Evaluating the In Vitro Activity and Safety of Modified LfcinB Peptides as Potential Colon Anticancer Agents: Cell Line Studies and Insect-Based Toxicity Assessments

[Image: see text] Anticancer peptides are increasingly being considered as alternative treatments for cancer due to their potency, selectivity, and low toxicity. Previously, the peptide LfcinB (21–25)(Pal) showed in vitro anticancer effects against the Caco-2 colon cancer cell line (half-maximal inhibitory concentration (IC(50)): 86 μM). In this study, we developed modifications to the peptide sequence to increase its anticancer activity. Sequence modifications were made such as the inclusion of amino hexanoic acid (Ahx), N-terminal biotinylation, acetylation, and substitutions of Orn for Arg and/or d-Arg by l-Arg. The molecules were synthesized using manual solid-phase peptide synthesis (SPPS), and their synthetic feasibility (SAScore) ranged from 6.2 to 7.6. The chromatographic purities of the synthesized peptides were greater than 89%. We found that Ahx-RWQWRWQWR and RWQWRWQW-Orn showed activity against both Caco-2 and HT-29 cell lines and decreased IC(50) values by approx. 50% in Caco-2 cells (IC(50): 40 μM) when compared to the parent peptide RWQWRWQWR. Moreover, the modified peptides demonstrated lower hemolytic effects, with values <10% at 200 μg/mL. Toxicity was assessed using the Galleria mellonella model and the half-maximal lethal dose (LD(50)) for the best peptides was >100 mg/kg, indicating that their toxicity is classified as moderately toxic or lower. In contrast, cisplatin showed an LD(50) of 13 mg/Kg. The designed anticancer peptides presented good in vitro activity and low toxicity, making them promising molecules for future drug development studies.