Biological Characterization of Natural Peptide BcI-1003 from Boana cordobae (anura): Role in Alzheimer’s Disease and Microbial Infections

Nature continues to be one of the most important sources of molecules for the development of novel therapeutic agents. The skin of anuran’s (frogs and toads) is a rich source of peptides with a great importance in the search of bioactive agents applying to human health. Alzheimer's disease (AD) is a complex disease associated with numerous pathological pathways, making their simultaneous modulation necessary. On the other hand, the increasing bacterial resistance against conventional antibiotics has made it essential to search for new antimicrobial drugs with different modes of action. Here in we report the natural peptide BcI-1003, isolated from Boana cordobae amphibian skin, as an agent capable to act on three key therapeutic targets of AD, inhibiting the activity of BChE (IC50 = 669 µM) and MAO-B (IC50 = 570 µM) enzymes, and showing a powerful and rapid antioxidant activity (EC50 = 7.24 µM). Besides, BcI-1003 showed antimicrobial activity against clinically drug-resistant gram-positive and gram-negative bacterial strains, with MIC values ranging from 8 to 127 µM against Staphylococcus aureus MR-1; S. aureus MR-2 and Escherichia coli MDR-1.


Introduction
Nature continues to be one of the most important sources of molecules for the development of novel therapeutic agents (Newman and Cragg 2012). Additionally, the relevance of peptides in drug discovery programs has significantly increased in the past years (Albericio and Kruger 2012;Kaspar and Reichert 2013;de la Torre and Albericio 2018). Antimicrobial peptides (AMPs) are produced by a variety of living organisms, including fungi, bacteria, plants, vertebrates, and invertebrates. These natural molecules represent the first line of defense against pathogens (Bulet et al. 2004).
The skin of amphibians serves as a complex defense system, producing a large variety of such bioactive substances, including AMPs (Zasloff 2002;Conlon et al. 2004), neurotoxic peptides (You et al. 2009), gastric disturbance peptides (Bevins and Zasloff 1990) and alkaloids (Daly et al. 2005). These molecules are stored in special glands called granular glands or poison glands scattered over the amphibian skin, and when the animal is injured or alarmed, its content is rapidly released as a defense weapon (Tyler et al. 2007).
Alzheimer's disease (AD) is a neurodegenerative disorder of the brain, affecting almost 48 million older adults, with unparalleled growth and projections of 131.5 million older adults affected by 2050 (Venkatraghavan et al. 2019). Although the exact etiology is still not fully understood, several factors including low levels of acetylcholine (ACh), the formation of β-amyloid (Aβ) deposits and oxidative stress, demonstrated to play significant roles in its pathogenesis (Tang et al. 2012). ACh is hydrolysed by acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) enzymes (Greig et al. 2002). Nowadays, cholinesterases (ChEs) inhibitors are the mainly symptomatic treatment indicated for AD, and the efforts in the development of new drugs continue focusing on this strategy (Bortolami et al. 2021). Also, the use of antioxidant agents that reduce the toxic 1 3 6 Page 2 of 10 effects of oxidative stress produced by the Aβ aggregates is capable of delaying and preventing these types of disorders (Santos et al. 2016). Furthermore, the inhibition of monoamine oxidase B (MAO-B) enzyme has also received increasing attention in recent years due to its role in improving the AD symptoms (Knez et al. 2017). Because of this, in order to modulate different pathological pathways simultaneously, the development of anti-AD drugs is now focused on a multi-target directed ligands (MTDLs) strategy. Chronic systemic bacterial infections may also be involved in the pathogenesis and progression of AD through a persistent inflammatory response in the brain, that produces upregulation of Aβ production (Little et al. 2004;Zhu et al. 2010). These conditions initiate the amyloid cascade leading to the self-aggregation of Aβ amyloid peptide as an antimicrobial response. Therefore, AMPs could delay the progression of AD, preventing bacterial infection and consequently the activation of the amyloid cascade (Moir et al. 2018).
On a separate note, the rise of bacterial resistance to conventional antibiotics is one of the major causes of high mortality rates and inefficient therapy. Regarding this, intensive research efforts are being channeled into the development of new antimicrobials agents (Costa et al. 2019;Han et al. 2021). Traditionally, the main targets for antibacterial drugs have been the bacterial cell walls and protein synthesis (Ye et al. 2020).
Argentina is a naturally rich area of amphibian species. Hylidae is a wide-spread anurans family well represented in the new world, with over 900 species (Faivovich et al. 2005). Boana cordobae (anura: Hylidae) (Faivovich et al. 2004) is an endemic species that inhabit only in the top of the mountain systems of Sierras Grandes, Sierra de Comechingones, and Sierras de San Luis, Argentina (Bionda et al. 2011). Previous work carried out by our research group, allowed to report the bioactivity of the B. cordobae complete skin extract (Spinelli et al. 2019). Herein we synthetized the peptide BcI-1003 isolated from B. cordobae by solid phase Fmoc peptide synthesis and carried out an in-depth biological characterization. BcI-1003 showed the capability to act against three AD targets (BChE, MAO-B and antioxidant) and also as an antimicrobial agent for gram positive and negative resistant bacteria strains.

