Allosteric modulators restore orthosteric agonist binding to mutated CB1 receptors

Abstract

Objectives: To determine if diminished orthosteric agonist binding due to mutations in extracellular loops 1 or 2 of the cannabinoid receptor 1 (CB1) can be overcome by an allosteric modulator and restore agonist binding. Methods: Binding assays were performed using a range of concentrations of orthosteric compound, in the presence or absence of a set concentration of the allosteric modulator PSNCBAM-1 to determine the EC50 in its absence or presence. Key findings: Single mutations in extracellular loop 1 or 2 of CB1 showed weak or no binding of agonist CP55940 to the receptor. Interestingly, upon addition of the allosteric modulator PSNCBAM-1, this binding was restored typically to wild-type CB1 levels. In a few cases, the allosteric modulator ORG27569 was compared with PSNCBAM-1 for CP55940 binding and it also restored binding. Further, wild-type levels of inverse agonist bound the CB1 mutants in the absence of modulator, suggesting the mutants were originally folded like the wild type. Conclusions: Based on our findings, we provide evidence of a therapeutic application for allosteric modulators in situations where a mutation in the receptor may hinder its function. By utilizing allosteric modulators, restoration of orthosteric binding may be possible.