PREreview of Structural basis for CCR6 modulation by allosteric antagonists

Summary

Chemokine receptors (CKRs) are a group of G protein-coupled receptors (GPCRs) that mediate many physiological processes. As such, CKRs are important drug targets for various indications, including cancer and autoimmune disorders. In this work, Wasilko et al focus on CC chemokine receptor 6 (CCR6), which is expressed particularly in T helper 17 (Th17) cells, B cells, and dendritic cells, and has been identified as a promising target for anti-inflammatory drug discovery. This manuscript extends previous structural studies performed by the group to understand the interactions of CCR6 with its native ligand (CCL20) and inform design of small molecules that function as allosteric antagonists.

The results presented include two inactive CCR6 structures in ternary complexes with allosteric antagonists from the squaramide (SQA) and oxomorpholine (OXM) series of small molecules as determined by single-particle cryogenic electron microscopy (cryoEM). Importantly, to solve these structures, the authors use an engineered CCR6 construct (Na7.1) which contains seven thermostabilizing alanine-substitutions, a point mutation to increase receptor expression, and several other modifications. The functional effects of these two molecules binding to the receptor are investigated, as well as the allosteric changes to the structures upon binding both the SQA and OXM molecules. To validate the binding modes identified in the structural studies, the authors synthesize additional OXM analogues (OXM3 and OXM4) that are inactive against CCR6 and use NMR studies to support a hypothesis that OXM analogues must be in a specific conformation in the free state in order to be active with CCR6. Further, the structural changes of the inactive and active forms of CCR6 are thoroughly discussed, with insights into the H-bond network governing this change from active to inactive.

Overall, this study presents an important contribution to understanding the structure of CCR6 in its activated and inactivated states, as well as strategies for identifying sites for allosteric modulation on other CKRs. The approach used to obtain these inactive CKR structures is clever and can be broadly applied to other receptor targets and the structural work is presented well.

Specific Points

-       The engineered form of CCR6 has been used for other studies, and the rationalization provided by the authors in the results - coupled with the functional results in Figure 1C and 1D - does not diminish the impact of these structures. However, as the molecular details of allosteric interaction are key findings of this study, some discussion of these changes to the protein structures relative to the WT CCR6 and the impact on the allosteric drug design would be useful.

-       Figure 1C shows thermal shifts for the WT CCR6 and Figure 1D shows the same for the thermostabilized CCR6. However, there are some differences in statistical significance as well as magnitude between the two sets – notably, the differences presented in the light blue and mid blue data points (OXM1 – OXM2 – SQA1 + vs OXM1 + OXM2 – SQA1 +). Some discussion or explanation of these differences would be meaningful.

-       The inhibition curves presented in Figure 1B of Log[OXM1] vs % inhibition look more linear than sigmoidal. Are there additional data below and above the current cutoffs (-10 and -4 M) that may strengthen the trend to establish the dose-response curve?

Competing interests

The authors declare that they have no competing interests.