Exploring the stereostructural requirements of peptide ligands for the melanocortin receptors and molecular mechanism study of GPCR based drugs

Abstract

A central goal of modern biology is to develop a detailed, predictive understanding of the relationships of three-dimensional structure and biological function. We are attempting build this relationship by combining interdisciplinary work. The dissertation is divided into two parts. In the first part of work, numerous structure-activitiy relationships (SAR) studies of different conformationally constrained peptides and peptide mimetics of human melanotropins have been accomplished and discussed. Through this very tedious hard work, selective agonists and antagonists for each subtype of melanocortin receptors have been obtained. We first started investigating the NMR 3D pharmacophore of agonist and antagonist of human melanotropin based on the NMR structure of AGRP ( PDB: 1HYK) and MTII. After a long struggle with appropriate force fields and calculation methodologies, almost identical structures were obtained for MTII as well as SHU-9119 by employing two different techniques of LLMOD (Large scale Lower Modes of Modeling) and NMR. Combining the existing SAR data with this new modeling approach, a series of linear and cyclized peptides (hybridization of the pharmacophore of AGRP and MTII) have been designed, synthesized and identified. We have been successful in obtaining selective agonists and antagonists of melanocortin receptors and these new discoveries shed new insight into peptide or nonpeptide selective drug design for the future. The second part of the dissertation mainly covers human melanocortin receptor (hMCRs) studies. For the purpose of screening the novel peptides and nonpeptides of melanotropins, a series of human melanocortin receptors have been stably transfected into HEK 293 cell line, and new high throughput screening methodologies have been set up. For the purpose of purification of the melanocortin receptors, hMC4R and hMC1R, -His-tag-flag stably transfected into HEK293 cell lines have been designed and applied in receptor purification. We also further studied the mechanism of selective pathway of melanotropin in the HEK293 cells. It was found that agonist mediated internalization of all subtypes of melanocortin receptors are dependent upon beta-arrestin mediated clathrin coated pits, and on the contrary, beta-Arrestin2-GFP recruitment is not dependent on PKA activation. The two-photon fluorescence laser scanning microscopy is a fast, powerful method to study the molecular mechanisms of G protein coupled receptor regulation. In addition, this technique also can serve as a rapid, real-time screening method to differentiate between agonists and antagonists irrespective of any knowledge of their intracellular functional properties (orphan receptors).