Related Experiment Video
Updated: Jun 11, 2025
Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers
Published on: September 19, 2022
Design and Biophysical Characterization of Second-Generation cyclic peptide LAG-3 inhibitors for cancer immunotherapy
Laura Calvo-Barreiro1, Longfei Zhang1, Yasir Ali2
1Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY 10065, USA.
Abstract:
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.
Insights
Researchers developed novel cyclic peptides to inhibit Lymphocyte Activation Gene 3 (LAG-3), a key target in cancer immunotherapy. While potent in vitro, the lead candidate failed to show anti-tumor effects in vivo, highlighting the need for combination therapies.
Area of Science:
- Immunology
- Pharmacology
- Biochemistry
Background:
- Lymphocyte Activation Gene 3 (LAG-3) is an inhibitory immune checkpoint that suppresses anti-cancer immune responses.
- Blocking LAG-3 can restore T cell cytotoxicity and reduce the impact of regulatory T cells.
- A previously identified cyclic peptide served as a basis for developing improved LAG-3 inhibitors.
Purpose of the Study:
- To design and synthesize novel cyclic peptide derivatives of a known LAG-3 inhibitor.
- To optimize tyrosine substitutions for enhanced LAG-3 inhibition.
- To evaluate the in vitro and in vivo efficacy of these peptide derivatives as potential cancer immunotherapies.
Main Methods:
- Design and synthesis of 19 cyclic peptide derivatives with modified tyrosine residues.
- Screening using time-resolved Förster resonance energy transfer (TR-FRET) assays to measure LAG-3 inhibition.
- Biophysical characterization using MicroScale Thermophoresis (MST) to determine binding affinity (KD).
- Molecular docking simulations to predict binding interactions and scores.
- In vivo studies to assess tumor growth inhibition.
Main Results:
- Eight peptide derivatives demonstrated superior LAG-3 inhibition compared to the original peptide.
- Cyclic peptides 12, 13, and 17 were identified as top candidates, with peptide 12 showing the highest inhibition (IC50 = 4.45 ± 1.36 µM).
- MST analysis revealed improved binding affinities for peptides 12 (KD = 2.66 ± 2.06 µM) and 13 (KD = 1.81 ± 1.42 µM) compared to the original.
- Docking simulations indicated enhanced binding for peptide 12 (score = -7.236 kcal/mol), attributed to the 3-cyano group's favorable interaction with MHC-II.
- Despite potent in vitro activity, cyclic peptide 12 did not inhibit tumor growth in vivo.
Conclusions:
- Novel cyclic peptides targeting LAG-3 were successfully developed with enhanced in vitro inhibitory activity and binding affinity.
- The 3-cyano substitution in cyclic peptide 12 significantly improved its interaction with LAG-3.
- The lack of in vivo efficacy for the lead peptide highlights the complexity of tumor immunity and the necessity of combination strategies targeting multiple immune checkpoints for effective cancer therapy.
