interest for biotechnological researches (Gomes et al. 2010).
Peptides with the ability to kill cancer cells both in vitro and in vivo are called anticancer peptides (Armbrecht et al. 2017). These peptides have great structural diversity but share similar characteristics. They are smaller than 50 amino acid residues, and present abundant cationic amino acid residues (arginine and lysine) and hydrophobic amino acids (Pérez-Peinado et al. 2020). Anticancer peptides occur in a wide range of snake species, particularly those belonging to the family Viperidae (Jain and Kumar 2012;Conlon et al. 2013). Snake venom peptides isolated from the Elapidae family, including dendrotoxin-κ from black mamba venom (Dendroaspis polylepis) (Jang et al. 2011) and α-cobratoxin from Thai snake Naja kaouthia venom (Grozio et al. 2008) are able to suppress the growth of lung human adenocarcinoma A549 cell line. Three venom peptides isolated from mamba Dendroaspis angusticeps are cytotoxic to human umbilical cord cells HUVEC, breast adenocarcinoma cells MDA-MB-231, and colorectal adenocarcinoma cells HT-29 (Conlon et al. 2014).
Bothrops genus' venoms have provided the peptide BPP13a isolated from Bothrops jararaca venom. This peptide exhibits cytotoxic activity against SK-MEL-28 and melanoma cell lines by inducing the increase of cell membrane permeability (Martins et al. 2010). A peptide from Bothrops atrox venom, with molecular mass lower than 6.5 kDa, is also capable of inhibit mitochondrial swelling due to changes in mitochondrial membrane permeability (Menaldo et al. 2015).
In this sense, venom from distinct snake species contains peptides with potential anticancer activity that merit to isolate and characterize for further investigation. These peptides represent a promising source for new bioactive molecules.
Thus, the objective of the present study was to perform isolation and functional analysis of peptides from B. atrox snake venom with antitumor activity against human hepatocarcinoma tumor cell line HepG2.

Material
The B. atrox venom was acquired from the serpentarium "Center for the Extraction of Animal Toxins" (CETA) registered in the Ministry of the Environment under no. 3002678. The human hepatocarcinoma tumor cell line (HepG2-HB-8065) was obtained from the American Type Culture Collection (ATCC-Rockville, MD, USA). Nontumor human cells (Human peripheral blood mononuclear cells-PBMC) were isolated from peripheral blood, as described by Menck et al. (2014). The blood human collection was approved by the Research Ethics Committee at the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil (CAAE: 40,741,720.1.0000.5403).
The cell viability was assessed by trypan blue dye method before biological assays. Only cells with viability equal or greater than 98% were used.
Molecular exclusion Sephacryl S-200 (Cytiva) and reverse phase C-18 (Shimazdu) chromatography columns were used. All reagents and solvents used in this work had analytical grade.

Molecular Exclusion Chromatography in Sephacryl S-200
The crystallized crude B. atrox venom (350 mg) was suspended in 0.2 M ammonium bicarbonate buffer, pH 7.8, and centrifuged. The clear supernatant was applied onto a chromatography column filled with Sephacryl S-200 resin (100 × 2.6 cm), previously equilibrated and eluted with same buffer. Fractions of 3 mL were collected per test tube, at a flow rate of 20 mL/h at room temperature, according to methodology described by Menaldo et al. (2015).

Reverse Phase Chromatography
The fraction capable of reducing the tumor cell viability, BaPIVa (5 mg), was suspended in 500 μL of 0.1% trifluoracetic acid (v/v) (TFA) and analyzed in a HPLC system equipped with a reverse phase C-18 column (2.0 × 250 mm) (Shimadzu). The column was equilibrated with the running solvents (solvent A -0.1% TFA; solvent B-70% acetonitrile) and eluted as follows: 10 min to 0% of B, concentration gradient from 0-50% of solvent B for 100 min, rising to 100% of B in 10 min, and maintaining for 10 min under a flow rate of 1 mL/min, totaling 130 min of running time at room temperature (25 °C). The eluates were monitored at 280 nm by a detector coupled to the system.
The BaPIVa I (1 mg) was suspended in 500 μL of 0.1% TFA (v/v) and analyzed by HPLC system using a reverse phase column to determine its purity, under the same conditions described above.

Quantification of Samples
Total peptide concentration in the samples was determined using the Pierce Peptide assay reagent, according to the manufacturer's instructions (Thermo Fischer Scientific, Waltham, MA, USA).

