Metadynamics simulations rationalize the conformational effects induced by N-methylation of RGD cyclohexapeptides

Cyclopeptides are a promising class of compounds with favourable pharmacokinetic characteristics that can be used as therapeutics in modulation of protein-protein interactions. Nevertheless their application has been limited due to the difficulties to predict in silico their three-dimensional structure and inhibitory activity. Because of these challenges, their optimization for specific biological targets has been mainly based on empirical approaches. Computational tools could be fundamental in accelerating the drug design process, reducing the efforts dedicated to expensive and time-consuming compound synthesis. In this scenario a detailed conformational search of ligands followed by docking calculations is highly recommendable to achieve reliable computational predictability. We have developed a multi-stage computational protocol[1] able to i. predict the affinity of a set of cyclopeptides for different integrins, ii. rationalize the interplay between conformational equilibria and receptor affinity. The protocol relies on the combination of enhanced sampling molecular dynamics technique (Bias-Exchange Metadynamics), docking calculations and re-scoring via Molecular Mechanics/Generalized-Born Surface Area. The reliability of our method was tested investigating the impact of single and multiple N-methylation on the equilibrium conformations of five RGD (Arg-Gly-Asp) cyclohexapeptides that were generated to increase their selectivity towards αIIbβ3 integrin.[2] We obtained excellent results: the conformational sampling was in good agreement with available NMR data and we were able to discriminate between binders and non-binders. Additionally we offered a structural rationale for why N-methylation increases peptides affinities towards a specific integrin. Herein we have shown that Metadynamics can represent a promising in silico screening strategy, opening new perspectives in the application of cyclopeptides as therapeutic inhibitors. We expect that this combination of techniques will be successfully exploited in future to predict the conformational effects of methylation and other chemical modifications in cyclopeptides.