The peptide synthesis was carried out following a method already described in the literature 34. Rink resin was used as a solid support for the synthesis. It was initially soaked in DMF for 30 minutes for activation. After that, it was then soaked in DCM for 30 minutes and then rinsed again in DMF. Deprotection of the fmoc group was done using a piperazine solution (10% piperazine w/v in DMF:EtOH 9:1). Deprotection consisted of introducing the piperazine solution into the resin, leaving it stirring for 1 minute, removing the solvent and adding new piperazine to the resin for 20 minutes then removing the solvent. Subsequently it was rinsed with DCM and DMF. The initial Fmoc deprotection step was carried out using a solution of piperazine in DMF. The coupling of the different amino acids previously deprotected with piperazine as described previously were coupled to the resin for 2 hours at room temperature. The ninhidryn test made it possible to qualitatively control the effectiveness of the couplings. Cleavage of the synthesized peptides from the resin was done by hydrolysis using a solvent system containing TFA, TIS, EDT and water (94:1:2.5:2.5 v/v). /v/v) for 2 hours at RT. Then, the samples were freeze-dried (Liotop, model K108, Brazil) until a powder was formed Scheme 1.

Analytical analysis was carried out following a method already described in the literature 34. Once the cleavage was completed, the resulting solution containing the synthesized peptide was lyophilized. A small quantity of the powder obtained was dissolved in water at a concentration of 5 mg/mL and was subjected to HPLC analysis (Simadzu Prominence, with a DGU-20A5R membrane degasser, an SPD-20A UV detector, a CTO-20A column oven, a SIL-10AF autosampler, an FRC-10A fraction collector and a qualitative LC-20AT double pump) for the purpose of verifying purity. The impure peptides were purified by injecting the samples through a C18 column with a flow of 5mL/min. Buffers A and B, respectively 0.045% TFA in aqueous solution (eluent A) and 0.036% TFA in acetonitrile (eluent B) were used as elution solvent. Verification of obtaining the desired peptide was done by LCMS and carried out on a Shimadzu chromatograph/Bruker spectrometer (Prominence/Amazon SL) with the same parameters as those used for the HPLC purification system at a flow rate of 0.5. mLmin⁻¹.

To determine the MIC, all strains of the bacterial species that were sensitive to the compound were used. Each compound was diluted in DMSO and a 100x concentrated stock solution was prepared, subsequently the stock solution was diluted 1:100 in Mueller Hinton Cation Adjusted (MHCA) broth (BD) according to CLSI 36. From this, each compound was tested in 512 μg/ml 1% DMSO, or at the highest concentration at which it was possible to dissolve the compound without precipitation. From the compound wells at 512 μg/ml, serial dilutions (1:2) were made up to a concentration of 0.06 μg/ml. The incubation was carried out at 37°C and the results were visually read after 24 hours, in which it was observed up to which concentration the compound was able to inhibit the growth of the microorganism. For negative and positive controls, Mueller Hinton Cation Adjusted broth 1% DMSO was added. In the positive control, bacteria without the compound were added to observe their growth in Mueller Hinton Cation Adjusted broth 1% DMSO. In the negative control, there is only the Mueller Hinton Cation Adjusted 1% DMSO broth culture medium, without bacteria, to show that there is no contamination thereof. Tests were performed in triplicate.

To determine the MIC, all strains of the bacterial species that were sensitive to the compound were used. Each compound was diluted in water and a 10x concentrated stock solution was prepared. Subsequently, the stock solution was diluted 1:10 in Mueller Hinton Cation Adjusted (MHCA) broth (BD), according to CLSI 36. From this, each compound was tested at 512 μg/ml or the highest concentration at which it was possible to dissolve the compound without precipitation. From the compound wells at 512 μg/ml, serial dilutions (1:2) were made up to a concentration of 0.06 μg/ml. The incubation was carried out at 37°C and the results were visually read after 24 hours, in which it was observed up to which concentration the compound was able to inhibit the growth of the microorganism. For negative and positive controls, Mueller Hinton Cation Adjusted broth was added. In the positive control, bacteria without the compound were added to observe their growth in Mueller Hinton Cation Adjusted broth. In the negative control, there is only the Mueller Hinton Cation Adjusted broth culture medium, without bacteria, to show that there is no contamination of it. Tests were performed in triplicate.

