Leuprolide [DLeu6, NHEt10]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr5(OMe), DLeu6, Aze9, NHEt10]GnRH (1) and [Tyr5(OMe), DLeu6, NHEt10]GnRH (2) have been studied in DMSO and H2O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2-5 segments of Leuprolide and its linear analogues in the case of using DMSO-d6 as solvent, whereas a β-turn type II in the 3-6 segments is indicated using D2O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.

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Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.