Comparative two-dimensional NKG2A/CD94 cell membrane chromatography for screening NK cell immune checkpoint inhibitors

The natural killer (NK) group 2 member A/C-type lectin domain family 4 member A (NKG2A/CD94) heterodimeric receptor is commonly recognized as a crucial immune checkpoint in NK cells. Currently, there is a notable lack of small-molecule inhibitors specifically targeting NKG2A that have progressed to clinical trials, and established screening methodologies for identifying such inhibitors remain limited. Cell membrane chromatography (CMC) is a biochromatographic technique that leverages the specific interactions between membrane receptors and their ligands. In this study, a comprehensive two-dimensional (2D) NKG2A/CD94 and HEK293 CMC comparative analysis system was developed to screen for selective NKG2A/CD94 ligands derived from Echinacea purpurea (L.) Moench and Alpinia katsumadai Hayata. The comprehensive 2D CMC comparative analysis system demonstrated superior selection performance, resulting in the successful screening and identification of five compounds. Of these compounds, chicoric acid and alpinetin exhibited greater binding affinity for the NKG2A/CD94 CMC column compared to the HEK293 CMC column, leading to their selection for further efficacy verification. Surface plasmon resonance (SPR) analysis revealed that chicoric acid and alpinetin exhibit binding affinities of 12.9 and 9.49 μM, to NKG2A/CD94. Molecular docking analyses and pharmacological investigations further demonstrated that both compounds could influence NK cell activation by interacting with the NKG2A/CD94. These findings suggest their potential as novel NKG2A/CD94 immune checkpoint inhibitors. Additionally, the comprehensive 2D CMC system serves as a robust and practical platform for drug discovery, and could be applied to other immune checkpoint receptor models.