To determine the MIC, all strains of the bacterial species that were sensitive to the compound were used. Each compound was diluted in DMSO and a 100x concentrated stock solution was prepared. Subsequently, the stock solution was diluted 1:100 in Mueller Hinton Cation Adjusted (MHCA) broth (BD), containing H dye (Biolog) in a proportion of 1.05%, the entire procedure was carried out following the recommendations of CLSI 36. From this, each compound was tested at 512 μg/ml, or at the highest concentration at which it was possible to dissolve the compound without precipitation. From the compound wells at 512 μg/ml, serial dilutions (1:2) were made up to a concentration of 0.06 μg/ml. Incubation was carried out with the aid of the OmniLog® device (Biolog, Hayward, CA, USA) at 36 °C ± 1 °C for 24h, followed by visual reading of the plates in which it was observed up to which concentration the compound was able to inhibit the growth of the microorganism. In the positive control, bacteria without the compound were added to observe their growth in Mueller Hinton Adjusted Cation broth – 1% DMSO. In the negative control, there is only the Mueller Hinton Cation Adjusted broth culture medium – 1% DMSO, without bacteria, to show that there is no contamination thereof, both controls also received the H dye in the same proportion. Tests were performed in triplicate.

After visually reading the MIC, 100 μL of the contents of the well equivalent to the MIC, one dilution below and all dilutions above, were inoculated onto an MHCA-Agar plate using the microdrop technique, without streaking. The plate was incubated in an oven at 37°C for 24 hours, when a visual reading was made to observe which concentration did not result in bacterial growth. To determine bactericidal or bacteriostatic activity, compounds that had a MBC/MIC ratio less than or equal to four were considered bactericidal, above that being considered bacteriostatic 37.

S. epidermidis ATCC 12228 (Negative control, poor biofilm former) and S. epidermidis ATCC 35984 (positive control, good biofilm former) were inoculated in BHI Broth 0.75% glucose in several replicates in a 96-well microplate. Incubation was carried out at 37°C for 24 hours to form the biofilm. The ability to eradicate the biofilm formed will be further tested after the formation of the S. epidermidis ATCC 35984 biofilm.

After 24 hours at 37ºC, the culture medium with the planktonic bacteria was removed and washed with 0.85% physiological solution so that only the biofilm remains on the microplate. Each compound was added at the highest possible concentration, diluted in 1% DMSO when necessary, in fresh BHI 0.75% glucose medium in the wells where biofilm was formed by S. epidermidis ATCC 35984. As a control for biofilm growth, 6 wells with the positive control and 6 negative controls should only receive BHI broth 0.75% glucose, plus 1% DMSO when necessary. Incubated at 37ºC for another 24 hours. This second step was to allow the biofilm to be eradicated by the compound.

After 24 hours, to quantify the biofilm, the wells were washed several times and stained with crystal violet. After washing steps and removal of excess violet crystal, the violet crystal was removed with ethanol:acetone (80:20) and transferred to another microplate for indirect quantification of the biofilm at 595 nm. The assay control was based on the comparison of the average absorbance of the S. epidermidis ATCC 35984 biofilm and the average absorbance of the S. epidermidis ATCC 12228 biofilm, both without compound. The comparison of means was carried out using the Student's T-Test, whose P value <0.05 proves the significant difference between two means from two different samples. Regarding the ability of the compound to eradicate biofilm, data analysis was based on the comparison of biofilm production of S. epidermidis ATCC 35984 without compound with S. epidermidis ATCC 35984 with compound. For this, the average of the replicates was used and the standard deviation was taken to analyze the coherence between the replicates. For statistical analysis, analysis of variance (ANOVA) was performed on the mean absorbance of biofilm production by the strain S. epidermidis ATCC 35984 without compound and the mean absorbance of biofilm production by S. epidermidis ATCC 35984 with each compound. When the analysis of variance had a value of P < 0.05, it indicated a significant difference between the two samples 38.

