Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe

Robert B. Laprairie, Abhijit R. Kulkarni, Pushkar M. Kulkarni, Dow P. Hurst, Diane Lynch, Patricia H. Reggio, David R. Janero, Roger G. Pertwee, Lesley A. Stevenson, Melanie E. M. Kelly, Eileen M. Denovan-Wright, Ganesh A. Thakur*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)


One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psycho-behavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CBIR allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [S-35]GTPyS and beta-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [S-35]GTP gamma S assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for beta-arrestinl recruitment, PLC beta 3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CBIR allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CBIR allosteric space.

Original languageEnglish
Pages (from-to)776-798
Number of pages23
JournalACS Chemical Neuroscience
Issue number6
Early online date4 Apr 2016
Publication statusPublished - 15 Jun 2016


  • Allosteric covalent probe
  • allosteric site
  • biased signaling
  • binding domain
  • cannabinoid 1 receptor
  • cysteine
  • functional selectivity
  • G protein-coupled receptors
  • homology modeling
  • isothiocyanate
  • ligand bias
  • ligand-binding motif
  • negative allosteric modulator
  • 7-transmembrane receptor
  • signal transduction
  • therapeutics discovery
  • protein-coupled receptors
  • cell-culture-model
  • CB1 receptor
  • drug discovery
  • endocannabinoid system
  • agonist activity
  • biased agonism
  • ligand-binding
  • covalent drugs


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