Identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p56 Lck SH2 domain via in silico screening against the pY + 3 binding site.

The protein p56 lymphoid T cell tyrosine kinase (Lck) is predominantly expressed in T lymphocytes where it plays a critical role in T-cell-mediated immune response. Lck participates in phosphotyrosine-dependent protein-protein interactions through its modular binding unit, the Src homology-2 (SH2) domain. Accordingly, virtual screening methods combined with experimental assays were used to identify small molecular weight nonpeptidic compounds that block Lck SH2 domain-dependent interactions. Virtual screening included scoring normalization procedures and postdocking structural clustering that is shown to facilitate the selection of active compounds. By targeting the well-defined hydrophobic binding pocket known to impart specificity on Lck-protein interactions (i.e., pY + 3 site), inhibitors of the Lck SH2 domain were discovered that omit the phosphotyrosine (pY) or related moieties. The 34 out of 196 computationally selected compounds were shown to inhibit Lck SH2 domain association with phosphorylated immunoreceptor tyrosine based activation motifs peptide. Twenty-four of the active compounds were further tested for their ability to modulate biological function. Thirteen of these compounds showed inhibitory activity in mixed lymphocyte culture assay. Fluorescence titration experiments on four of these active compounds further verified their binding to the SH2 domain. Because of their simple chemical structures, these small organic compounds have the potential to act as lead compounds for the development of novel immunosuppressant drugs.