Discovery of Homobivalent Bitopic Ligands of the Cannabinoid CB₂ Receptor**

Abstract

Single chemical entities with potential to simultaneously interact with two binding sites are emerging strategies in medicinal chemistry. We have designed, synthesized and functionally characterized the first bitopic ligands for the CB 2 receptor. These compounds selectively target CB 2 versus CB 1 receptors. Their binding mode was studied by molecular dynamic simulations and site-directed mutagenesis.

N-Methyl-D-aspartate (NMDA) and cannabinoid CB2 receptors form functional complexes in cells of the central nervous system: insights into the therapeutic potential of neuronal and microglial NMDA receptors

“…Multiple modes of cannabinoid binding to CB 2 R lead to specific receptor conformations underlying functional selectivity (biased agonism) [ 40 ] and, ultimately, differentially regulating NMDAR function. As an example, we have designed bitopic ligands that bind to an exosite located at the entrance of the structure that connects the orthosteric site with the lipid bilayer [ 41 ]. These findings constitute a selective advantage since the expression of CB 2 -NMDA-Het increases in neurons and microglia of APP Sw/Ind transgenic mice.…”

The Binding Mode to Orthosteric Sites and/or Exosites Underlies the Therapeutic Potential of Drugs Targeting Cannabinoid CB2 Receptors

“…This report shows that CBD can bind to an allosteric cavity close to the receptor entrance in a transmembrane portal defined by transmembrane helices 1 and 7. As aforementioned, concomitant binding at orthosteric and allosteric/exosites has been shown at CB 2 R with chromenopyrazole bitopic ligands ( Morales et al, 2020 ). Site-directed mutagenesis and molecular dynamic studies determined key interacting residues at transmembrane helices 1 and 7 which define the entry portal for these ligands ( Figures 1C,D ).…”

“…Bitopic ligands designed according to these findings improve subfamily selectivity ( Medina et al, 2014 ; Masureel et al, 2018 ); they also offer signaling bias and better off-rates ( Valant et al, 2012 ; Lane et al, 2013 ). Knowing that unlike GPCRs for polar compounds, CBRs do not have the orthosteric center accessible from the extracellular milieu, we designed bitopic ligands able to enter into the CB 2 R orthosteric site but also able to interact with amino acids located at the receptor transmembrane portals ( Morales et al, 2020 ). Signaling assays in the CB 2 R wild-type and specific mutants led us to discover the first CB 2 R bitopic ligands.…”

“…(B) View from the outside of the cell of the CB 2 R in complex with the agonist AM12033 (PDB-ID 6KPF); ligand access from extracellular is blocked by the N-terminus and the EC loops. (C) General view of the binding mode of a CB 2 R bitopic ligand [molecule 22 in ( Morales et al, 2020 )] into the orthosteric site and the vestibule of the CB2R-Gi complex (depicted as cylinders for CB 2 R and grey surfaces for Gi). (D) Detailed view of the binding mode of ligand 22 into the receptor vestibule obtained during the MD simulations.…”

“…TMs 1 and 7 are shown in orange and blue, respectively; and the pharmacophore units and spacer of bitopic ligands are shown in green and yellow tubes, respectively. (C,D) have been reproduced from our previously reported article ( Morales et al, 2020 ); permitted reproduction under the terms and conditions of the Creative Commons Attribution (CC BY) license ( https://creativecommons.org/licenses/by/4.0/ ).…”