G Protein-coupled Receptors (gpcr)

Introduction: G protein-coupled receptors (GPCR) are a well-known broad group of membrane receptors which are among the most significant therapeutic targets. Activation of GPCRs by extracellular stimuli, initiates the signal transduction that eventually leads to the response. Crystal structure of GPCRs by crystallization protocol is hard to obtain, because it is hard to remove and purify them from the membranes. One of the GPCRs that has crystal structure determined is P2Y12 (PDB: 4PXZ). P2Y12 is one of the main GPCR whose work is to stabilize the platelet aggregation in blood cloth forming, making this receptor as an essential target for current antiplatelet therapy [1]. In my proposed research, I have targeted G protein-coupled receptor 171 (GPR171), which is recently discovered as a receptor for modulation of appetite and metabolism in mice. The biological role of GPCR171 and gene that is coding for it (GPR171), is received a lot of attention lately, and it is found to involve feeding and metabolism in mice [2] and myeloid differentiation [3]. It was also found that GPR171 is overexpressed in the lung cancer tissue, suggesting him to be a target of promise for the development of antineoplastic drugs. [4]

Since GPR171 is expressed in the part of the brain that is responsible for psychological disorders, recently, it was found that it may be used as a novel target to develop anxiolytics.[5] Up until recently, GPCR171 was thought to be an orphan receptor, because its natural agonist BigLen is discovered not long ago. BigLen is a 16-amino acid neuropeptide (LENSSPQAPARRLLPP) which acts as a natural agonist of GPCR171 in hypothalamus. Its binding affinity for the GPCR171 is high (low Kd= 0.5 Nm). The structural requirement of the BigLen that is essential for the binding to its target was found by the drug design approach named drug simplification i.e. by cutting the endopeptide piece by piece. Finally, after many tests, it was found that only four amino acids (LLPP) of the C-terminus are enough to trigger the receptor [2]. These findings initiated the development of small chemical ligands which can selectively activate or deactivate the GPR171. Since there are no crystal structure of the GPR171, homology modeling is an alternative option. For cooperation was used P2Y 12 receptor, because they are phylogenetically related as found by comparing their homologous sequences.

The mechanism of action of the P2Y12 receptor demonstrates that the ADP binding in the extracellular domain of the receptor initiates the cascade of events inside the cell. The examination of the P2Y12 crystal structure revealed the binding pocket of this receptor has Histidine side chain as an essential for receptor activity. This was proven by the fact that when Histidine is replaced by Glutamine, the activity of the receptor is disturbed.[1] In ligand binding assays, it was found that ligands like 2MeSADP, ADP, and ADPβS which binds the P2Y12 receptor are not active towards the GPCR171 as they do not displace its natural ligand BigLen. These observations suggested that there is a significant structural difference in the binding site of GPR171 and P2Y12. However, the atomic coordinates from PDB file 4PXZ are all that is available to thread the GPR171 amino acid sequence. Fig. 1. Chemical structure of MS0015203 (Left) and its possible binding sites in the GPR171 receptor (Right).

There are various amino acid side chains present in the binding pocket of GPR171 as shown in the above figure which plays a major role in the bond making with MS0015203. (Adopted from Ref.6) Molsoft ICM-Pro Software was used to find out best fit of small chemical structure to the binding site of GPR171 by analyzing thousands of molecules from the database. After this rigorous screening process, MS0015203 was found as a promising candidate who can act as an agonist on GPR171. [6] Figure 1 shows a chemical ligand binding interaction between GPR171 and compound MS0015203. Molecule MS0015203 was found to be a partial agonist of GPR171 when checked through in vivo experiment. It was found that after the peripheral injection of MS0015203, food intake in mice is significantly increased. This effect is shown to be reversible through shRNA-mediated knockdown of hypothalamic GPR171. This result suggested that targeting the GPR171 may be of use for managing body weight and metabolism in mammals [2].