N-methylmorpholine incorporation into the structure of biphenyl leads to the bioactive inhibitor of PD-1/PD-L1 interaction.

We present new small-molecular probes targeting the human PD-L1 protein. The molecules were designed by incorporating a newly discovered N-methylmorpholine substituent into a known biphenyl-based structure. Four prototype derivatives of 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine-7-carbonitrile (STD4), comprising a morpholine substituent fused with a biphenyl core at different orientations were first verified for their potential binding to PD-L1 using the molecular docking method. A more favorable 7-phenyl derivative of STD4 was then equipped with an amide bond, pyridine, and either a tris(hydroxymethyl)aminomethane or serinol tail leading to two final molecules. Among them, compound 1c showed activity in three bioassays, i.e., the homogenous time-resolved fluorescence (HTRF) assay, immune checkpoint blockade (ICB) assay, and T-cell activation (TCA) assay. Our work shows that morpholine can substitute for dioxane and becomes a promising component in PD-L1-targeting molecules. This finding unlocks new avenues for optimizing PD-L1-targeting compounds, presenting exciting prospects for future developments in this field.

Exploring the Surface of the Ectodomain of the PD-L1 Immune Checkpoint with Small-Molecule Fragments.

Development of small molecules targeting the PD-L1/PD-1 interface is advancing both in industry and academia, but only a few have reached early-stage clinical trials. Here, we take a closer look at the general druggability of PD-L1 using in silico hot spot mapping and nuclear magnetic resonance (NMR)-based characterization. We found that the conformational elasticity of the PD-L1 surface strongly influences the formation of hot spots. We deconstructed several generations of known inhibitors into fragments and examined their binding properties using differential scanning fluorimetry (DSF) and protein-based nuclear magnetic resonance (NMR). These biophysical analyses showed that not all fragments bind to the PD-L1 ectodomain despite having the biphenyl scaffold. Although most of the binding fragments induced PD-L1 oligomerization, two compounds, TAH35 and TAH36, retain the monomeric state of proteins upon binding. Additionally, the presence of the entire ectodomain did not affect the binding of the hit compounds and dimerization of PD-L1. The data demonstrated here provide important information on the PD-L1 druggability and the structure-activity relationship of the biphenyl core moiety and therefore may aid in the design of novel inhibitors and focused fragment libraries for PD-L1.