William Gustavo Lima

Stereochemical modification represents a powerful strategy for optimizing the therapeutic potential of antimicrobial peptides. Herein, we present a comprehensive structural and biophysical characterization of ecPis-2s and its epimerized analog, d-ecPis-2s, featuring a single d-F substitution. Although both peptides possess identical net charges and global hydrophobicity, our results reveal that the d-substitution acts as a conformational switch altering membrane interaction dynamics and antimicrobial efficacy. CD and solution NMR spectroscopy demonstrate that d-ecPis-2s maintains a similar overall helical character but adopts a distinctive N-terminal structural bend. Structural refinements and topology studies based on solid-state NMR elucidated that d-configuration at position 2 promotes a deeper sequestration of the N-terminal aromatic triad (F1−F3) into the hydrophobic membrane core. This aromatic anchor drives the peptide backbone and consecutively the aromatic side chains into the lipid bilayer, enhancing membrane tension, pore-forming kinetics and lipid packing disruption, which correlates directly with increased antimicrobial activity.

The emergence of multidrug-resistant (MDR) bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA), represents a major global health challenge by limiting the current therapeutic options. In this context, antimicrobial peptides (AMPs) have been widely studied for their potent antimicrobial properties. In this study, we evaluated the anti-MRSA effect of a novel AMP, LyeTx I mnΔKL, derived from a toxin of Lycosa erythrognatha. Its activity was evaluated in vitro by minimal inhibitory and bactericidal concentrations (MIC and MBC), antibiofilm effect, membrane interaction, cytotoxicity, synergistic interaction with vancomycin, and in vivo in an MRSA murine wound/abscess infection model. LyeTx I mnΔKL showed enhanced antimicrobial activity against clinical MRSA isolates compared to its prototype (LyeTx I mnΔK), with MIC₅₀ and MBC₅₀ of 2 and 8 µM and 16 and 32 µM, respectively. Furthermore, LyeTx I mnΔKL exhibited a rapid bactericidal effect and a pronounced ability to inhibit biofilm formation and disrupt mature biofilms. LyeTx I mnΔKL interacts with bacterial membranes, adopts an α-helical structure, and induces membrane disruption and leakage of intracellular material. In vivo, topical treatment with LyeTx I mnΔKL reduced MRSA burden compared with LyeTx I mnΔK and untreated controls (log₁₀ CFU/g of wound of 2.4, 4.4, and 6.5 for LyeTx I mnΔKL, LyeTx I mnΔK, and the saline group, respectively). However, increased cytotoxicity remains a significant limitation. Overall, LyeTx I mnΔKL is a promising anti-MRSA candidate for topical use with potent antibiofilm and in vivo activity.

Background: Sporotrichosis is a fungal infection caused by species of the Sporothrix schenckii complex. Antifungal treatment with itraconazole and amphotericin B is limited by increasing resistance, adverse effects, and prolonged treatment courses, highlighting the need for novel antifungal strategies. Methods: The antifungal activity of seven antimicrobial peptides derived from arthropod toxins against Sporothrix species was evaluated: six synthetic peptides derived from the spider Lycosa erythrognatha toxin and one peptide isolated from apitoxin (melittin). Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were determined. Mechanistic assays were performed to investigate membrane damage, oxidative stress induction, and interactions with ergosterol and the fungal cell wall. Synergistic activity with itraconazole was assessed, and the effect of melittin on selected virulence factors was studied. To explore potential therapeutic applications, a melittin-based formulation for local (intralesional) usage was developed, and its cytotoxicity was tested in HEK-293 and HepG2 cell lines, as well as its short-term safety in murine models. Results: All peptides inhibited S. schenckii and S. brasiliensis, with MIC and MFC values ranging from 0.5 to 32 µM. Melittin displayed the strongest antifungal effect, acting predominantly through a membranolytic mechanism associated with oxidative stress. Combined with itraconazole, melittin demonstrated synergistic activity against both species. Melittin selectively reduced pyomelanin production, while urease activity remained unaffected. The melittin-based formulation showed lower cytotoxicity compared to melittin alone, and subcutaneous administration in mice was well tolerated at the lowest dose tested (0.1 mg/kg). Conclusion: Melittin exhibits potent antifungal activity against Sporothrix spp. and synergism with itraconazole, supporting further investigation as an antifungal candidate. Although therapeutic efficacy was not evaluated in infected animal models, the development of a safer melittin-based formulation provides a proof-of-concept foundation for future studies focusing on the local treatment of cutaneous sporotrichosis.

