News & Events
Posted on October 19, 2016
Date - October 19, 2016
Ph.D. Thesis Defense – 752
Title: “Construction and Validation of GPR55 Active and Inactive State in Silicomodels through the Use of Biological Assays, Mutation Data and Structure Activity Relationships”
The number of experimentally solved structures of G protein-coupled receptors (GPCRs) has increased rapidly in the last few years with the advent of numerous technical advancements in membrane protein crystallization. Though the structures of the two known cannabinoid receptors have yet to be crystallographically determined, prior work done in the Reggio lab over the last fifteen years has produced highly accurate homology models of both the CB1 and CB2 receptors. GPR55, newly deorphanized and ostensibly the third cannabinoid receptor, has neither been crystallized nor has its contradictory pharmacology been completely untangled. To date, few GPR55 specific, low-nanomolar potency ligands have been produced to elucidate the interactions which occur within the binding pocket of this receptor. This receptor is of therapeutic interest because it has been shown to have a role in an array of physiological and pathological processes, including inflammation and pain synaptic transmission, bone development and cancer.1
This research details construction of both a GPR55 R and R* model to be used as tools to address the aforementioned issues. Validation of the current GPR55R* model will be discussed in detail using a combination of in silico experimentation, in vitro single point mutations and ligand docking. The model was initially constructed using the 1.8 Å crystal structure of the human delta opioid receptor (hDOR) as the template. Modifications were made to refine the model to reflect any important GPR55 sequence differences from hDOR. These models have been instrumental in helping to predict the overall three dimensional structure of GPR55 and the specific residues it uses for ligand recognition. The present representation of this receptor has several residues which, pointing into the interior of the binding pocket, we had hypothesized would interact with GPR55 agonist ML184 (3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide, CID2440433). The importance of these key residues, H170, K2.60, E3.29, Y3.32, M3.36 , F6.48 and F6.55 to ML184 signaling were tested in mutation studies performed in Dr. Mary Abood’s lab. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 at GPR55. Y3.32F, H(170)F and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32L, M3.36A and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. Taken together, these results provide the first set of discrete data on residues important for ML184 signaling and for GPR55 activation.
The entirety of this research will aid in the rational design of next generation ligands for GPR55 and should lead to the creation of the first high affinity radioligand for this receptor. There is a profound need for a GPR55 radioligand, and creation of such, coupled with the employment of the homology models as predictive tools, will allow research to move forward more rapidly towards the development of this receptor as a therapeutic target.