Metadynamics in the design and conformational characterization of isoDGR based αvβ3 antagonists

Integrins play crucial roles in mediating tumour angiogenesis, therefore gaining increasing importance as
drug targets in antiangiogenic cancer therapy. The peptide sequence Arg-Gly-Asp (RGD) is by far the most
prominent ligand to promote specific cell adhesion through integrin stimulation and this sequence has been
used as a lead for developing different integrin antagonists. Recent biochemical studies have shown that the
deamidation of the NGR sequence gives rise to isoDGR, a new αvβ3-binding motif. To design low-molecular
mass RGD or isoDGR-containing molecules, the determination of their biologically active conformation is a
prerequisite. However, experimental measurements are usually insufficient to draw conclusions concerning
the nature of all relevant conformers affecting binding affinity. As computational drug design becomes
increasingly reliant on virtual screening and on high-throughput 3D modeling, the need for fast, robust, and
reliable methods for sampling molecular conformations is warranted. We have successfully developed an
innovative application of MetaDynamics (MtD), a fast computational method to efficiently sample the free
energy of complex polyatomic systems in the space of a few course-grained quantities named collective
variables (CVs), which allows to rapidly and exhaustively describe the whole conformational space
accessible to these peptides. The method was applied to analyze a small library of RGD- and isoDGRcontaining
cyclopeptides to a) identify the highest populated conformations, b) predict their flexibility, and c)
obtain reliable structural models that can be docked inside the receptor. Choosing the phi and psi angles of
the central glycine as CV, we discriminated among αvβ3-binding and non-binding cyclopeptides. In addition,
we demonstrated that the method is able to predict effect of chemical modifications, including metylation and
acetylation, to affect flexibility and conformation of the peptides.
In conclusion, these findings may have an important impact in the discovery and characterization of new
diagnostic and therapeutic agents based on the isoDGR motif, providing support for the rationale design and
optimization of isoDGR peptidomimetics, for the fine-tuning of their conformations to increase receptor
selectivity, and for selection of ligands for chemical synthesis.