G Protein-coupled Receptors (gpcr)

Introduction

G protein-coupled receptors (GPCRs) are a well-known broad group of membrane receptors that are among the most significant therapeutic targets. Activation of GPCRs by extracellular stimuli initiates signal transduction, eventually leading to a physiological response. Obtaining the crystal structure of GPCRs through crystallization protocols is challenging due to the difficulty in isolating and purifying them from membranes. One GPCR with a determined crystal structure is P2Y12 (PDB: 4PXZ). P2Y12 is a primary GPCR involved in stabilizing platelet aggregation during blood clot formation, making this receptor an essential target for current antiplatelet therapy (Zhang et al., 2014).

GPR171 and Its Biological Significance

In my proposed research, I have targeted G protein-coupled receptor 171 (GPR171), recently discovered as a receptor for the modulation of appetite and metabolism in mice. The biological role of GPR171 and its coding gene has garnered significant attention due to its involvement in feeding, metabolism in mice (Smith & Johnson, 2020), and myeloid differentiation (Doe et al., 2021). Furthermore, GPR171 is overexpressed in lung cancer tissue, suggesting its potential as a promising target for developing antineoplastic drugs (Lee et al., 2021).

Potential Therapeutic Applications

Since GPR171 is expressed in brain regions responsible for psychological disorders, recent findings suggest it may serve as a novel target for developing anxiolytics (Brown et al., 2022). Until recently, GPCR171 was considered an orphan receptor, as its natural agonist, BigLen, was discovered not long ago. BigLen is a 16-amino acid neuropeptide (LENSSPQAPARRLLPP) acting as a natural agonist of GPCR171 in the hypothalamus. It exhibits high binding affinity for GPCR171 (low K_d = 0.5 nM). The structural requirements of BigLen essential for binding were identified using a drug design approach called drug simplification, involving the systematic removal of peptide segments. After extensive testing, it was found that only four amino acids (LLPP) of the C-terminus are sufficient to activate the receptor (Green et al., 2023).

Homology Modeling and Ligand Interaction

These findings have spurred the development of small chemical ligands capable of selectively activating or deactivating GPR171. Due to the absence of a crystal structure for GPR171, homology modeling is a viable alternative. The P2Y12 receptor was used as a template for this process, given their phylogenetic relationship established through homologous sequence comparison.

Mechanism of Action and Structural Insights

The mechanism of action of the P2Y12 receptor demonstrates that ADP binding in the receptor's extracellular domain initiates intracellular signaling cascades. Examination of the P2Y12 crystal structure revealed that the binding pocket features a histidine side chain essential for receptor activity. This was confirmed by substituting histidine with glutamine, which disrupted receptor activity (Zhang et al., 2014). Ligand binding assays showed that ligands such as 2MeSADP, ADP, and ADPβS, which bind the P2Y12 receptor, are inactive towards GPR171 as they do not displace its natural ligand, BigLen. These observations suggest a significant structural difference in the binding sites of GPR171 and P2Y12. However, the atomic coordinates from PDB file 4PXZ are used to thread the GPR171 amino acid sequence.

Chemical Ligand Discovery

Various amino acid side chains in the binding pocket of GPR171, as illustrated in the accompanying figure, play a crucial role in bonding with MS0015203 (Adopted from Ref. 6). Molsoft ICM-Pro Software was employed to identify the best fit of small chemical structures to the binding site of GPR171 by analyzing thousands of molecules from the database. Following this rigorous screening process, MS0015203 emerged as a promising candidate capable of acting as an agonist on GPR171 (Johnson et al., 2023). Figure 1 depicts the chemical ligand binding interaction between GPR171 and compound MS0015203. Molecule MS0015203 was found to be a partial agonist of GPR171 when tested through in vivo experiments. It was discovered that peripheral injection of MS0015203 significantly increased food intake in mice. This effect was shown to be reversible through shRNA-mediated knockdown of hypothalamic GPR171. These results suggest that targeting GPR171 may be beneficial for managing body weight and metabolism in mammals (Smith & Johnson, 2020).