Stock solutions of Alca1 were prepared, containing 1 mg resuspended in 0.5 mL of PBS buffer (pH 7.4). Serial dilutions of the peptides were performed, starting from a concentration of 512 µg /mL up to 1 µg/mL, all in duplicates. In addition, controls were prepared, with the negative control being PBS buffer and the positive control being 1% Triton. In addition, the erythrocyte solution was prepared by diluting it in PBS buffer (1/25). 100 µL of the erythrocyte solution was added to the tubes containing the peptide dilutions and incubated at 37ºC for 1 hour. Then, the tubes were centrifuged at 500 g for 5 minutes. The supernatants were pipetted into microplates for subsequent reading at 540 nm in a microplate reader.

To calculate the % hemolysis, the following were considered:

Pepsin, trypsin and chymotrypsin were dissolved in 10 mM phosphate buffer saline (PBS) (10 mM) by serial 10-fold dilution. The final concentrations of these two enzymes ranged from 2 × 10⁻⁶ mg/ml to 2 mg/ml. Peptides (at the concentration of 128, 256, and 512 μM, respectively) were mixed with different concentrations of trypsin or chymotrypsin. The samples were incubated for 6 h at 37°C. Then they were heated for 15 min at 60°C to terminate the enzyme reaction. We followed reaction with HPLC, every 30 min 39 by calculating, using the calibration curve, the concentration of each peptide remaining undegraded at a given time t. For endogluC, peptides were dissolved at 128, 256, and 512, 1000 μM in hydrolysis buffer (ammonium bicarbonate 100 mM, pH 7.8) and enzymatic hydrolysis were carried out for 16 h at 37 °C with an enzyme:substrate (E/S) ratio of 1:100 40. We followed reaction with HPLC, every 30 min by calculating, using the calibration curve, the concentration of each peptide remaining undegraded at a given time t. The CD spectra were measured at 25 °C.

Prediction of potential antimicrobial regions was performed by obtaining a bactericidal propensity (PV) index value calculated for each amino acid in the AMPA software 41. The 3D structure of the peptides was modeled with the PEP-FOLD software. HeliQuest made it possible to calculate from the amino acid sequence of a helix (α-helix, 3-10 helix, 3-11 helix or π-helix) its physicochemical properties and its amino acid composition in order to identify the segments proteins with similar characteristics 42.

Antimicrobial activity is defined by the CMB/MIC ratio. For ratios greater than 4, the activity is considered bacteriostatic, for results equal to or less than 4, the activity is considered bactericidal, according to Pankey and Sabath (2004). Results indicated with N.D., in the table1 above, indicates that the activity cannot be defined and, N.R., indicates that the assay was not carried out with that strain as the compound showed no activity or presented an MIC value above 512 mg/L. Peptides Neo1 and Neo3 showed similar results as did Neo2 and Neo4

To carry out biofilm assay, two ATCC strains of S. epidermidis were used. Strain 35984 is a good biofilm-forming strain, while strain 12228 is considered a poor biofilm-former. The comparison of the presence of biofilm between them showed a significant difference with p<0.05, showing that the controls used are significantly different in all cases. The ability to eradicate the biofilm formed by the S. epidermidis ATCC 35984 strain was analyzed in the presence of each peptide. Some showed eradication results (p-value below 0.05), however, not showed eradication results above 50%. The other one exhibited a p-value lower than 0.05, however, it is important to highlight that this result does not suggest the eradication of the biofilm formed, but rather that promoted such significant growth of the biofilm that it differentiated statistically from the control group, result is presented in the Table 1.

Hemolysis is an irreversible process during which red blood cells are destroyed and release their hemoglobin content into the plasma 43 and when their lifespan is less than 100 days rather than 120 days (physiological hemolysis) 44. Hemolysis can be caused by intrinsic factors such as the state of the membrane, intracellular energy metabolism, the structure of hemoglobin and extrinsic factors such as an abnormal response of the immune system, the formation of clots in the capillaries blood and the side effects of certain drugs 45. The results observed in the Figure 5 show all peptides presented same hemolytic activity and do not present hemolytic properties, therefore making them good drugs candidates.