Conformational and docking studies of Gonadotropin releasing hormone and its analogsby NMR spectroscopy and molecular modelling

Doctoral Thesis


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University of Cape Town

Gonadotropin releasing hormone (GnRH) is a decapeptide with blocked amino and carboxy termini and plays a central role in reproduction. Mammalian GnRH has a positively charged Arg8 residue and binds to the mammalian receptor with high affinity. However, the neutral analog Cln8GnRH has very low affinity. The affinity is restored when the Gly6 is replaced by the achiral D-Trp6, or the Gly6 and Leu7 are modified to form a 6,7 y-lactarn. His5Trp7Tyr8GnRH also shows reasonably high affinity. A comprehensive conformational search was carried out using Nuclear Magnetic Resonance spectroscopy and Simulated annealing to identify the bio-active conformations and to explain the different binding affinities of these peptide analogs. The interproton distances and backbone torsion angles obtained from the NMR data were used to constrain the peptides during the simulated annealing. A large number of structures were generated for each peptide and their conformations analyzed. All live peptides showed some degree of flexibility of conformation, mainly in the terminal domains. The four high affinity analogs all had similar backbones with a β type bend around the Glys residue and the two termini in proximity. The Arg8 in GnRH was involved in several hydrogen bonds that stabilized the folded conformation. In contrast, the inactive Gln8GnRH had a markedly different conformation, with the termini pointing away from each other. The lowest energy structures identified from the simulated annealing were used in subsequent receptor-ligand docking studies. All the high affinity analogs were found to fit neatly into the binding pocket. Arg8 of GnRH formed several H-bonds with residues on the receptor. However, Gln8GnRH showed a poor fit and considerable repulsion between ligand and receptor was evident. The Gln8 did not form H-bonds with the receptor. The calculated binding energies were consistent with the relative binding potencies observed for all five analogs studied.

Bibliographical references (pages 125-132)