Aaron A. Thompsona; Michael B. Harbut; Pei-Pei Kung; Nathan K. Karpowich; Jeffrey D. Branson; Joanna C. Grant; Deborah Hagan; Heather A. Pascual; Guoyun Bai; Reza Beheshti Zavareh; Heather R. Coate; Bernard C. Collins; Marjorie Côte; Christine F. Gelin; Kelly L. Damm-Ganamet; Hadi Gholami; Adam R. Huff; Luis Limon; Kevin J. Lumb; Puiying A. Mak; Kohki M. Nakafuku; Edmund V. Price; Amy Y. Shih; Mandana Tootoonchi; Nadeem A. Vellore; Jocelyn Wang; Na Wei; Jeannie Ziff; Scott B. Berger; James P. Edwards; Agnès Gardet; Siquan Sun; Jennifer E. Towne; Jennifer D. Venable; Zhicai Shi; Hariharan Venkatesan; Marie-Laure Rives; Sujata Sharma; Brock T. Shireman; Samantha J. Allen Proc. Natl. Acad. Sci. USA, 2023, 120(18), e2216342120 https://doi.org/10.1073/pnas.2216342120
Abstract
Significance The disruption of protein–protein interactions (PPI) represents a major opportunity for therapeutic intervention, as illustrated by many FDA-approved antibodies. However, the discovery of small-molecule PPI inhibitors remains challenging due to the complex nature and large size of most PPI interfaces. We report two distinct small-molecule classes targeting NKG2D (Naturalkiller group 2D), an immune receptor considered to be intractable for small-molecule inhibition. Inhibitor discovery and optimization were facilitated by large chemically diverse libraries combined with a comprehensive biochemical and cell-based assay suite. Both chemical series inhibited ligand-induced signaling by allosterically altering the NKG2D dimer conformation. This indicates that by targeting protein–protein interfaces involving oligomers, it is possible to increase the probability of discovering small-molecule PPI modulators. Abstract NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, 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.