A novel 3D Model of the human Cannabinoid receptor 2 in its active state helps to develop new radiotracers for positron emission tomography (PET)

PET is a method, which provides images of functional processes in living humans. Thus, there is a strong need for highly selective positron emitting radiotracers. In order to develop such compounds for imaging of human cannabinoid receptors type 2 (hCB2) we constructed a 3 dimensional model of the human cannabinoid receptor in its active state based on the recently published X-ray structure 3qak (Xu et al., Science 2011;332:322-327) of the human adenosine receptor A2a (hAA2R). The model served as a guide for the rational design of hCB2-selective N-aryl oxadiazoles, which were synthetized, tested for their receptor affinity and evaluated concerning a suitable site for labeling with 18F, the most preferred PET radionuclide.

Methods: The sequence of the hCB2 receptor was aligned to that of hAA2R by multiple sequence alignment. 10 models of hCB2 were constructed based on structure 3qak using the MOE software package (Molecular Operating Environment, CGC Inc., Montreal). The best model was simulated in a solvated lipid bilayer for 15 ns using the Amber03 force field as implemented in the software package Yasara (Yasara Biosciences GmbH, Wien). The N-aryl oxadiazoles were synthesized in a two-step approach. Hydroxylamine hydrochlorides and benzonitriles reacted for 24 h at 80 °C under diffuse light. After drying the intermediate, addition of succinic acid anhydrides at room temperature under diffuse light yielded the final products. Ki values of the compounds were determined in competitive radioligand displacement studies on hCB1- and hCB2-CHO cell homogenates in the presence of [3H]CP55940.

Results: 1) The 3D model of hCB2 is in good agreement with experimental data and
2) remains in its conformation during a 15 ns MD simulation. 3) The synthesized N-aryl oxadiazoles bind selectively on hCB2 receptors with a ratio of Ki(CB1):Ki(CB2) of up to 10,000. 4) Based on molecular docking studies, the compounds most likely bind with their N-aryl-moiety (R) inside the proposed binding pocket indicating, that 5) introduction of 18F on the ring system at position 3 of the oxadiazole is most promising.

Conclusions: Our model of the hCB2-receptor provides insights into binding of ligands and allows improvement and design of hCB2-selective ligands by means of rational design. N-aryl oxadiazoles can serve as scaffolds for the synthesis of selective hCB2 PET radioligands.
Acknowledgment: This work was supported by DFG (Br 1360/12-1).