Design and Biophysical Characterization of Second-Generation cyclic peptide LAG-3 inhibitors for cancer immunotherapy

[Display omitted] •Nineteen cyclic peptide derivatives designed to improve LAG-3 inhibition.•Cyclic peptide 12 showed the highest LAG-3/MHC-II inhibition in TR-FRET assay.•Biophysical screening validated the LAG-3 binding affinity of the cyclic peptide.•Docking simulations confirmed improved LAG-3 binding for cyclic peptide 12. Lymphocyte activation gene 3 (LAG-3) is an inhibitory immune checkpoint crucial for suppressing the immune response against cancer. Blocking LAG-3 interactions enables T cells to recover their cytotoxic capabilities and diminishes the immunosuppressive effects of regulatory T cells. A cyclic peptide (Cys-Val-Pro-Met-Thr-Tyr-Arg-Ala-Cys, disulfide bridge: 1–9) was recently reported as a LAG-3 inhibitor. Based on this peptide, we designed 19 derivatives by substituting tyrosine residue to maximize LAG-3 inhibition. Screening via TR-FRET assay identified 8 outperforming derivatives, with cyclic peptides 12 [Tyr6(L-3-CN-Phe)], 13 [Tyr6(L-4-NH2-Phe)], and 17 [Tyr6(L-3,5-DiF-Phe)] as top candidates. Cyclic peptide 12 exhibited the highest inhibition (IC50 = 4.45 ± 1.36 µM). MST analysis showed cyclic peptides 12 and 13 bound LAG-3 with KD values of 2.66 ± 2.06 µM and 1.81 ± 1.42 µM, respectively, surpassing the original peptide (9.94 ± 4.13 µM). Docking simulations revealed that cyclic peptide 12 exhibited significantly enhanced binding, with a docking score of −7.236 kcal/mol, outperforming the original peptide (−5.236 kcal/mol) and cyclic peptide 5 (L-4-CN-Phe) (−5.131 kcal/mol). A per-residue decomposition of the interaction energy indicated that the 3-cyano group in cyclic peptide 12 contributes to a more favorable conformation, yielding an interaction energy of −9.22 kcal/mol with Phe443 of MHC-II, compared to −6.03 kcal/mol and −5.619 kcal/mol for cyclic peptides 0 and 5, respectively. Despite promising in vitro results, cyclic peptide 12 failed to inhibit tumor growth in vivo, underscoring the importance of dual immunotherapies targeting several immune checkpoints to achieve anti-tumor efficacy.