Fragment-based identification of phosphatase inhibitors

Chapter 1. A new fragment-based method for the identification of phosphatase inhibitors, Substrate Activity Screening, is described. Application of the method to Mycobacterium tuberculosis protein tyrosine phosphatase PtpB resulted in the identification of novel, nonpeptidic substrate scaffolds that were optimized by rapid analog synthesis and evaluation. These substrate scaffolds were then converted to low molecular weight inhibitors for PtpB by incorporation of a variety of established phosphate mimetics, resulting in nanomolar affinity inhibitors that were highly selective for PtpB over mycobacterial and human phosphatases.;Chapter 2. The design and synthesis of new inhibitor analogs based on the scaffold identified in Chapter 1 is described. The synthesis of more challenging inhibitor scaffolds was achieved, resulting in a panel of low molecular weight, nanomolar to micromolar affinity inhibitors for the Mycobacterium tuberculosis protein tyrosine phosphatase PtpB. These compounds represent chemical tools for further dissection of the biochemical role of PtpB in tuberculosis infection.;Chapter 3. Application of the method described in Chapter 1 to Mycobacterium tuberculosis protein tyrosine phosphatase PtpA is described. Inhibitors incorporating a well established phosphate mimetic were explored, resulting in compounds with low micromolar affinity for PtpA. Modeling studies provided a rationale for the observed structure-activity relationships and guided further compound optimization. The most potent compound was additionally shown to be selective for PtpA over a variety of human enzymes as well as the other Mycobacterium tuberculosis phosphatase PtpB. This inhibitor represents a chemical tool that can be used in conjunction with the inhibitors described in Chapters 1-2 to further probe the role of PtpA and PtpB in tuberculosis infection, and to examine potential synergistic effects.;Chapter 4. The application of inhibitors developed in Chapters 1-3 to the pathogenic target Staphylococcus aureus, the causative agent of Staph infection, is described. Several of the inhibitors described in Chapter 1 were found to have activity versus Staphylococcus aureus bacteria, prompting further probing of structure-activity relationships. The synthesis of new analogs was realized by developing a new synthetic strategy to allow for rapid analog synthesis and evaluation. The cellular target is postulated to be Staphylococcus aureus protein tyrosine phosphatases SaPtpA and SaPtpB, newly discovered enzymes which may play a role in pathogenesis. Compounds were evaluated directly in cell assays, and the mechanism of action of these compounds, which show activity in Staphylococcus aureus strains that are resistant to traditional beta-lactam antibiotics, is under investigation.