Solution conformations of potent bicyclic antagonists of oxytocin by nuclear magnetic resonance spectroscopy and molecular dynamics simulations

The solution conformations of two potent bicyclic antagonists of oxytocin (H-Cys¹-Tyr²-Ile³-Gln⁴- Asn⁵-Cys⁶-Pro⁷-Leu⁸-Gly⁹-NH₂, OT). [Mpa¹,cyclo(Glu⁴,Lys⁸)]OT, and [dPen¹, cyclo(Glu⁴,Lys⁸)]OT were studied by a combined use of ¹H and ¹³C NMR spectroscopy in DMSO and molecular dynamics (MD) simulations. NMR data have suggested a model for the three- dimensional (3D) structure of the bicyclic analogues of OT (OT-BC) with a β- turn at the Tyr² and Ile³ residues, and with a cis amide bond between Cys⁶ and Pro⁷. A 3D structure containing a type III β-turn at Tyr²-Ile³ has been shown to be consistent with NMR data. This structure was proposed as a model of the solution conformation of OT-BC and extensively tested by MD simulations with the AMBER force field. MD simulations at 300 K with NMR derived distance and Φ torsion angle constraints demonstrated the consistency of this model with NMR data, and its stability was further demonstrated by non-constrained MD simulations. Dynamic properties of the 3D structure were explored by high-temperature MD at 500 K. Conformational transitions induced by a constrained rotation around the S-S bond revealed relatively low potential energy barriers 130 to 50 kJ/mol) between equilibrium left-handed and right-handed conformers of the disulfide bridge in OT-BC. A dynamic model of the solution structure of OT-BC with the relatively stable backbone conformation and a fast conformational equilibrium in the disulfide bridge and lactam bridge moieties was proposed as a result of the extensive MD simulations. The solution structure of OT-BC is consistent with structure-activity relations of peptide and non-peptide antagonists of OT. In particular, a β-turn at Tyr²-Ile³ seems to be the common feature responsible for antagonist interaction with the uterine receptor of OT. On the other hand, the 3D structure of OT-BC differs considerably from the crystal and solution structures of OT analogues with agonist activity. Therefore, this study supports the hypothesis of different modes of receptor binding for agonists and antagonists of OT. The model of 3D structure of OT-BC proposed in this study may be used as a template for the rational design of peptide and non-peptide antagonists of oxytocin.