Design, Synthesis, Evaluation, and Structural Studies of <i>C</i>₂-Symmetric Small Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein–Protein Interaction

Abstract

A series of <i>C</i>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. <i>C</i>₂-symmetric inhibitors <b>2a</b> (LH1306) and <b>2b</b> (LH1307) exhibited IC₅₀ values of 25 and 3.0 nM, respectively, in the HTRF assay. While <b>2a</b> was ∼3.8-fold more potent than previously reported inhibitor <b>1a</b>, <b>2b</b> could not be differentiated from <b>1b</b> due to their high potency and the limit of our HTRF assay conditions. In one cell-based coculture PD-1 signaling assay, <b>2a</b> and <b>2b</b> were 8.2- and 2.8-fold more potent in inhibiting PD-1 signaling than <b>1a</b> and <b>1b</b>, respectively. NMR and X-ray cocrystal structural studies provided more structural insights into the interaction between <b>2b</b> and PD-L1; <b>2b</b> 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.