Secondary Structure Prediction
The amino acid sequences was analyzed through web resources for 3D structure prediction named PEP-FOLD (http:// biose rv. rpbs. univ-paris-dider ot. fr/ cgi-bin/ PEP-FOLD). This approach allowed us to obtain a predictive structural model for the studied amino acid sequence (Camproux et al. 1999(Camproux et al. , 2004Zhang et al. 2008;Yang and Chen 2011).

Cholinesterases Inhibition Assay
The cholinesterase inhibition was determined following Ellman's method with modifications (Ellman et al. 1961). Briefly, increasing concentrations of the peptide were prepared in phosphate buffer (pH 7.5). For AChE, 50 μL of extract were added to a 96 wells microplate followed by 50 μL of Electrophorus electricus AChE (0.25 U/mL) in phosphate buffer (pH 7.5). After 30 min of incubation at 25 °C, 100 μl of a solution of 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (0.2 mM) and acetylthiocholine iodide (ATCI) (0.24 mM) were added. The absorbance was measured at 405 nm by microplate reader Thermo Scientific FC. The same procedures were applied for BChE assay, with modification of the enzyme and substrate used, which were human serum BChE and S-butyrylthiocholine iodide (BTCI) (7 mM), respectively. In both cases, Rivastigmine was used as positive control. Percentage of inhibition was calculated as (1-EAS/EAB) × 100, where EAS and EAB represent the absorbance enzymatic activity of the sample and basal, respectively.

MAO-B Inhibition Assay
The inhibition of MAO-B (obtained from the mitochondrial fraction of rat brain) was determined following the method described by Soto-Otero et al. with slightly modification (Méndez-Álvarez et al. 1997). Briefly, 20 µL of increasing concentrations of the peptide in phosphate buffer were mixed with 45 µL of MAO-B. After 30 min of incubation at 30 °C, 10 μL of Horse Radish Peroxidase (5 U/mL), 20 μL of a solution of benzylamine (20 mM) and 20 μL of a solution of o-dianisidine (5 mM) were added and incubated at 30 °C for 60 min. Selegiline was used as positive control. The absorbance was measured at 405 nm. Percentage of inhibition was calculated as (1-EAS/EAB) × 100, EAS and EAB represent the absorbance enzymatic activity of the sample and basal, respectively.

