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.

Nonsymmetrically Substituted 1,1'-Biphenyl-Based Small Molecule Inhibitors of the PD-1/PD-L1 Interaction.

Therapeutic antibodies directed against either programmed cell death-1 protein (PD-1) or its ligand PD-L1 have demonstrated efficacy in the treatment of various cancers. In contrast with antibodies, small molecules have the potential for increased tissue penetration; better pharmacology; and therefore, improved antitumor activity. A series of nonsymmetric C2 inhibitors were synthesized and evaluated for PD-1/PD-L1 interaction inhibition. These compounds induced PD-L1 dimerization and effectively blocked PD-L1/PD-1 interaction in a homogeneous time-resolved fluorescence (HTRF) assay with most inhibitors exhibiting IC50 values in the single-digit nM range and below. Their high inhibitory potency was also demonstrated in a cell-based coculture PD-1 signaling assay where 2 exhibited an EC50 inhibitory activity of 21.8 nM, which approached that of the PD-L1 antibody durvalumab (EC50 = 0.3-1.8 nM). Structural insight into how these inhibitors interact with PD-L1 was gained by using NMR and X-ray cocrystal structure studies. These data support further preclinical evaluation of these compounds as antibody alternatives.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.