Modelling protein-protein interactions

Protein-protein interactions are fundamental to many biological processes, from signal transduction to cytoskeleton assembly and are therefore important targets for pharmaceutical research1. Particular attention is given here to the network of interactions occurring between tubulin dimers, in order to understand the nature of their association to form microtubules (MTs), polymeric cellular structures involved in mitosis. Given their primary importance in cell division, microtubules are interesting targets for antimitotic agents to be used in cancer therapies2. Currently, most of the MTs targeted drugs in use derive from screenings of natural compounds or their modifications. Following a different approach, we present here an in silico design of novel peptides able to interfere with MTs dynamic, thus acting as inhibitors of cancer cells proliferation. A molecular dynamics (MD) simulation was carried out on a system composed of two tubulin dimers in explicit water solvent, followed by interdimer binding energy evaluations and computational alanine scanning3 of the interface residues. It resulted that the binding energy is not evenly distributed over the protein-protein interface, but is concerntrated on some crucial amino acids, determinant for subunits association, defined as “hot-spots”. A subsequent simulation of a tubulin tetramer in complex with a vinblastine molecule in its active site pointed out how this antimitotic agent strongly reduces the number of interactions, thus explaining its microtubule destabilizing properties. Protein subsequences including a number of hot-spots were then used as a starting point for the development of peptides that could target different sites on tubulin with respect to vinblastine and other traditional chemoteherapeutic agents. The so designed peptides underwent further MD simulations in complex with tubulin, in order to evaluate if they conserved binding ability even when no longer inserted in the protein structure. The most promising ones were then sinthesized and underwent biological tests proving their ability to effectively alter MTs morphology and stability.