Biochemical Characterization
Molecular mass analysis was performed on a MALDI-TOF/ TOF UltraFlex II mass spectrometer (Bruker Daltonics-DE), under positive ionization mode, using α-cyano-4-hydroxycinnamic acid as matrix. One μL of aqueous solution containing the samples was co-sedimented with the matrix (1:1). Experiments were performed by scanning from a mass-to-charge ratio (m/z) of 700 to 5000. De novo peptide sequencing was carried out in positive ionization mode with selected ions that were submitted to collision-induced dissociation (CID), using the LITF™ cell equipment. The precursor ion and fragments were selected and re-accelerated to a kinetic power of 19 keV. The laser potency was set to 60% with more than a thousand shots of incidence, which provided in a satisfactory signal/noise ratio. The resulting spectra were analyzed using BioTools 3.1 and FlexAnalysis 3.0 softwares (Bruker Daltonics software, DE) to deduce the amino acid sequence of the peptides.

LC-MS/MS and "De Novo" Peptide Sequencing
Briefly, previously lyophilized samples were dissolved in 0.5% formic acid and deposited into the 96-well plate of the SIL-20A auto-sampler (Shimadzu, Kyoto, Japan) for LC-MS/MS analysis in an IT-TOF mass spectrometer system (Shimadzu, Kyoto, Japan), equipped with ESI source. Twenty μL sample aliquots were injected and subjected to binary RP-HPLC (20A Prominence system, Shimadzu, Kyoto, Japan) separation through a Discovery C18 (2 × 50 mm) column employing as solvents 0.5% formic acid (A) and 90% acetonitrile containing 0.5% formic acid (B) in a linear gradient of B over A from 0 to 100% in 15 min, under a constant flow rate of 0.2 mL/min. Instrument control, data acquisition and processing were performed by the LCMS Solution suite (Shimadzu). PEAKS studio 7.0 (Ma et al. 2002) software was used for de novo peptide sequencing as well as complementary proteomic analyses on public databases. All MS/MS assignments were manually revised for correctness as well as the quality of the mass spectra of peptides from near-threshold identification.

Cells and Cell Culture
Cultivation of the Tumor Cell Line HepG2 HepG2 cell line were stored in liquid nitrogen (− 195 °C) in freezing solution (10% DMSO and 90% fetal bovine serum). For the experiments, the cells were thawed and cultivated in monolayer, in a 25 cm 2 flask with 5 mL of DMEM culture medium (Gibco 31,600-034, USA) supplemented with 10% fetal bovine serum, in a humidified incubator under 95% air and 5% CO 2 , at 37 °C. The assays were performed with cells between the third and sixth subculture.

Isolation of Peripheral Blood Mononuclear Cells (PBMC)
Peripheral blood was collected by venous puncture from six healthy individuals using heparin (143 U/10 mL of blood) as anticoagulant. This sample size improved reproducibility of quantitative data and provided blood volumes sufficient to perform all the experiments, with minimal discomfort to the donors caused by repeated blood collection. PBMC was isolated using density centrifugation methods, as described by Menck et al. (2014). Briefly, the peripheral blood collected was layered onto Histopaque 1077 and centrifuged, according to the manufacturer's instructions (Sigma-Aldrich-St. Louis, USA). After centrifugation, the phase containing the leukocytes was removed with a Pasteur pipette, pooled and centrifuged. The supernatant was discarded and the pellet was suspended in approximately 5 mL of RPMI medium (Gibco, 31,800-022) supplemented with 10% fetal bovine serum (Gibco, 12,657) for use in the experiments.

Assessment of Cell Viability
The viability of cells, treated with the fractions or isolated compounds, was determined using the MTT reduction method (Mosmann 1983). HepG2 and PBMC cells were plated in 96-well plates at a density of 5 × 10 4 cells/well and incubated for 24 h in a humidified incubator under 95% air and 5% CO 2 , at 37 °C, for total adhesion. After this period, the cells were treated with 50 μL of sample, PBS buffer (negative control) or cisplatin (Incel, Darrow®-positive 20 Page 4 of 13 control), and reincubated in a 5% CO 2 incubator. After 24 h of treatment, 20 μL of 3-(4,5-dimethyl thiazole 2-il) 2,5-diphenyl tetrazolium bromide (MTT; Sigma M2128, USA) were added to each well and the plates were incubated for 4 h at 37 °C, under 5% CO 2 .
The formazan crystals formed were solubilized in 100 μL of DMSO (Sigma D2650, USA), and the absorbance was recorded at 570 nm in a Spectramax 190 microplate reader (Molecular Devices). The mean absorbance values of the negative control corresponded to 100% cell viability. The evaluations were performed in three independent experiments, in triplicate for each experimental group (n = 9). The GraphPad Prism 8 software (GraphPad, USA) was used to analyze data, plot the graphs and determine the IC 50 values by nonlinear regression of a concentration-response curve of the cytotoxic peptide.

