Identification of small-molecule protein-protein interaction inhibitors for NKG2D

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, & gamma;& delta;+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure- activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Automated summary

NKG2D is a key receptor involved in immune response to infections and stress, but its abnormal signaling is associated with inflammatory and autoimmune diseases. In this study, we identified two series of protein-protein interaction inhibitors for NKG2D using a comprehensive small-molecule hit identification strategy. These inhibitors disrupt ligand binding through allosteric modulation of the NKG2D receptor dimer, resulting in improved potency and physicochemical properties. Our findings demonstrate the potential of targeting NKG2D for immune intervention through allosteric modulation.