Functional Effects of Cyclization on Li1 Peptide Activity against Metacyclic Leishmania amazonensis Internalization

Leishmaniases are caused by protozoa of the genus Leishmania, whose metacyclic promastigote forms initiate infection in the mammalian host. Building upon previous work with Leishmania infantum, this study evaluated the cyclic peptide Li1 and its linear analogue Li1nc regarding their capacity to interfere with Leishmania amazonensis 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 L. infantum, 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 Leishmania transmission.