Molecular Dynamic and Docking Studies
The molecular model of the BcI-1003 obtained by the Pep-Fold 3.5 server (http:// biose rv. rpbs. univ-paris-dider ot. fr/ cgi-bin/ PEP-FOLD) to predict its structure (Camproux et al. 1999(Camproux et al. , 2004Zhang et al. 2008;Yang and Chen 2011). Following molecular docking was performed both for BChE and MAO-B, in other to acquired knowledge about the interaction with the peptide. The protein structures were obtained from the Protein Data Bank, human butyrylcholinesterase 2PM8 (Sehnal and Bittrich 2021), and MAO-B 2V5Z (Binda et al. 2007). For BChE and MAO-B PDB ligands were removed, and polar hydrogens were added (Discovery Studio Visualizer 2021 -Biovia). The peptide was docked with each enzyme using HADDOCK 2.4 software (Dominguez et al. 2003;van Zundert et al. 2016). 10 independent runs of flexible docking calculations were performed, and all the predicted structures were clustered through a k-means clustering algorithm (k = 10) developed in the CPPTRAJ module of the Amber 18.0 package. Finally, the structure with the lowest cumulative distance to every other point within the most populated cluster was used to evaluate the ligand-protein interactions and generate a 2D interaction diagram with Discovery Studio Visualizer 2021 (BIOVIA). The resulting structures with the best score were promoted to the MD simulations. For this, initially, missing hydrogen atoms were added using LEaP from Amber 18.0 (Case et al. 2020), and ionizable residues were protonated according to their standard protonation state at pH 7.0. This structure was solvated with a periodic octahedral TIP3P water box with a margin distance of 10.0 Å. System charge was neutralized adding counterions. To perform these processes, tLEaP module of Amber 18.0 package and the ff14SB force-field were used (Maier et al. 2015). Equilibration and production: the system was prepared for the production run performing first a two-step energy minimization, a thermalization, and an equilibration run. Briefly, the solvent molecules were first relaxed around the complex, restraining its backbone with a harmonic restraint of 10 kcal/(mol Å2). Then, the geometry of the whole system was optimized without constraints. Finally, the temperature was slowly raised from 0 to 298 K before the 1 ns equilibration run of constant temperature (Langevin thermostat) and pressure (Berendsen barostat) (Berendsen et al. 1984;Pastor et al. 1988), in which the system density was adjusted. The 100 ns production run was subsequently started using the GPU implementation of the PMEMD program (Götz et al. 2012;Salomon-Ferrer et al. 2013) in Amber18, with the same simulation conditions of the equilibration process, that included the use of the SHAKE algorithm (Miyamoto and Kollman 1992) for the constraining of the bonds involving hydrogens, periodic boundary conditions and the Ewald algorithm (de Souza and Ornstein 1997) for the inclusion of the long-range electrostatic interactions. Newton motion equations were integrated using a 2 fs timestep, and snapshots of the system were saved every 1 ps during all the production runs.

DPPH Radical Scavenging Activity
The antioxidant activity was determined by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay according to the method of Memarpoor-Yazdi with minor modifications (Memarpoor-Yazdi et al. 2013). 0.4 mL of increasing concentrations of the peptide were mixed with 1.2 mL of methanol and 0.4 mL of DPPH (0.15 mM in methanol). The mixture was incubated for 30 min in the dark. Ascorbic acid was used as positive control. The absorbance of the resulting solution was measured at 517 nm in a microplate reader Thermo Scientific FC. Percentage of DPPH radical scavenging activity (RSA) was calculated as (1-As/Ab) × 100, As and Ab representing the absorbance of the sample and basal, respectively.

Antimicrobial Activity
Minimal Inhibitory Concentration (MIC) determination against bacterial strains were performed by the broth microtiter dilution assay, according to the procedures proposed by R.E.W. Hancock Laboratory for testing antimicrobial peptides, with few modifications (Siano et al. 2011). The clinical isolated strains Staphylococcus aureus MR-1 and Staphylococcus aureus MR-2, both methicillin resistant; Escherichia coli MDR-1 resistant to ampicillin, cefazolin, ampicillin sulbactam and gentamicin and Klebsiella pneumoniae Kb-1 were used to these assays. All strains belong to the Culture Collection of Clinical Bacteriology Section of FBCB-UNL. All the strains were activated by culture (24 h at 37 °C) in Mueller-Hinton Broth (MHB) (Biokar Diagnostics). Each inoculum was adjusted in order to achieve a cellular concentration of 5 × 10 7 CFU/mL. All the peptides were dissolved in bovine serum albumin buffer with 0.01% acetic acid in order to favor their solubilization; 450 µL of each inoculum was added to 50 µL of peptide solution in serial twofold dilutions and were incubated for 18-24 h at 37 ºC. The MIC was determined as the lowest peptide concentration that inhibited the growth of each bacterial strain 6 Page 4 of 10 completely. The tests were done in triplicate. For determination of minimal bactericidal concentration (MBC), 10 µL from each tube without visible grown were taken and seeded on CLDE agar plaques and then were incubated for 24 h at 37 °C. The lowest concentration of peptide without visible grown on the plaque was considered the MBC.