Apoptosis Quantification
The mechanism by which BaPIVa I reduced cell viability of tumor cells was investigated by marking cells with fluorescein isothiocyanate (FITC)-labeled Annexin V and the fluorescent viability marker propidium iodite (PI), using the Anexin V-FITC/PI kit, according to the manufacturer's recommendations (Invitrogen). HepG2 tumor cells (5 × 10 5 cells) were stimulated with peptide (0.72 μg/ mL), cisplatin (8 μg/mL) or PBS buffer for 24 h, at 37 °C, and under 5% CO 2 atmosphere. After treatment, the cells were collected from the wells and transferred to cytometry tubes. Ten thousand events were analyzed in a FACScanto equipment (Becton Dickinson, Franklin Lakes, NJ, USA) and the analyzed data by FlowJo software (BD Biosciences). The evaluations were performed in three independent experiments, in triplicate for each experimental group (n = 9).

Western Blot Analysis of Caspase Expression
To examine which apoptosis pathway BaPIVa I activated, we performed a western blot assay to analyze expression of the caspases 3, 8, and 9. HepG2 tumor cells (1 × 10 6 cells) were stimulated with the peptide (0.72 and 1.44 μg/mL), cisplatin (8 μg/mL) or PBS buffer during 24 h, at 37 °C, and under 5% CO 2 atmosphere. Afterwards, the cells were collected and suspended in the western blot lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and phosphatase and protease inhibitors). Total protein concentration in the samples was determined using the BCA protein assay reagent, according to the manufacturer's instructions (Thermo Fischer Scientific, Waltham, MA, USA). To separate the proteins of interest according to their molecular weight, equal amounts of protein were analyzed by 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Amersham, GE Healthcare Life Science, Pittsburgh, PA, USA). The membranes were blocked for 2 h with 5% non-fat dry milk prepared in TBS-Tween (20 mM Tris, 137 mM NaCl, 0.01% Tween 20). Next, the membranes were incubated overnight with the following primary monoclonal antibodies: anti-caspase 8 (BD Pharmingen, 551,243), anti-caspase 9 (BD Pharmigen 63,103), anti-caspase 3 (BD Pharmingen, T3320), and anti-beta actin (PIERCE, MA515739) as endogenous protein marker. After overnight incubation, the samples were treated with secondary antibodies for 2 h. Next, the membrane was washed three times in PBS-T buffer and revealed using the Kit Amersham ECL Western Blotting Detection Kit and the ChemiDoc™ MP System Image equipment (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
Graphs were plotted and statistical analyses were performed using the GraphPad Prism software version 8.0 (GraphPad Software Inc, San Diego, USA). One-way analysis of variance (ANOVA) followed by the Dunnett's post-test was used to compare each group with the control. The Student's t-test was used to compare differences between two groups. Values of p were considered statistically significant for *p < 0.5, **p < 0.05, and *** p < 0.005.

Identification and Purification of the Cytotoxic BaPIVa I
Fractionation of Bothrops atrox crude venom by molecular exclusion chromatography in Sephacryl S-200 column afforded six fractions (Fig. 1A). The IVa, IVb and V fractions containing molecules of low molecular mass (below 10 kDa)-which we named BaP (B. atrox snake venom portion) IVa, IVb, and V-were tested for their ability to reduce cell viability of HepG2 cells and PBMC, using the MTT assay.
All fractions strongly reduced the viability of HepG2 tumor cells (Fig. 1B), but only the BaP-IVa fraction was not toxic to normal PBMC cells (Fig. 1C), which indicated that it contained potential cytotoxic molecules with biotechnological application. The compounds from this fraction was isolated by reverse phase chromatography in a C18 column, which resulted in 27 subfractions, named as BaPIVa I-XXVII ( Fig. 2A). The ability of these subfractions to reduce cell viability of HepG2 cells was assessed using the MTT method. Our results suggested the presence of several molecules capable of modulating cell viability; however, only the subfraction BaPIVa I strongly reduced cell viability (approximately 94.6% reduction), demonstrating that it contained potent cytotoxic molecules (Fig. 2B). A new step of reverse phase chromatography confirmed that such strongly active subfraction was composed of only one compound that eluted in a single peak (Fig. 3).

