Discovery of small molecules that regulate the activity of protein tyrosine phosphatases
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes responsible for dephosphorylating phosphotyrosine residues through a phosphocysteine intermediate. PTPs work with protein tyrosine kinases to regulate a number of essential signal transduction pathways. The inhibition or inactivation of these enzymes can lead to profound biological consequences. So PTP inactivators are of widespread interest in medicinal chemistry and cellular biology. PTP1B is a protein tyrosine phosphatase involved with insulin and leptin signaling and plays a key role in down-regulating both the insulin and leptin receptor signaling pathways. Thus, PTP1B inactivation is expected to be useful in the treatment of type 2 diabetes, obesity, and the metabolic syndrome. Hydrogen peroxide is now recognized as an important cellular signaling agent. Peroxide-mediated inactivation of target protein tyrosine phospatases (PTPs) regulates the duration and intensity of cellular response to various stimuli including growth factors, cytokines, and hormones. Loss of PTPs activity in cells during H2O2-mediated signal processes is rapid. Curiously, however, kinetic measurement on isolated PTPs suggest that, at the low concentration expected to be generated during cell signaling processes (0.1-1 μM), H2O2 is a rather sluggish PTP inactivator. We have suggested that the discrepancy might be explained by spontaneous or enzymic conversion of H2O2 to a more reactive peroxidizing agent that is capable of rapid PTP inactivation. Along these lines, in Chapter 1 we set out to explore whether equilibrium amounts of peroxymonocarbonate generated by the reaction of H2O2 with the biological bicarbonate/CO2 buffer system is an efficient PTP inactivator that might be involved in signal transduction. Indeed, we provide evidence that peroxymonocarbonate is a potent PTP inactivator. The reaction with physiological concentrations of bicarbonate/CO2 allow low micromolar concentrations of H2O2 to inactivate PTP enzymes within the 10-15 min timeframe that is relevant to cell signaling events. Heterocyclic N-oxides have the potential to serve as phosphotyrosine mimics binding at the active site of PTPs. In addition, N-oxides have the potential to serve as mild, cell-stable oxidants that can inactivate PTP1B by reaction with active site thiol. Chapter 2 will discuss the synthesis of N-oxides and assessment of their ability to inactivate PTPs. Some analogs of 3-alkyl-1,2,4-benzotriazine 1,4-dioxide show inactivation towards PTP1B. At the same time, these agents show good stability under physiological conditions. A summary of structure-activity relationships and a proposed mechanism for the inactivation of PTP1B by 3-alkyl-1,2,4-benzotriazine 1,4-dioxide was provided. In Chapter 3, we found that nucleophiles (2,6-dimethylaniline, Nhydroxyphthalimide, methoxylamine, benzylalcohol, aniline, 4-aminophenylacetic acid, phenylhydroxamic acid and benzylamine) can react with the sulfenyl amide form of PTP1B and that capture of the enzyme prevents subsequent reactivation by thiol. We proposed the reaction mechanism between different necleophiles and PTP-sulfenyl amide forms. During this project, we also found some inactivators (disulfide of 2- mercaptoimidazole, sodium thiolsulfate, 4-mercaptobenzoic acid and thioacetic acid) of PTP1B and we proposed possible mechanism for the inactivation. Also we found two PTP1B inhibitors (sodium thiophosphate and thiosalicylic acid) when we carried out the thiol reactivation assays.
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