Fragments of gD Protein as Inhibitors of BTLA/HVEM Complex Formation - Design, Synthesis, and Cellular Studies

Abstract

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N-terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions.

Peptide-based inhibitors targeting the PD-1/PD-L1 axis: potential immunotherapeutics for cancer

“…3 S. The decrease in the concentration of the peptides at 0 h is not the effect of their degradation, but it is probably because of the interaction of these peptides with medium components such as albumin contained in FBS. We previously observed similar effects for other peptides targeting the BTLA/HVEM axis [35] , [36] , [37] . These studies show that the peptides are present in the samples under the conditions in which cellular assays are conducted and have the potential to exert a biological effect.…”

“…All these data indicate that slight changes in the amino acid sequence or disulfide bond position may affect the binding strength. We have previously investigated the importance of the position of the disulfide bond in the peptide amino acid sequence in studies focusing on the inhibitors of B- and T-lymphocyte attenuator and herpes virus entry mediator, and these prior studies reached the same conclusions on the importance of the position of stapling disulfide bonds [ [35] , [36] , [37] , 55 ].…”

Computational Design of a Highly-Specific HVEM-Based Inhibitor of LIGHT Protein

“…Moreover, our results can be used for further experimental verification as in our previous work. (Kuncewicz et al ., 2023) Overall, the comprehensive multi-step study of structural properties, binding affinity, selectivity, and mechanisms establishes the gold standard for the computational design of new peptide drugs.…”

“…We performed detailed analyses of binding free energies, dynamics, and stability of the obtained complexes based on extensive molecular dynamics (MD) simulations. This method proved to be an effective tool for peptide design in other HVEM ligand systems, those peptides proved to bind strongly in vitro (Kuncewicz et al ., 2023). We also determined amino-acid residues and disulfide bonds that have a crucial influence on the formation of the HVEM-LIGHT complex and identified the binding mechanism to LIGHT for the designed peptides.…”

Emerging targets for anticancer vaccination: PD-1

“…In addition to PD-1 and PD-L1, peptides that block other immune checkpoints such as B- and T-lymphocyte attenuator (BTLA) might also have potential in immunotherapeutic approaches. 46 …”