Deciphering the class B GPCR activation mechanism

Abstract

Abstract of thesis entitled DECIPHERING THE CLASS B GPCR ACTIVATION MECHANISM

Submitted by Singh Kailash

For the degree of Doctor of Philosophy At The University of Hong Kong In February 2019

G-protein coupled receptors (GPCRs) represent the largest gene family functioning as molecular signaling devices. Evolutionary significance of these receptors can be explained by their early existence in unicellular eukaryotes to the most evolved human genome. Our lab has recently identified the most primitive form of pituitary adenylyl cyclase activating polypeptide (PACAP)/glucagon (GLUC) and parathyroid hormone (PTH) receptor subfamilies as well as their endogenous ligands from an invertebrate, amphioxus. A comparative approach is commonly used to investigate GPCR evolution. Comparison of the tissue distribution and the structures between these receptors of invertebrate and vertebrate species provides an insight into how communicating systems are built to support the complicated operation of multiple tissues. In the human genome, PACAP, GLUC and PTH receptors belong to class B1 GPCR family. Therefore, in the quest for a better comprehension of the evolution of this GPCR family from a structural perspective, we have developed the full receptor homology models of amiphioxus (PACAP)/glucagon receptor, as well as human secretin, glucagon and calcitonin receptors by the homology modeling approach. Using this approach, we provide the full receptor models of an invertebrate and mammalian Class B GPCRs, since full receptors structure for the same have not yet been solved by X-ray crystallization and/or NMR. Furthermore, the binding site of the peptide agonist was identified through a virtual docking algorithm. Our results suggest that the ligand binding motif has shifted more into the extracellular domain of the invertebrate receptor, while ligands of the mammalian receptors interact with both the extracellular domain and the transmembrane region of the GPCR. Based on this structural information, we hypothesize that such a shift of ligand binding site minimizes the sequence variation required to enable the evolution of primitive receptor into diverse functioning receptors. As a result, diverse physiological functions of several Class B1 GPCR were evolved from a single ancestral receptor. After reviewing the key phylogeny taxa-conserved motifs or domains for ligand binding and intracellular signaling of the receptors, invaluable information for drug design was obtained. We then utilized this structure-activity relationship data along with the full receptor model to develop small compound modulators for secretin receptor (SCTR). SCTR is known to be involved in many physiological functions including HCO3- release from the pancreas, fatty acid absorption, and water homeostasis. It is also suggested to play role in improving cardiac function and can mediate insulin secretion. Therefore, secretin receptor is considered as a potential therapeutic target to be exploited for several diseased states including gastritis, acidity, and gastrointestinal ulcers, pancreatitis and related disorders, liver cirrhosis and hepatoma, asthma and bronchitis, water homeostasis, as well as obesity. We have selected one small compound allosteric agonist for SCTR through series of in-silico experiments followed by in-vitro and in-vivo functional activity testing. We have validated the physiological effect of the compound by intra-cerebroventricular injection which leads to an increase in plasma vasopressin levels after 30 minutes of treatment. Similarly, its effect on the reduction of blood pressure was observed by intravenous administration of drug in spontaneously hypertensive rat (SHR) models, signifying the antihypertensive potency of the drug similar to that of secretin peptide, but with longer biological half-life and response.