*4.1. Molecular Modeling and Molecular Properties Calculation*

The coordinates of the polypeptide crotamine from the Brazilian rattlesnake *C. durissus terrificus* [24] were retrieved from Protein Data Bank (PBD ID 4GV5; resolution at 1.70 Å) [30] and used as starting geometry to extract the fragment Cys30-Gly42 (13 aa), which were employed to build up the peptides' 3D molecular models. The Cys residues were mutated into Ser, generating the peptide PS1. The PS6 peptide was obtained by extracting Ser37-Gly42 from PS1. PS6 (7 aa) corresponds to the minor sequence designed and assayed in this study. The other fragments were constructed using the tool build-mutate or build-grow available in the Discovery Studio Visualizer 4.0 software (Accelrys Software Inc., 2005–2013). The geometries were optimized, and partial atomic charges were assigned using the CHARMM force field [63], included in Discovery Studio (Accelrys Software Inc., 2005–2013).

Furthermore, the electrostatic potential (EP) property was calculated for the PS3 and PS5 peptides to visualize the changes in electronic density distribution concerning the amino acid substitution patterns (PS5 has one more residue, Trp, in comparison to PS3). The charges from electrostatic potential using a grid-based method (CHELPG [64]) were calculated employing the ab initio method Hartree-Fock/3-21G\* basis set (Gaussian 03W software; Gaussian, Inc., Pittsburgh, PA, USA, 2003). The EP maps were calculated onto the peptides' molecular surfaces using GaussView 05 software (Gaussian, Inc., Pittsburg, PA, USA, 2002–2008). The interpretation of EP maps is based on a color scheme, where regions having higher electronic density distribution are presented as an intense red color (negatively charged), whereas regions with lower electronic density distribution are shown as an intense blue color (positively charged). Since the EP property has been calculated onto the PS3 and PS5 molecular surfaces, their molecular shapes were also assessed. Moreover, the molecular volume (intrinsic molecular property) of each peptide considering the van der Waals radii was also calculated employing Discovery Studio Visualizer 4.0 software (Accelrys Software Inc., 2005–2013). Of note, the molecular shape and electronic properties are among the primary molecular properties in the ligand–receptor recognition process.

#### *4.2. Antibacterial Activity*

The antibacterial activity of the alkylated peptides was tested against Gram-negative (G−) bacteria (*Klebsiella pneumonia*, *Serratia marcescens*, *Morganella morganii*, *Providencia rettgeri*, *Citrobacter freundii*, *Escherichia coli* ATCC 25922 and Gram-positive (G+) bacteria (*Micrococcus luteus* A270 and *Staphylococcus aureus*) using microbroth assay [28]. In a 96-well plate, 90 <sup>μ</sup>L of 10% tryptone soy broth (TSB) with 4 × <sup>10</sup><sup>5</sup> colony-forming unit (CFU/mL) were mixed with serial twofold dilutions of β-defensins duplicate in concentrations from 512 to 1 μg/mL. The derived peptides coded as PS1, PS2, PS3, PS5, and PS6 (Table 1) were tested against these bacteria in serial twofold dilutions with concentrations bellow 700 μg/mL. After overnight incubation at 37 ◦C, the turbidity of the bacterial culture was read at 600 nm (Epoch microplate reader, Biotek). The minimal inhibitory concentration (MIC) of each peptide was determined as the lowest concentration that results in no visible

bacterial growth. The MIC resulted from three independent experiments. The bacteria *K. pneumonia*, *S. marcescens*, *M. morganii*, *P. rettgeri*, *C. freundii*, *M. luteus* A270 and *S. aureus* were isolated from *Bothrops jararaca* buccal flora and kindly provided by Dr. Márcia R. Franzolin (Laboratory of Bacteriology—Instituto Butantan). *M. luteus* was kindly provided by Dr. Pedro I. da Silva Jr. (Laboratory of Applied Toxinology—Instituto Butantan).

### *4.3. Antifungal Activity*

The antifungal activity was evaluated by the microdilution assay as previously described [31] on clinical strains of *Candida albicans* (IOC 4525), *Cryptococcus neoformans* (IOC 4528), *Trichophyton rubrum* (IOC 4527) and *Aspergillus fumigatus* (IOC 4526). The fungi were cultivated in potato dextrose agar at 28 ◦C according to the Clinical and Laboratory Standards Institute recommendations [75]. Fungal suspensions were prepared in RPMI 1640 culture media; the CFU/mL was adjusted to 0.5–2.5 × 103 for yeasts (*C. albicans* and *C. neoformans*) and 0.4–5 × <sup>10</sup><sup>4</sup> CFU/mL for filamentous fungi (*T. rubrum* <sup>e</sup> *A. fumigatus*), by comparison with a standard curve previously stablished in our laboratory.

The peptides PS1, PS2, PS, PS3, PS4, PS5 and PS6 at 1 mM in acetic acid at 0.01% were diluted in RPMI 1640 culture media at concentrations ranging from 1 to 250 μM. Amphotericin B (AMB) at concentrations below 15 μg/mL (16 μM) and acetic acid from 0.0002 to 0.0025% was used as control.

The fungi suspensions were added to a 96 well plate (100 μL/well) and the samples (100 μL) in different concentrations were added to each well. The plate was incubated at 28 ◦C for 24–72 h, and 24 h before the end of the assay 25 μL of the rezazurin dye at 0.02% were added to each well. The minimal inhibitory concentration (MIC90) was defined as the lowest concentration that prevents the rezazurin's change in color from blue to pink due to the inhibition of at least 90% of the microorganism's growth. The assays were performed in three independent experiments.

**Author Contributions:** Conceptualization: K.F.M.P. and N.O.; Investigation: P.G.C., I.L.R., A.O.d.S.; Formal analysis: K.F.M.P., N.O.; Funding acquisition: N.O.; Writing—original draft: N.O.; Writing review & editing: A.O.d.S., K.F.M.P., N.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by São Paulo Research Foundation (www.fapesp.br)—FAPESP: 2015/00003-5 (N.O.), 2017/11735-2 (fellowship of I.L.R.); 2008/06524-3 (A.O.S).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

