Identification of an Endogenous Allosteric Modulator's Binding Site at the Human Cannabinoid-1 Receptor, Using Forced-Biased Metropolis Monte Carlo Simulated Annealing Method (MMC) and Molecular Dynamics

Abstract

The CB1 endogenous, positive allosteric modulator, lipoxin A4, increases the equilibrium binding and efficacy of CP55,940 and anandamide (orthosteric agonists), yet has no significant effect when applied alone. We have reported that ORG27569 (a negative CB1 allosteric modulator) binds in the THM3/6/7 region (Shore et al., JBC, 2013); here, ORG27569 sterically blocks movements of the second and third extracellular (EC) loops, as well as those of TMH6, that are necessary for G protein-mediated signaling. Because lipoxin A4 is a positive allosteric modulator, one would not expect it to sterically block these functionally-important conformational changes.To identify lipoxin A4's binding site(s) at CB1, we used the Forced-Biased Metropolis Monte Carlo simulated annealing program, MMC. In this method, lipoxin A4 was separated into 4 fragments. Four MMC runs were performed, in which our in silico CB1 receptor model (with CP55,940 docked) was immersed in a box filled with copies of one of these fragments. The system chemical potential was then systematically annealed, causing only those fragment copies with the best free energy of binding to the protein to remain. MMC results were used as a starting point for Glide automated-docking studies of lipoxin-A4. Molecular dynamics simulations were also performed to study how lipoxin A4 may enter CB1. Here, CB1 was placed in a fully hydrated, POPC bilayer; 14 lipoxin A4 molecules (7 per leaflet) were placed with random orientations, around the receptor. Altogether, these results suggest that lipoxin A4 may bind in the TMH3/6/7, extending extracellularly. Lipoxin A4 may act as a positive allosteric modulator by forming electrostatic interactions with the EC-1 and EC-3 loops, promoting an active loop conformations. [Support: RO1 DA003934 and KO5 DA021358 (PHR)]