New thiazole-biaryls as small molecule immunomodulators (SMIs) targeting PD-1/PD-L1 axis.

Immune Checkpoint Inhibitors (ICIs) targeting the PD-1/PD-L1 axis have improved the clinical outcomes and revolutionized oncology treatment. However, monoclonal antibody (mAb)-based ICIs are limited by high production and treatment cost, poor oral bioavailability, limited tumor penetration, and immune-related adverse effects. To address this, here we have designed, synthesized, and evaluated a series of new thiazole-biphenyl-containing small molecules (15a-j, 16a-n, and 17a-y) targeting the PD-1/PD-L1 interface. Among all, compounds 17t and 17h were identified as potential PD-1/PD-L1 inhibitors, restoring T-cell signalling with EC50 values of 513 nM and 215 nM in a cell-based PD-1/PD-L1 blockade functional assay. Isothermal titration calorimetry (ITC) confirmed direct binding of both compounds to PD-L1, with 17h exhibiting stronger affinity (Kd = 1.43 μM) compared to 17t (Kd = 19.8 μM). Consistently, competitive PD-1/PD-L1 ELISA demonstrated dose-dependent disruption of the interaction, with 17h showing higher potency (EC₅₀ = 6.06 μM) than 17t (EC₅₀ = 23.83 μM), confirming effective target engagement. Further, co-culture cytokine induction assay involving MDA-MB-231 and activated PBMCs demonstrated the robust upregulation of pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1β) and the cytolytic protein (perforin), suggesting an effective rescue of the small molecule-mediated T-cell exhaustion. The T-cell cytotoxicity assay revealed that 17t and 17h enhanced T-cell-mediated lysis of cancer cells without intrinsic cytotoxicity against MDA-MB-231 cells, confirming a selective immunomodulatory effect. In addition, structure-based molecular simulations of 17t and 17h revealed a key π-π interaction with Y56, as well as hydrogen bonding with the Y123 and I116 residues of the PD-L1 dimer.