Circular Dichroism (CD) Analyses
Far-UV CD measurements were taken on a Jasco J-810 CD spectrometer (Tokyo, Japan) in a 0.1-cm path quartz curvet (Hellma, Mullheim, Germany) and recorded after the accumulation of five runs. CD analyses were recorded in the presence of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) vesicles. For the preparation of small unilamellar vesicles (SUVs), the lipid dispersion in Milli Q H 2 O was sonicated, using a tipsonicator, until the solution became transparent. In all samples, the peptide concentration was 0.2 mg ⁄mL, and the final lipid concentration was 3 mM. To correct for background scattering caused by the vesicles, the spectrum of a single vesicle solution was subtracted from that of the peptide in the presence of vesicles. Additional spectra were obtained in the presence of Trifluoroethanol (TFE) [50% TFE (v⁄v)] and in H 2 O (Sreerama et al. 2000(Sreerama et al. , 2001.

Hemolytic Activity
The hemolytic activity was determined by previously reported methodology (Siano et al. 2011;Spinelli et al. 2020). Briefly, human erythrocytes were isolated from heparinized blood by centrifugation (1000 g for 10 min) after washing three times with saline solution. Erythrocyte solutions were prepared to a concentration of 0.4% (v⁄v) in isotonic saline solution. Test tubes containing 200 μL of erythrocyte solution were incubated at 37 °C for 60 min with increasing concentrations of the peptide. After centrifugation at 1000 g for 5 min, the supernatant absorbance was measured at 405 nm. Lysis induced by 1% Triton X-100 was taken as 100%.

Data Analysis
All experiments were assayed in triplicates. The statistical analysis was performed using the computing environment R (Team 2010). A confidence interval of 95% was considered. Furthermore, the package GRmetrics (Clark et al. 2017) from the Bioconductor project was used (Gentleman et al. 2004) for the creation of the dose-response models and the calculations of the IC 50 . The plots were created with the package ggplot2 (Wickham 2016).

Results and Discussion
In this work we synthesized and performed an in-depth biological and biophysical characterization of the peptide BcI-1003 previously identified from B. cordobae specie (see supplementary and Figure S1). The synthesis was carried out using the solid phase synthesis by the Fmoc method, as C-terminal amide, given that amidation is a common feature of many AMPs isolated from frogs (Dourado et al. 2007;Gomes et al. 2007).

In Silico Analysis of Structural, Physicochemical and Stability Parameters of the Synthetic Peptide BcI-1003
The amino acid sequence and additional characteristics of peptide BcI-1003 are listed in Table 1. BcI-1003 consists of a polypeptide chain of 9 amino acid, with a net charge of + 5 at pH 7, and has a hydrophobic amino acids percentage (Hy/ total aa) of 11.11%. According to Protparam tool (Gasteiger et al. 2005), BcI-1003 has a grand average of hydropathicity (GRAVY) of -1.911 and an aliphatic index of 0.00, so it is considered as a hydrophilic peptide. These results are in concordance with the low retention capacity of BcI-1003 in a C 18 column (R t : 6.57 min) observed in the RP-HPLC analysis ( Figure S2). The proteolytic stability prediction of BcI-1003 in an intestine environment was analyzed in silico by Protparam (Guruprasad et al. 1990) and HPL online tools (Gorris et al. 2009). For both methods, BcI-1003 showed a high proteolytic stability, given by an instability index of < 40 and a half-life (sec) > 1.0. The prediction of the secondary structure of BcI-1003 is shown in Fig. 1. The result showed that is a not structured peptide random coil.

