Abstract

Inhibition of Protein Tyrosine Phosphatase 1B (PTP1B) has been proposed as a novel therapy to treat type 2 diabetes by sensitising the patients to the effect of insulin while protecting them against diet-induced obesity. A mutant screen in yeast was devised to facilitate the identification of residues involved in inhibitor binding. The screen was based on the observation that overexpression of v-Src, a tyrosine kinase, in yeast leads to lethality through mitotic dysfunction. This was reversed by co-expression of PTP1B whereas co-expression of the catalytically inactive enzyme failed to rescue the v-Src phenotype. Treatment with specific PTP1B inhibitors reversed, in a dose-dependent manner, the rescue effect of the phosphatase and indicated that this screen could be used to identify inhibitors with enhanced bioavailability properties. A library of PTP1B mutants was co-expressed with v-Src in yeast. The clones obtained were tested for their ability to grow in the presence of inhibitor: only PTP1B mutants that had conserved their catalytic activity and were resistant to inhibition grew. This screen revealed resistant mutations absent from the active site loops to concentrate on the {460}7 helix and its surrounding region. This region was further characterized using a mutation of serine 295 to phenylalanine resulting in resistance to a panel of structurally diverse inhibitors. Inhibitor determinants previously identified using X-ray crystallography and site-directed mutagenesis have all been located to the active site. This is the first report that an active-site distal region can modulate inhibition of PTP1B. This novel approach to identify structural determinants of inhibition can be applied to other tyrosine phosphatases and has the advantage of not being biased towards primary shell residues.