Cytotoxic Effect and Determination of IC 50
Cytotoxicity of the peptide BaPIVa I to the tumor cell line was assessed at concentrations from 0.25 to 16 μg/mL, using the MTT method (Fig. 4A). BaPIVa I reduced the viability of HepG2 cells at concentrations from 1 to 16 μg/mL, with calculated IC 50 value of 0.72 ± 0.31 μg/mL.
The IC 50 concentration was also used to examine cytotoxicity of the peptide to PBMC cells. BaPIVa I at 0.72 μg/mL reduced the viability of PBMC by only 7%, confirming the hypothesis that it has low toxicity to normal cells and high potential for biotechnological application (Fig. 4B).

Determination of the Cytotoxic Mechanism of Action of BaPIVa I
To examine how BaPIVa I reduced cell viability, we carried out flow cytometry assays using the apoptosis marker Annexin V-FITC (AV) and the necrosis marker PI. The early and late apoptotic cell populations were marked as AV + / PI − and AV + /PI + , respectively. The region of highest confluence of cells plotted by FSC-H x SSC-A (data not shown) was selected in the gates for data analysis.
To determine which apoptosis pathway (intrinsic or extrinsic) BaPIVa I activated in HepG2 cells, we examined activation of caspases by Western blot. After 24 h of stimulus of HepG2 cells with BaPIVa I, increased levels of procaspases 3, 8 and 9 were detected, but only the activated form of caspase-9 was detected, indicating activation of apoptosis by the intrinsic pathway (Fig. 6).

Determination of Intact Mass and De Novo Peptide Sequencing
The m/z of peptide BaPIVa I was 1384,818 (1,385) (Fig. 7). MS/MS fragmentation of this ion (Fig. 8) resulted in the sequence reported in Table 1. Fig.2 A Isolation of compounds from BaPIVa fraction and identification of BaPIVa I by reverse phase chromatography (Column C18). Elution with solvent A (0.1% TFA) and solvent B (70% acetonitrile) was performed as follows: 10 min to 0% B, concentration gradient of 0-50% of solvent B for 100 min, rising to 100% of B in 10 min, remaining for 10 min, under a flow rate of 1 mL/min, totaling 130 min of running. B Cytotoxicity of the isolated compounds to HepG2 tumor cells, as assessed by the MTT method. PBS buffer was used as negative control and cisplatin (40 μg/mL) as positive control (cisp). The results are expressed as percentage of cell viability (%) ± SEM. Statistical analysis was performed by One-way ANOVA, where *p < 0.5, **p < 0.05, and ***p < 0.005 when the group treated with sample was compared with control

