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

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. Unlike ORG27569 (a negative allosteric modulator of CB1), lipoxin A4 does not significantly displace SR141716A (an orthosteric inverse agonist) in equilibrium binding assays. In addition, we have previously reported that ORG27569's binds in the THM3/TMH6/TMH7 region (Shore et al., ICRS, 2012); at this site, ORG27569 sterically blocks important movements of the second and third extracellular 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. Together, these results may suggest that lipoxin A4 binds in a topographically different region of CB1 than ORG27569. To identify lipoxin A4's binding site(s) at CB1, we are using the Forced-Biased Metropolis Monte Carlo simulated annealing program, MMC. In this method, lipoxin A4 was separated into 4 fragments. Four MMC runs are currently being 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 is then systematically annealed, causing only those fragment copies with the best free energy of binding to the protein surface to remain. Ultimately, analysis of these four MMC runs will suggest region(s) in which all four fragments cluster in the correct spatial proximity, thus suggesting a possible binding site(s). [Support: RO1 DA003934 and KO5 DA021358 (PHR)]