Alzheimer's Multi-Target Analysis
To study the capability of the peptide BcI-1003 to act as a multi-target agent on AD, we evaluated its activity against four representative pathological pathways. In this sense, the inhibition assay against cholinesterases (AChE and BChE) and MAO-B enzymes, and the antioxidant activity were performed. The biological activity was expressed as IC 50 or EC 50 values, defined as the concentration of sample that produces 50% of biological activity. The results are showed in the Table 2 and Figure S3. Regarding, the cholinesterases inhibitory activity, the peptide showed to be active against BChE with an IC 50 value of 669 ± 12 µM, while no evidence of activity against AChE was found at the tested range. The results are in concordance with previous work, where we reported the activity of the complete skin extract of B. cordobae against cholinesterases enzymes (Spinelli et al. 2019).
On the basis of these results, growing evidence suggests that BChE rather than AChE plays a key role in the hydrolysis of the neurotransmitter acetylcholine, because during AD progression, the AChE level decreases extensively in the brain, while BChE activity is maintained in a higher level. Hence, discovering BChE inhibitors is a strategy that may increase the amount of acetylcholine (Greig et al. 2005;Hartmann et al. 2007). Cholinesterases and MAOs are enzymes that are closely associated with the AD symptomatology and progression. The modulation of both enzymes offers an important chance to alter the course of AD. More than 15 years of intensive research has led to the identification of an important number of dual inhibitors, where some present positive outcomes in clinical trials (Knez et al. 2017). In 2019 we reported the first study of amphibian extracts with the capability of inhibiting the activity of MAO-B and cholinesterases enzymes (Spinelli et al. 2019(Spinelli et al. , 2021. In this  The in-vitro enzymatic inhibition studies were backed by molecular dynamic studies. For BChE, the results showed that BcI-1003 residues interact by hydrogen bound with both Asp70 and Tyr332 (Fig. 2), which constitute the peripherical anionic site (PAS) (Bajda et al. 2013). The PAS site is the region involved in non-catalytic processes and non-cholinergic functions of ChEs (Kryger et al. 1999). Through the PAS, ChEs act as a chaperone in the aggregation process of Aβ, accelerating the deposition of the amyloid plaques (Sadeghi et al. 2020). The inhibition of the PAS site is considered to be a promising strategy for the treatment of AD, since PAS inhibitors can potentially reduce the hydrolysis of ACh and deaccelerate the aggregation of Aβ at the same time (Pohanka 2011). Regarding the MAO-B in sillico studies, they showed that the enzyme-peptide complex is stabilized by several interactions. Notoriously, the Cys7 of the peptide interacts with Tyr435 of the MAO-B by a π-sulphur interaction (see Fig. 2). The residue Tyr435 is located at the aromatic cage of the MAO-B and plays a key role in the catalytic process (Li et al. 2006). Recently, Mathew et al. (2021) reported the strong competitive MAO-B inhibitor TM1 (a chalcone-thioester derivate) which interacts mainly with Tyr435 (Mathew et al. 2021). On the other hand, the antioxidant activity of the peptide was evaluated using the DPPH assay (Memarpoor-Yazdi et al. 2013). The ROS (reactive oxygen species) are one of the most important stress factors for amphibian naked skins. As an organ directly exposed to ROS from the environment as well as endogenous sources, skin is a candidate to oxidative stresses, so as result has developed an efficient antioxidant defense system ). In this work, BcI-1003 showed a potent antioxidant activity with an effective concentration 50 (EC 50 ) value of 7.24 ± 0.5 µM, a value that it is in the order of activity of the ascorbic acid. In addition, we observed a rapid colour change when performing the DPPH bioassay due to a fast capture of the free radical. These results could be attributed to the Cys7 in the sequence of BcI-1003. Several reports have demonstrated that the presence of cysteine, a reductive residue, in the peptide sequence strongly contributes to the antioxidative activity, but also is the determinant residue for rapid radical scavenging (Liu et al. 2010;Wang et al. 2017). On the other hand, the presence of basic residues such as Lysine or Arginine have shown improvements in the antioxidant capacity of peptides ). Out of the four AD therapeutic target studied, this was the most modulated by BcI-1003. The central nervous system cells are considered as one of the more vulnerable to oxidative stress by ROS (Olmez and Ozyurt 2012). Extensive studies demonstrated that the Aβ aggregates, as well as the MAO-B, exacerbated oxidative deamination increasing the ROS levels in neuronal cells, therefore intensifying the damage in AD. Accumulated evidence showed that the antioxidant agents act as neuroprotectors, reducing the toxic effects of oxidative stress and helping to improve cognition in AD patients (Knez et al. 2018 (Elias et al. 2008;Arasu et al. 2017;Shalgum et al. 2019;Prabha et al. 2022). Here, out of the four AD therapeutic targets studied, the antioxidant activity was the most strongly modulated by BcI-1003, being this peptide a hopeful neuroprotective agent. The peptide BcI-1003 has been shown to have the ability to act on three key therapeutic targets of AD, inhibiting the activity of BChE and MAO-B enzymes, and showing a powerful and rapid antioxidant capability.
Previous studies have shown that the modification of key residues to natural sequences managed to increase inhibitory activities of enzymes, as well as confer new activities (Sanchis et al. 2020(Sanchis et al. , 2022. This kind of strategy could be a useful tool to improve the activity of the natural peptide BcI-1003 against BChE and MAO-B.

