Background: Peptides are promising tools in medicine for viral, bacterial, and parasitic infections. MAP1 and MAP2 are peptides with antimicrobial activity. Aiming to increase selectivity, enhance antimicrobial activity, and reduce its cytotoxicity, two bioconjugates (GVL1-MAP2 and GVL1-MAP1) with the guanidine derivative group in the N-terminus position were synthesized. Methods: The peptides and bioconjugates were synthesized by SPPS. The biological activity and inhibition of the CPB enzyme were evaluated. Results: Antileishmanial activity was evaluated, and the bioconjugates exhibited higher activity in both the promastigote and amastigote forms than isolated GVL1 and peptides alone. Discussion: GVL1-MAP2 bioconjugate demonstrated not only the greatest activity against L. amazonensis in the promastigote (IC₅₀ = 3.2 μM) and amastigote (IC₅₀ = 0.6 μM) forms but also prevented the parasite from infecting new host cells, reducing the infection rate by 3-fold compared to the untreated control. Similar results were obtained in L. infantum, with IC₅₀ = 2.5 and 1.0 μM for promastigote and amastigote forms, respectively. In active serum, GVL1-MAP2 continued to show high activity. GVL1-MAP2 also showed bactericidal activity against most strains tested. The bioconjugate GVL1-MAP2 showed lower cytotoxicity than GVL1-MAP1 and amphotericin B in macrophages. Permeabilization studies and enzyme inhibition revealed that the peptide acts via at least two distinct mechanisms, with the primary mechanism targeting the membrane and inner targets as an inhibitor of the CPB enzyme. Conclusions: These data demonstrated that the synthesis of bioconjugates can be a tool for the development of bactericidal and antileishmanial compounds with improved potency and selectivity.

Leishmaniasis remains an important neglected tropical disease, and current treatments are limited by toxicity, resistance, and low bioavailability. In this study, novel guanidine derivatives were evaluated through an integrated approach, combining in silico physicochemical profiling with in vitro biological assays using Leishmania (Viannia) braziliensis, the etiological agent of American Tegumentary Leishmaniasis (ATL). Most compounds exhibited favorable drug-like properties, though variations in lipophilicity and solubility influenced biological performance. Among the tested molecules, FURL-G5 emerged as the most promising candidate, showing potent activity against promastigote forms and low cytotoxicity in murine macrophages, resulting in high selectivity indices (SI > 10), comparable to those of LQOF-G1, a compound with previously established leishmanicidal effects. These compounds were also tested on intracellular amastigotes, drastically reducing the infection rate of macrophages. The integration of an in silico approach and biological validation enabled rational compound prioritization and supports the early-stage development of these scaffolds. Overall, this study reinforces the potential of guanidine-based compounds as leads for innovative ATL drug discovery and demonstrates the value of multidisciplinary strategies for identifying selective and safe therapeutic candidates.