Design, Synthesis, Evaluation, and Structural Studies of C2-Symmetric Small Molecule Inhibitors of Programmed Cell Death1/Programmed Death-Ligand 1 Protein-Protein Interaction

A series of C-2-symmetric inhibitors was designed and evaluated for inhibitory activity against the programmed cell death-1/programmed death-ligand 1(PD-1/PD-L1) protein protein interaction (PPI) in a homogenous time-resolved fluorescence (HTRF) assay and PD-1 signaling in cell-based coculture assays. CZ symmetric inhibitors 2a (LH1306) and 2b (LH1307) exhibited ICso values of 25 and 3.0 nM, respectively, in the HTRF assay. While 2a was similar to 3.8-fold more potent than previously reported inhibitor la, 2b could not be differentiated from lb due to their high potency and the limit of our HTRF assay conditions. In one cell-based coculture PD-1 signaling assay, 2a and 2b were 8.2- and 2.8-fold more potent in inhibiting PD-1 signaling than la and lb, respectively. NMR and X-ray cocrystal structural studies provided more structural insights into the interaction between 2b and PD-L1; 2b binds to PD-L1 at the PD-1 binding site and induces the formation of a more symmetrically arranged PD-L1 homodimer than that previously reported for other inhibitors.