Discussion
The present study describes the biomonitored isolation and characterization of a new BPP with cytotoxic activity, named Batroxin I, through bioprospection of anticancer activity in the low molecular mass fractions of B. atrox venom. Snake venoms are rich sources of biologically active molecules with high therapeutic values. The proteins and peptides present in venoms can act on important physiological systems such as coagulation cascade, hemostasis, and tissue repair. In the last decades, there is increasing interest in studying peptides from these venoms due to their selective toxicity to bacteria and a broad spectrum of tumor cells, but not to normal mammalian cells. Thus, these molecules have become interesting molecular models for the development of new therapeutic and/or experimental agents for basic and applied research.
The first step of the present work consisted in the isolation of low molecular mass fractions using molecular exclusion chromatography, according to the methodology described by Determination of BaPIVa I peptide IC 50 and toxicity to PBMC. A Cytotoxicity of BaPIVa I isolated from reverse phase chromatography in C18 column, tested at different concentrations (0.25-16 μg/ mL) for determining the IC 50 value. The calculated IC 50 for BaPIVa I was 0.72 μg/mL (95% confidence interval: 0.40-1.3 μg/mL). (B) Cell viability of PBMC treated with the peptide BaPIVa I at 0.72 μg/ mL, as assessed using the MTT method. Statistical analyses were performed by One-way ANOVA, where *p < 0.5, **p < 0.05, and ***p < 0.005 when the group treated with sample was compared with control Menaldo et al. (2015). The BaPIVa fraction reduced the cell viability of HepG2 tumor cells by 80%, as assessed using the MTT assay. Purification of the BaPIVa fraction by reverse phase chromatography afforded the peptide called BaPIVa I (Fig. 3). This peptide was strongly cytotoxic to HepG2 tumor cells (90% reduction of cell viability) (Fig. 4A), with IC 50 = 0.72 μg/mL, and reduced cell viability of normal human PBMC by 7% (Fig. 4B); this was a determining parameter to attest its low toxicity, which is a fundamental characteristic of a candidate antitumor drug.
We examined the apoptosis-inducing capacity of BaPIVa I to confirm its cytotoxicity and elucidate the mechanisms by which it reduced cell viability. In the first approach using flow cytometry, BaPIVa I significantly increased annexin V labeling, indicating apoptosis induction. Compared with the positive control, cisplatin, a known anti-neoplastic drug used in the treatment of various types of cancer, BaPIVa I induced apoptosis more strongly (Fig. 5).
The functional characterization of BaPIVa I sought to elucidate the mechanism by which it induced apoptosis. Apoptotic signal transduction involves two main pathways: the extrinsic pathway, which is initiated by the FAS receptor, where caspase-8 dimerizes with the FASassociated death domain; and the intrinsic pathway, initiated by mitochondrial protein cytochrome c, with the involvement of caspase-9 (Westaby et al. 2022). In the present study, the peptide BaPIVa I increased expression of pro-caspases-3, -8 and -9, but we identified only the presence of the active form of caspase-9 (cleaved caspase 9- Fig. 6); it suggested that this compound acted and late apoptosis (AV + /PI + ) indices in HepG2 cells treated with BaPIVa I at IC 50. Cisplatin (8 μg/mL) was used as positive control for apoptosis induction (PC) and cells without any treatment as negative control (NC). The population with apoptotic cells was marked as AV + /PI − and the population with both necrotic and apoptotic cells was marked as AV + /PI + . Results expressed by the mean ± SD for three independent experiments (n = 9). The statistical analysis was performed by One-way ANOVA, where *p < 0.5, **p < 0.05, and ***p < 0.005 when the group treated with sample was compared with control on the intrinsic pathway, with side effects on the extrinsic pathway of apoptosis. Although activation of caspases is divided into different pathways, triggering an apoptotic signal leads to a threshold level that induces a feedback mechanism between the pathways, leading to cross-promotion in the apoptotic network (McComb et al. 2019). Our results indicated that the intrinsic pathway had prominent action. Structural characterization of the peptide by MALDI-TOF and ESI-MS/MS mass spectrometry revealed that the molecular mass of BaPIVa I was 1,385 Da (Fig. 7). The amino acid sequence was elucidated by de novo sequencing, determining that the peptide had a structure composed of 12 amino acid residues at sequence: < EKWPRPDA-PIPP, where < E is pyroglutamic acid. Other structural determination techniques, such as N-terminal sequencing, were not performed due to the presence of a pyroglutamic acid residue in the amino terminal position that makes it impossible to react through the Edman method.
An in silico analysis was performed to compare the peptide structure from this work with the sequences deposited on the NCBI (National Center for Biotechnology Information-BLAST) database. No deposits containing the structure elucidated for the peptide BaPIVa I were identified, attesting that this is an unprecedented compound; therefore, we named it according to the patterns of peptide nomenclature as Batroxin I.
We searched for Batroxin I homology based on its amino acid sequence and other peptides isolated from the venom of snakes from the genus Bothrops. Our search resulted in 12 sequences that produced significant alignments, all of which belonged to BPP class (Table 1).
