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Inside-out receptor inhibition

Nature volume 540, pages 344345 (15 December 2016) | Download Citation

Structures of two chemokine receptor proteins in complex with small molecules reveal a previously unknown binding pocket that could be a drug target for treating a range of diseases involving this receptor family. See Letters p.458 & p.462

A family of cell-membrane proteins known as G-protein-coupled receptors mediates transmembrane signal transduction. One subset of this family is the chemokine receptors, which regulate cell migration and whose activation has been implicated in a range of diseases, including immune disorders and cancer. But finding drugs that inhibit these receptors has been challenging. Two papers in this issue1,2 now describe crystal structures of two different chemokine receptors in complex with small-molecule inhibitors. Two of these antagonists bind to pockets near to the receptors' intracellular surfaces, pointing to a previously unidentified pathway that can be targeted for drug discovery.

Most drug molecules that target G-protein-coupled receptors (GPCRs) mimic the binding activity of a native activator, enhancing or inhibiting receptor signalling to achieve a therapeutic effect. The drugs typically occupy a binding pocket called the orthosteric site in the transmembrane region of the receptor that is accessible to the outside of the cell. But the affinity with which activating ligands bind to receptors can be increased by the binding of a G protein. This phenomenon, known as an allosteric effect, is well established in GPCR pharmacology3 and provides an alternative avenue for drug discovery.

Unlike G proteins, which bind to the intracellular side of the receptor, other allosteric molecules tend to bind to sites that are within the membrane region or at the extracellular surface, sometimes even overlapping with the orthosteric pocket. However, a few drug candidates and antibodies seem to bind to the cytoplasmic surface of GPCRs (including chemokine receptors) and affect function4,5,6,7. An allosteric drug that binds at the cytoplasmic surface of a GPCR has not been described in detail, until now.

In the first study, Oswald et al.1 report the crystal structure of the chemokine receptor CCR9 in complex with a small-molecule drug called vercirnon, which acts as an antagonist to CCR9 activity (Fig. 1a). Inhibition of CCR9 is desirable as a possible way to treat inflammatory bowel disease, but vercirnon did not pass a phase III trial in people with Crohn's disease. Crystallization of the CCR9–vercirnon complex required the use of a CCR9 variant that had eight amino-acid substitutions and truncated amino- and carboxy-terminal tails, but the authors confirmed that none of these changes affected vercirnon binding.

Figure 1: Visualizing allosteric inhibition.
Figure 1

Two papers report structures of chemokine receptor proteins in complex with small-molecule antagonists, which inhibit receptor activity. In both structures, the receptor comprises seven transmembrane helical domains and an eighth helix that lies along the cytoplasmic surface of the membrane (different domains denoted by gradually changing colours, from blue and white to pink). a, Oswald et al.1 report that the small molecule vercirnon binds in a pocket on the intracellular side of the CCR9 receptor. The binding pocket for the drug maraviroc in another chemokine receptor, CCR5, is shown in purple for comparison12. b, Zheng et al.2 report the structure of CCR2 in simultaneous complex with two different antagonists: BMS-681, which binds in a pocket that overlaps with the maraviroc binding site, and CCR2-RA-[R], which binds in an intracellular pocket almost identical to that in CCR9.

The structure reveals the seven transmembrane helical segments of CCR9 connected by three cytoplasmic loops, with an eighth helix that seems to rest on the intracellular surface of the membrane. Vercirnon is an asymmetric, inverted-V-shaped structure that binds in a pocket comprising five of the seven helices, and it peeks out directly into the cytoplasm. The vercirnon binding site is about 33 ångströms from the presumed orthosteric pocket, which lies towards the extracellular surface of the seven-helical bundle.

In the second study, Zheng et al.2 report the structure of CCR2 — which has been implicated in various chronic inflammatory and autoimmune disorders and in antitumour immunity — in simultaneous complex with two small-molecule antagonists dubbed CCR2-RA-[R] and BMS-681 (Fig. 1b). The authors enabled crystallization by truncating the carboxy-terminal tail of CCR2, and by fusing a stabilizing protein called T4 lysozyme into an altered third cytoplasmic loop, which is a common strategy to facilitate GPCR crystallization.

BMS-681 binds in a pocket that overlaps with the presumed orthosteric binding site near the extracellular surface of CCR2, whereas CCR2-RA-[R] binds in a site remarkably like the allosteric pocket in CCR9. The simultaneous binding of the two antagonists traps CCR2 in a conformation that seems to be completely inactive, on the basis of the tight helical arrangement of the protein and the absence of regional conformations characteristic of receptor activation. The helix bundle looks similar to that of rhodopsin, a light-activated GPCR, when the protein is in the dark — the gold standard for an inactive GPCR structure.

Although vercirnon and CCR2-RA-[R] are different chemical entities, they occupy binding pockets that lie in the same location and have a three-dimensional lining made up of amino-acid side chains from helices 1, 2, 3, 6 and 7. These similarities indicate that the allosteric pocket might be present in other chemokine receptors. If this supposition holds true, medicinal and computational chemists will have a field day using structure-aided design strategies to develop drugs that target the pocket. Vercirnon, for example, was not optimized for the CCR9 pocket, and it is likely that some minor alterations would improve its drug properties.

The structures also provide insights into the mechanism of intracellular allosteric antagonism. Both show that the bound antagonists prevent outward movement and rotation of helices (especially helix 6), which is the hallmark of the active-state structure. Particularly in the CCR9–vercirnon structure, in which the cytoplasmic loops are not modified for crystallization and are reasonably well resolved, it is clear that vercirnon occupies a space that would normally be filled by the carboxy-terminal tail of a bound G protein during receptor activation8. Binding by the protein β-arrestin, which inhibits signalling and causes receptors to be internalized into the cell, would also certainly clash with bound vercirnon9.

Although structures have been produced for the human chemokine receptor CXCR4 in complex with a chemokine from a virus10, and for a viral chemokine receptor in complex with the human chemokine CX3CL1 (ref. 7), there is not yet a structure of a human chemokine receptor in complex with a human chemokine. Chemokines themselves are relatively complex protein ligands, generally comprising about 70 amino acids. What is clear is that multiple regions of the extracellular surface of chemokine receptors have roles in docking the chemokine before it can engage the orthosteric pocket in the helix bundle11. Identification of a cytoplasmic allosteric binding pocket in chemokine receptors is especially valuable, because it provides an alternative strategy for structure-based drug discovery before the precise binding mode of chemokines has been fully elucidated.

Progress in developing useful small-molecule drugs for chemokine receptors has been slow. There have been just two successes — maraviroc, which targets CCR5 to prevent HIV-1 from entering cells, and plerixafor, which targets CXCR4 to mobilize bone-marrow stem cells for transplantation in people with cancer. Another dozen or so chemokine receptors are drug targets for diseases ranging from autoimmune disorders to cancer metastasis. The intracellular binding pocket identified in the current studies might provide a new strategy for inhibiting these receptors, by turning drug-discovery efforts inside out.



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  1. Thomas P. Sakmar and Thomas Huber are in the Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065, USA.

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Correspondence to Thomas P. Sakmar or Thomas Huber.

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