Science 357, 503–507 (2017)

Credit: AAAS

DREADD technology is widely used to manipulate neuronal activity in whole animals, and it involves 'designer' receptors based on human muscarinic receptors hM3Dq or hM4Di that have been mutated to be specifically activated by an inert and inactive metabolite of clozapine, CNO, but not endogenous ligands. Unfortunately, CNO is converted to clozapine in vivo, thus complicating analysis and the clinical use of DREADDs. To assess the extent of this liability and to understand the mechanism of action of CNO at DREADD receptors, Gomez et al. have tested CNO binding and activity in cells and rodent brain tissue by using ligand-binding assays and autoradiography. Clozapine bind and activate DREADDs, but CNO do so only at concentrations much greater than those generally used in DREADD experiments. PET imaging has revealed substantial brain uptake of clozapine but not CNO, thus suggesting that a combination of CNO conversion to clozapine, high clozapine brain permeability and high-affinity clozapine binding accounts for DREADD activation. Further experiments supporting this model have shown that the biological effects of DREADD activation require an ∼100-fold-higher dose of CNO than clozapine, an effect aligned with the extent of conversion of CNO to clozapine. These results suggest that DREADDs require subthreshold clozapine levels for activation and may be useful as CNS disease therapeutics.