BPPs are small hypotensive peptides rich in proline residues, and consist of a structure of 5 to 14 amino acid residues, arranged in a structural formula " < E-X n-P-X n -P-X n -I-P-P-P", where N-terminal presents a pyroglutamic acid residue, followed by one or more XP sequences, where X represents one or more amino acids, except cysteine, bound to a proline, and a C-terminal composed of the isoleucine-proline-proline tripeptide (Sciani and Pimenta 2017). These peptides are known inhibitors of the angiotensinconverting enzyme, an important component of the reninangiotensin system that mediates regulation of blood pressure. Initially, BPPs were isolated from B. jararaca venom (FERREIRA 1965), but they have already been identified in numerous venoms from the family Viperidae and Crotalidae.
For decades, the hypotensive effect and mechanism of action of BPPs have been the targets of studies. They have already served as prototypes for the development of angiotensin-converting enzyme inhibitor drugs, such as Captopril and its analogues, used in the treatment of hypertension (Ondetti et al. 1977;Cushman and Ondetti 1991). Currently, great advances have been made to identify other actions played by these peptides, such as antimicrobial and antitumor activity, and the associated mechanisms. Fourteen new BPPs composed of 3 to 13 amino acid residues were identified in B. atrox and B. jararacussu venom; five of these molecules were cytotoxic towards fungi and bacteria of clinical importance (da Silva Caldeira et al. 2021). The peptide BPP 13A, isolated from B. jararaca venom, is cytotoxic towards melanoma SK-Mel-28 and A2058 cells; it acts on the intracellular system of nitric oxide and demonstrates the ability to specifically penetrate the cell membrane of tumor cell lines, similarly to a cell-penetrating peptide (CPP) .
CPPs are a new class of peptides of up to 30 amino acid residues, cationic in character and resistant to the action of proteases due to the presence of various proline residues (Pujals and Giralt 2008;Xie et al. 2020). The mechanism of cell penetration of these peptides is varied, and may occur mechanically in the pathways involving endocytosis, dependent or not on receptors. It may also occur by direct translocation through a mechanism known as "snorkeling", where regions rich in hydrophobic amino acids interact with the lipid bilayer of membranes, anchoring the charged region on the cell surface; it facilitates the interaction of cationic amino acid residues with the proteoglycans Cleaved Caspase 9 Fig. 6 HepG2 apoptosis was induced by BaPIVa I via intrinsic pathway activation. HepG2 cells were treated with 0.72 or 1.44 μg/mL of the cytotoxic peptide BaPIVa I. After cell lysis, the protein extract was submitted to Western blot for detection of the main enzymes involved in the apoptotic pathways (Caspases 3, 8 and 9). Cisplatin (8 μg/mL) was used as positive control for apoptosis induction (PC) and cells without treatment as negative control (NC). The immunoreactivity was revealed by chemiluminescence, with an exposure time of 60 s 20 Page 10 of 13 and anionic phospholipids in the cell membrane, leading to the diffusion of these peptides from the surface of negatively charged membranes into cells, and consequently causing cell permeability (Feni and Neundorf 2017).
In general, tumor cell membranes have a negative residual charge due to the presence of phosphatidylglycerol, cardiolipin, or phosphatidylserine lipids. In contrast, the membranes of normal human cells are rich in zwiterionic phospholipids, such as phosphatidyletolamine, phosphatidylcholine and sphingomyelin, which maintain the membrane with a neutral liquid charge; therefore, this class of compound presents an activity with high specificity to tumor cells (Yount and Yeaman 2013). Given their specific character, CPPs have been used in a wide range of biotechnological applications, such as tumor treatment, carrier of molecules through the blood brain membrane or the intracellular environment (Feni and Neundorf 2017;Xie et al. 2020).
There are reports of CPPs isolated from venoms of bees, wasps, spiders, scorpions, snakes, fishes, and frogs. The reports of CPPs in snake venoms are concentrated in the Elapidae and Viperidae families, with peptides up to 40 amino acid residues such as crotamine, crotalicidine, AMPs, and cathelicidins (Rádis-Baptista 2021). BPP13a is a unique peptide with both characteristics of BPP and CPP ). BPP13a (< EGGWPRPGPEIPP) is a BPP with molecular mass of 1370 Da, and composed of 13 amino acid residues. Batroxin I and BPP13a share an approximate molecular mass and structural length, which is an important impact factor on the activity of CPPs. CPPs with a sequence length of 12 amino acid residues have better penetration capacity than shorter peptides (Lee et al. 2021).
Although the relationship between Batroxin I and CPPs is not experimentally characterized, we believe that the homology between this molecule and BPP13a (about 70% sequential alignment) indicates that it belongs to this class. It is necessary to expand our studies with this new molecule, in order to elucidate its potential drug delivery system, expanding its "hall" of anticancer biotechnological applications, which already has the promotion of programmed cell death, with selectivity to tumor cells compared to normal blood cells. Fig. 7 MALDI-TOF analysis of BAPIVa I peptide. Analysis was performed under positive ionization mode, using α-cyano-4-hydroxycinnamic acid as matrix. One μL of aqueous solution, containing the samples, was co-sedimented with the matrix (1:1). Experiments were performed by scanning from a mass-to-charge ratio (m/z) of 700 to isoleucine-proline-proline tripeptide. The characteristics of Batroxin I demonstrate that it is a promising model for the study and development of new drugs with antitumor action, and opens perspectives on the still unexplored activities of the main class of snake venom peptides.