Intracellular allosteric antagonism of the CCR9 receptor

Abstract

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

What Do Structures Tell Us About Chemokine Receptor Function and Antagonism?

“…5e). A similar trend is observed with Vercirnon, the crystallized allosterically acting antagonist of CCR9 (not shown) (153)…”

“…The binding site of several CXCR1 and CXCR2 antagonists was also mapped to a homologous region through mutagenesis (93, 113) and competition binding studies (27). Moreover, an X-ray structure has recently been solved for CCR9 with an allosteric antagonist Vercirnon bound at a homologous site (153)…”

“…Nevertheless, the favorable druggability properties of the allosteric pocket, and the fact that it appears to be present in other receptors, may open a new avenue for chemokine receptor inhibition. The structures of CCR2:BMS-681:CCR2-RA-[R] and CCR9:Vercirnon complexes (147, 153) will undoubtedly facilitate rational drug discovery efforts for these and possibly for other chemokine receptors, through ligand-informed homology modeling (65, 90). …”

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…Finding that two similar but distinct receptors bind the same chemokine, and that CXCR1 almost exclusively binds to IL-8 whereas CXCR2 also binds to several other chemokines, provided an early indication of the complexity of the chemokine defense system. At present, the structures of six chemokine receptors have been reported: the crystal structures of CXCR4 (24,25), CCR2 (26), CCR5 (27), CCR9 (28), and viral US28 (29), and our NMR structure of CXCR1 in lipid bilayers (23), which is shown in Fig. 1 B. Although none of the structures represent complexes of a receptor with one of its native chemokine agonists, the structures of receptors bound to viral chemokines provide information that is complementary to the many mutagenesis and binding studies of their interactions.…”

Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations

“…As this site typically corresponds to variable and flexible loop regions, the bound conformation of an allosteric ligand may not be readily identified from a crystal structure with only an orthosteric ligand. For instance, while a positive allosteric modulator (PAM) was found to be bound at the extracellular side of the M2 muscarinic receptor crystal structure (PDB: 4MQT [1]), the negative allosteric modulators (NAMs) cocrystallized with the chemokine receptors CCR9 (PDB: 5LWE [2]) and CCR2 (PDB: 5T1A [3]) were found at the intracellular side. Notably, allosteric GPCR ligands are of great interest from a drug discovery perspective due to their expected greater subtype selectivity, their ability to maintain temporal and spatial characteristics of endogenous signals, and their potentially limited on-target overdosing risks [4].…”