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Updated: Jan 22, 2026
Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology
Published on: May 2, 2025
Structure-based derivation and intramolecular cyclization of peptide inhibitors from PD-1/PD-L1 complex interface as
Kun Zhou1, Ji Lu2, Xiaoxin Yin2
1Department of General Surgery, Huashan Hospital Affiliated to Fudan University, Shanghai 200040, China.
Abstract:
The interaction event between programmed death receptor-1 (PD-1) and its ligand (PD-L1) functions as an essential immune checkpoint against cytotoxic T effector cell activation. Previously, a number of small-molecule inhibitors and antibody drugs have been successfully developed to block the PD1/PDL1 signaling axis for breast cancer immunotherapy. Here, we attempt to directly disrupt the formation of PD-1/PD-L1 complex by using a self-inhibitory peptide (SIP) strategy. In the procedure, the complex crystal structure is examined systematically with energetic analysis and alanine scanning. Two double-stranded segments I and II in PD-L1 active finger are identified as hotspot regions; they directly interact with the amphipathic pocket of PD-1 to form the complex system. The segments are derived from PD-L1 to define two SIP peptides, namely, DS-I and DS-II, which are thought to have capability of rebinding at the complex interface, thus disrupting PD-1/PD-L1 interaction as a new immune checkpoint blockade. A further analysis reveals that the free linear DS-I and DS-II peptides are highly flexible without protein context support, which would incur a large entropy penalty (unfavorable indirect readout effect) when rebinding to PD-1. Next, intramolecular cyclization is applied to constraining the intrinsically disordered conformation of free DS-II peptide into native ordered double-stranded configuration, which can be substantiated by molecular dynamics simulation and circular dichroism spectroscopy. Several cyclized counterparts of linear DS-II peptide are designed and their affinities to PD-1 are determined using fluorescence polarization assays. As might be expected, three designed cyclic peptides DS-II[c111-127], ΔDS-II[c111-127] and ΔDS-II[c110-128] exhibit considerably increased potency (Kd = 28.0 ± 4.2, 17.5 ± 3.1 and 11.6 ± 2.3 μM, respectively) relative to linear DS-II peptide (Kd = 109 ± 15 μM).
Insights
This study introduces self-inhibitory peptides (SIPs) to disrupt the programmed death receptor-1 (PD-1) and programmed death-ligand 1 (PD-L1) interaction, offering a novel strategy for cancer immunotherapy by blocking immune checkpoints.
Area of Science:
- Immunology
- Structural Biology
- Drug Discovery
Background:
- The programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) interaction is a key immune checkpoint that suppresses anti-tumor immune responses.
- Existing therapies like small-molecule inhibitors and antibodies target this axis for cancer immunotherapy, particularly in breast cancer.
Purpose of the Study:
- To develop a novel therapeutic strategy by directly disrupting the PD-1/PD-L1 complex using self-inhibitory peptides (SIPs).
- To investigate the structural basis of PD-1/PD-L1 interaction and design peptides that can interfere with complex formation.
Main Methods:
- Systematic analysis of the PD-1/PD-L1 complex crystal structure, including energetic analysis and alanine scanning.
- Design and synthesis of self-inhibitory peptides (DS-I and DS-II) derived from PD-L1.
- Molecular dynamics simulations and circular dichroism spectroscopy to assess peptide conformation.
- Intramolecular cyclization to stabilize peptide structure and enhance binding affinity.
- Fluorescence polarization assays to determine binding affinities of cyclic peptides to PD-1.
Main Results:
- Two hotspot regions (segments I and II) in PD-L1 were identified as crucial for PD-1 binding.
- Linear peptides (DS-I, DS-II) showed limited binding affinity due to conformational flexibility.
- Cyclized peptide counterparts of DS-II demonstrated significantly enhanced binding potency to PD-1.
- Specifically, DS-II[c111-127], ΔDS-II[c111-127], and ΔDS-II[c110-128] showed Kd values in the micromolar range, outperforming the linear DS-II peptide.
Conclusions:
- Self-inhibitory peptides, particularly when cyclized, represent a promising approach to disrupt the PD-1/PD-L1 immune checkpoint.
- Stabilizing the conformation of peptides through cyclization enhances their ability to inhibit PD-1/PD-L1 interactions.
- This strategy offers a new avenue for developing targeted cancer immunotherapies.
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