Functional Effects of Cyclization on Li1 Peptide Activity against Metacyclic <i>Leishmania amazonensis</i> Internalization

Leishmaniases are caused by protozoa of the genus <i>Leishmania</i>, whose metacyclic promastigote forms initiate infection in the mammalian host. Building upon previous work with <i>Leishmania infantum</i>, this study evaluated the cyclic peptide Li1 and its linear analogue Li1nc regarding their capacity to interfere with <i>Leishmania amazonensis</i> infection. Both peptides were synthesized by solid-phase peptide synthesis (SPPS-Fmoc) and tested after pre-exposure of metacyclic promastigotes prior to infection of murine peritoneal and THP-1-derived macrophages. In contrast to the previous study conducted by our group, which evaluated only Li1 against <i>L. infantum</i>, the present work introduces the first direct comparison between the cyclic peptide and its linear analogue, allowing a structure-activity assessment not previously available. Pre-exposure to Li1 (0.5 mg mL^-1 ≅ 0.318 mmol L^-1) significantly reduced the infection rate and infection index in both macrophage models, while Li1nc (0.324 mmol L^-1) exhibited moderate inhibition. Neither peptide displayed cytotoxicity toward host cells (CC₅₀ > 0.5 mg mL^-1) nor direct antipromastigote activity (IC₅₀ > 0.5 mg mL^-1). Confocal microscopy revealed stronger and more defined binding of Li1 to the parasite surface than Li1nc, particularly along the flagellum, supporting structure-dependent interaction with surface molecules involved in parasite internalization. In a murine model of cutaneous leishmaniasis, preincubation of metacyclic promastigotes with Li1 (0.2 mg mL^-1 ≅ 0.127 mmol L^-1) reduced parasite burden by 35.2%, whereas Li1nc (0.130 mmol L^-1) achieved a 20.4% reduction relative to untreated controls. No significant alterations in hepatic or renal biochemical parameters were observed, indicating the absence of systemic toxicity. Notably, neither peptide showed activity under a postinfection treatment regimen (2 mg Kg^-1), suggesting that their effects are restricted to early host-parasite interactions rather than therapeutic clearance. Collectively, these findings demonstrate that Li1 acts through a structure-dependent mechanism that interferes with host-parasite recognition and reduces infectivity without inducing detectable toxicity. The results support the translational potential of Li1 as a safe peptide scaffold for prophylactic or paratransgenic strategies aimed at preventing <i>Leishmania</i> transmission.