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

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.

Secondary Sites of the C‐type Lectin‐Like Fold

“…(g) NKG2D (PDB ID: 8EA6) has a binding site for a small‐molecule ligand in the interaction interface of the physiologically functional homodimer (both small molecule and second monomer not shown). Amino acids within 5 Å of the small molecule are colored blue [135] . The small molecule ligand prevents binding of the NKG2D homodimer to endogenous protein ligands containing an α1α2 platform domain.…”

“…Likewise, high‐throughput screenings (HTS) yielded biologically active and chemically tractable lead structures [89,129–131] . Follow‐up studies and mode of action analysis suggested some of these secondary sites to impact CLR function via unprecedented mechanisms, including allosteric modulation of the canonical CBS and changes in quaternary conformation [13,89,130–135] . For instance, a recently identified small molecule ligand was shown to inhibit LOX‐1‐dependent oxLDL uptake by cross‐linking two dimers in a head‐to‐head fashion, sterically blocking the basic spine region, and locking the receptor in an inactive tetramer state (Figure 3c, d and e).…”

“…The ligand expands a small pocket between the NKG2D domains and acts as a wedge between the monomers by replacing reciprocal hydrogen bonds between L148 A and K150 B /L148 B and K150 A (Figure 10d). This way, the ligand disrupts the quaternary protein structure, distorts the protein‐protein interaction interface, and abrogates binding of MHC‐I‐like protein ligands [131,135] . The small‐molecule ligands for NKG2D are a good demonstration of how allosteric and, in this case, cryptic sites can be used to bypass the usually low druggability of protein‐protein interaction interfaces [259] …”

Exploring the Application of SiteMap and Site Finder for Focused Cryptic Pocket Identification

“…The systems used in this work are a combined benchmark dataset from two previous studies. , The Cryptosite set includes 93 apo-holo protein structures, and the PocketMiner set includes 39 apo-holo proteins. We also included a known challenging cryptic pocket from our internal work on natural killer group 2D (NKG2D) in our test set. The Cryptosite set contains more systems with cryptic pockets of different categories.…”

The Role of NKG2D and Its Ligands in Autoimmune Diseases: New Targets for Immunotherapy

“…The NKG2D receptor consists of two disulfide-linked type II transmembrane glycoproteins whose extracellular region contains a C-type lectin-like structural domain [8].…”