Antimicrobial Activity
To evaluate the antibacterial activity of peptide BcI-1003, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were determined against clinically isolated drug-resistant strain of gram positive and negative bacteria. The results are shown in This peptide presented a high cationicity (+ 5), but low percentage of hydrophobic amino acids (35%) and helicity, showing was more active against gram negative bacteria than gram positive ones (Siano et al. 2014). In general, the major AMPs contain a significant proportion of hydrophobic amino acid residues and have a positive net charge (ranging from + 2 to + 9). The cationic residues are electrostatically attracted to negative components of the bacterial wall and membrane, and the hydrophobic residues facilitate interactions with fatty acyl chains and subsequently caused the disrupting of the integrity of the bacterial membrane (Zasloff 2002). However, several reports indicate that for antimicrobial activity against gram-negative bacteria, structural requirements of AMPs are less rigorous, while a stabilized amphipathic alpha-helical conformation is an important parameter for activity against grampositive bacteria (Giangaspero et al. 2001). In addition, numerous strains of gram-negative bacteria are susceptible to non-helical and unstructured cationic peptides, suggesting that inhibitory activity against gram-negative bacteria is mostly influenced by electrostatic interactions (Dathe et al. 1997;Giangaspero et al. 2001). In 2015, Nacif-Marçal reported the isolation and characterization of a novel antibacterial peptide from Hypsiboas semilineatus, Hs-1 (FLPLILPSIVTALSSFLKQG) (Nacif Marçal et al. 2015). This peptide had 55% of hydrophobic amino acids and a net charge of + 1 at pH 7, with a propensity to form an amphipathic alpha helix. It showed a selective spectrum of action against gram positive bacteria (MIC values ranging from 11.7 to 46 μM). The authors suggested that the lack of inhibitory activity against gram negative bacteria could be due to the low cationicity of Hs-1.

Circular Dichroism (CD) Analyses
BcI-1003 CD spectra recorded in different media are shown in Fig. 3. In H 2 O the peptide adopted a random coil conformation, as expected for short linear peptides. These data are consistent with a minimum at 198 nm. In 2,2,2-trifluoroethanol (TFE)/H 2 O (50% v/v), an alpha-helix inductive solvent, and in presence of neutral dipalmitoylphosphatidylcholine (DPPC) vesicles, that simulate eukaryotic membranes, the peptide was preferentially unordered. However, in presence of dipalmitoylphosphatidylglycerol (DPPG) vesicles, that mimic the bacterial membrane, BcI-1003 was partially structured, with a spectrum consistent with a β-structure, showing a minimum around 213 nm. This suggests that the peptide interacts electrostatically with the negatively charged DPPG vesicles, which induce the adoption of a secondary structure conformation.

Toxicological Profile
The knowledge of the cytotoxic activity of a compound is of great importance because this could be a limiting factor for its applicability. In this sense, the hemolytic activity against human healthy erythrocytes was determined ( Figure S4). Notably, BcI-1003 demonstrated to be non-hemolytic, with hemolysis values below 10% at the assayed concentrations. In addition to the hemolysis results, the in-silico analysis by ToxinPred (Gupta et al. 2013) and ToxIBTL (Wei et al. 2022) confirmed that the peptide BcI-1003 is non-toxic (see Table 1).

Conclusions
The present study reports the potential of the natural peptide BcI-1003 isolated from the skin of the frog B. cordobae, to act on three Alzheimer´s disease targets (BChE, MAO-B, and antioxidant), attending to the new anti-Alzheimer strategies. Also, the peptide showed the capability to inhibit the growth of clinical drug-resistant strains of gram positive and negative bacteria. On the other hand, BcI-1003 demonstrated to be an unstructured and non-hemolytic peptide. Therefore, this work represents the starting point for future in-depth studies that allow progress towards the discovery of a new drug for application in human health.