Bixin is a lipophilic apocarotenoid abundant in the aril of Bixa orellana L. seeds. Widely used as a natural colorant in food, textile, and cosmetic industries, its pharmacological potential remains underexplored. This study aimed to extract and purify bixin, characterize its physicochemical properties, develop and validate an HPLC method for quantification, and evaluate its antifungal activity, cytotoxicity, and selectivity in vitro. Extraction was performed by Soxhlet using hexane and chloroform, followed by recrystallization in acetone. Characterization (TG/DTA, FTIR, and ¹H NMR) confirmed predominance of the 9'-cis isomer. The HPLC method (λ=470 nm) was validated per ICH Q2-(R2), showing specificity, linearity (R² = 0.9993), low LOD/LOQ (0.638 and 1.934 μg mL^-1), and high precision and accuracy (RSD < 2%; recovery 99.4-100.8%). Antifungal assays against eight Candida strains revealed moderate activity, with MICs from 2 to 256 μg mL^-1. The best results were observed for C. glabrata and C. tropicalis (MIC = 2 and 4 μg mL^-1). Bixin also inhibited virulence factors inC. albicans Cytotoxicity on Vero cells showed a CC₅₀ of 30.71 μg mL^-1, with selectivity indices of 15.36 and 7.68 forC. glabrata andC. tropicalis, respectively. These results support bixin's potential as a natural antifungal agent.

Klebsiella pneumoniae can acquire resistance mechanisms to colistin and present a pan-resistant phenotype. Therefore, new alternative agents are imperative to control this pathogen, and the peptide Jelleine-I stands out as a promising prototype. Here, the antibacterial activity of Jelleine-I against clinical isolates of colistin-resistant K. pneumoniae (CRKP) was investigated. Antimicrobial activity was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill-curve assay. The release of 260 nm-absorbing materials (DNA/RNA) and the release of proteins were used in the lysis assay. Anti-biofilm activity was studied in microplates. In vivo activity was determined by the lethality assay using Tenebrio molitor larvae. The results show that the MIC of Jelleine-I ranged from 16 to 128 µM and the MBC was on average 128 µM. Jelleine-I at 200 µM killed all CRKP cells in suspension (10⁶ colony-forming units (CFU)/mL) after 150 min of incubation. Jelleine-I acts on the CRKP cell membrane inducing lysis. Biomass and viability of CRKP-induced biofilms are reduced after treatment with Jelleine-I, and the use of this peptide in T. molitor larvae infected with CRKP reduces lethality and improves overall larval health. In conclusion, Jelleine-I is a potential prototype for the development of new antimicrobial agents.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a multidrug-resistant (MDR) gram-negative bacterium frequently involved in hospital-acquired pneumonia. The infection caused by this superbug has spread quickly in health centers worldwide, leading to high mortality rates. Due to this emerging scenario, the World Health Organization has categorized CRKP as the highest-priority species for the development of new compounds. In this context, antimicrobial peptides (AMPs) stand out as prototypes for alternative antimicrobials against superbugs, including CRKP. Objectives: We aimed to describe the antibacterial effect of an AMP (LyeTx I), derived from the venom of the spider Lycosa erythrognatha, against CRKP in vitro and in a murine pneumonia model. Results: LyeTx I showed antibacterial effects against all the CRKP clinical isolates tested, with a minimum inhibitory concentration (MIC) range of 2–8 µM and a minimum bactericidal concentration (MBC) range of 2–16 µM. The microbial anionic membrane was the primary target of LyeTx I, which acts by displacing divalent cations bound to this structure in a manner similar to that of polymyxins. Notably, LyeTx I displayed significant lytic activity against mimetic membranes, indicating its potential to disrupt bacterial cell integrity. In in vivo assays, the LyeTx I peptide proved to be safe at a dose of 10 mg/kg. In addition, intraperitoneal use of LyeTx I reduced the bacterial load and inflammation in the lungs of animals infected with a hypervirulent strain of CRKP. Conclusions: These results indicate that LyeTx I is a potential prototype for the development of new antibacterials against MDR species, such as CRKP.

The urgent global health challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) infections demands effective solutions. Antimicrobial peptides (AMPs) represent promising tools of research of new antibacterial agents and LyeTx I mn∆K, a short synthetic peptide based on the Lycosa erythrognatha spider venom, is a good representative. This study focused on analyzing some antimicrobial activities of LyeTx I mn∆K, including minimum inhibitory and bactericidal concentrations, synergy and resensitization assays, lysis activity, the effect on biofilm, and the bacterial death curve in MRSA. Additionally, its characterization was conducted through isothermal titration calorimetry, dynamic light scattering, calcein release, and finally, efficacy in a mice wound model. The peptide demonstrating remarkable efficacy against planktonic cells and biofilms of MRSA, outperforms vancomycin in terms of rapid bactericidal action and anti-biofilm effects. The mechanism involves significant membrane damage. Interactions with bacterial model membranes, including those with lysylphosphatidylglycerol (LysylPOPG) modifications, highlight versatility and selectivity of this compound. Also, the peptide has the ability to sensitize resistant bacteria to conventional antibiotics, showing potential for combinatory therapy. Furthermore, using an in vivo model, was showed that a formulated gel containing the peptide proved superior to vancomycin in treating MRSA-induced wound in mice. Together the results highlight LyeTx I mnΔK as a promising prototype for the development of effective therapeutic strategies against superficial MRSA infections.