Study ligand binding to protein-tyrosine phosphatase 1B and pp60c-src SH2 domain by free energy simulations

Signal transduction by means of ligands containing phosphorylated tyrosine is an important biological cycle found in all eukaryotic organisms. Multiple proteins participate in it. Malfunctions of this cycle are responsible for various illnesses, including some forms of cancer. For the investigation of this cycle, as well as for drug development, selective inhibitors (artificial ligands) that do not disrupt the complete signaling pathway are of the utmost importance. The physical parameter for the quantitative description of the affinity of a ligand or inhibitor for a protein receptor is the binding free energy. Understanding the rules how this binding free energy depends on chemical modifications of the ligand is of great theoretical, as well as practical interest since the knowledge of these relationships permits the systematic development of potent and selective inhibitors. In this project it is planned to investigate the binding affinity of several mimetics of phosphorylated tyrosine to two different receptor proteins (protein tyrosine phosphatase 1B (PTP1B) and pp60c-src SH2 domain) by computer simulations. Using molecular dynamics (MD) simulations makes it possible to calculate the difference of binding free energies of two ligands, as well as the difference of their solvation free energies. The analysis of such computer experiments permits a better understanding of the relationships between the free energy of a complex and its structure. The detailed questions addressed by the project concern (i) the effect of selective fluorination on the binding affinity and selectivity of ligands, and (ii) the factors determining the selectivity of ligands for different receptors. Further, for PTP1B it is planned to investigate (iii) the conformational change of the so-called WPD loop, which is observed upon binding of most ligands, as well as (iv) to investigate the binding affinity of phosphotyrosine and two mimetics to a recently discovered secondary binding site. Aside from this application oriented part the project also encompasses methodological development: For a number of calculations it is necessary to simulate conformationally highly flexible ligands (in particular peptides). For such systems special techniques are required to guarantee sufficient sampling of phase space while keeping the computational effort acceptable. Although the theory of coupling such methods with the MD based calculation of free energy differences has been described in the literature, it has not yet been applied in practice to complex, biologically relevant systems. The development of the required computer code, as well as of reliable protocols represents an important part of the project. Such methods are of great interest for various problems; their relevance reaches far beyond the concrete applications planned for this project.