Efficient Method for High-Throughput Screening of Compound Libraries Targeting Human PD-L1.

Area of Science:

• Immunology
• Molecular Biology
• Drug Discovery

Background:

• The PD-1/PD-L1 pathway is a critical target for cancer immunotherapy.
• Current PD-1/PD-L1 inhibitors (monoclonal antibodies) have limitations including cost, tumor penetration, and immunogenicity.
• There is a need for alternative therapeutic strategies, such as small molecule inhibitors.

Purpose of the Study:

• To develop and validate a high-throughput screening (HTS) platform, the DNA-linked Inhibitor Antibody Assay (DIANA), for identifying low-molecular-weight inhibitors of human PD-L1.
• To assess the suitability of DIANA for characterizing inhibitor binding affinity (K_d) and screening compound libraries.
• To address limitations associated with traditional antibody-based therapies and screening methods.

Main Methods:

• Developed DIANA, a platform integrating competitive binding assays with quantitative PCR (qPCR) detection.
• Validated DIANA using known PD-1/PD-L1 binders, including FDA-approved monoclonal antibodies (mAbs) and a macrocyclic peptide.
• Screened two compound libraries (5,280 and 1,298 compounds) using DIANA in pooled and individual formats.

Main Results:

• DIANA accurately determined dissociation constants (K_d) for known inhibitors, consistent with literature values.
• The assay demonstrated a wide dynamic range (>4 orders of magnitude), excellent robustness (Z'-factor = 0.94), and high DMSO tolerance (10%).
• Initial screening of compound libraries yielded weak hits that were not confirmed in subsequent validation assays.

Conclusions:

• DIANA is a sensitive, scalable, and robust HTS platform for discovering low-molecular-weight PD-L1 inhibitors.
• The platform overcomes key limitations of conventional screening methods for antibody-based targets.
• Further optimization and application of DIANA are warranted for accelerating the development of next-generation cancer immunotherapies.