Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase.

Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.

Strategies for developing protein tyrosine phosphatase inhibitors.

Protein tyrosine phosphatases (PTPs) play vital roles in numerous cellular processes and are implicated in a growing number of human diseases, ranging from cancer to cardiovascular, immunological, infectious, neurological, and metabolic diseases. Here we present methods for developing small molecule inhibitors for these enzymes, starting with how to set up a high throughput chemical library screening for PTP inhibitors, how to confirm and prioritize hits, and how to circumnavigate possible pitfalls. Next, we present the relatively new hit generating method of in silico or virtual screening. We give an overview of existing software tools, describe how to choose and generate protein target structures and illustrate the procedure with examples. We then discuss how three-dimensional PTP structures can be analyzed in terms of their potential to bind small molecule inhibitors selectively over homologous proteins and how computer tools can be applied for lead optimization efforts. We finish with a perspective of how well these PTP inhibitors might perform as future drugs to treat human disease.

PTPome-wide functional RNA interference screening methods.

The elucidation of the entire complement of genes encoding protein tyrosine phosphatases (PTPs) in human genome, the human 'PTPome', has made it possible to experimentally address the entire family in an unbiased manner. Here we describe a functional RNA interference-based assay, in which we evaluate 87 of the known 107 PTPs for effects on cell survival in a high throughput manner. The details of assay rationale and design, instrumentation, pitfalls, data analysis, and further validation steps are described. We also discuss the suitability of this technology for further assay development and application to other functional read-outs and signaling pathways.

NMR-based techniques in the hit identification and optimisation processes.

In this review, the use of general NMR spectroscopy techniques to detect ligand binding and to monitor enzyme kinetics and inhibition, which appear particularly useful in hit identification and validation, is reiterated. Furthermore, the use of NMR-based strategies for lead optimisations that are based on either iterative derivatisations of an initial core structure or on linking fragments that occupy adjacent pockets in the target's binding site will also be described. Several recent examples will be reported and the use of these techniques in cases when the three dimensional structure of the target protein is known will be discussed.

VHY, a novel myristoylated testis-restricted dual specificity protein phosphatase related to VHX.

The human DUSP15 gene encodes an uncharacterized 235-amino acid member of the subfamily of small dual specificity protein phosphatases related to the Vaccinia virus VH1 phosphatase. Similar to VHR-related MKPX (VHX) (DUSP22), the predicted protein has an N-terminal myristoylation recognition sequence, and we show here that both are indeed modified by the attachment of a myristate to Gly-2. In recognition of this relatedness to VHX, we refer to the DUSP15-encoded protein as VH1-related member Y (VHY). We report that VHY is expressed at high levels in the testis and barely detectable levels in the brain, spinal cord, and thyroid. A VHY-specific antiserum detected a protein with an apparent molecular mass of 26 kDa, and histochemical analysis showed that VHY was readily detectable in pachytene spermatocytes (midstage of meiotic division I) and round spermatids and weakly in Leydig cells (somatic cells outside of the seminiferous tubules). When expressed in 293T or NIH-3T3 cells, VHY was concentrated at the plasma membrane with some staining of vesicular structures in the Golgi region. Mutation of the myristoylation site Gly-2 abrogated membrane location. Finally, we demonstrate that VHY is an active phosphatase in vitro. We conclude that VHY is a new member of a subgroup of myristoylated VH1-like small dual specificity phosphatases.

Aurintricarboxylic acid blocks in vitro and in vivo activity of YopH, an essential virulent factor of Yersinia pestis, the agent of plague.

Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to Bubonic Plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. YopH is an essential virulence factor whose protein-tyrosine phosphatase (PTP) activity is required for Yersinia pathogenicity. Consequently, there is considerable interest in developing potent and selective YopH inhibitors as novel anti-plague agents. We have screened a library of 720 structurally diverse commercially available carboxylic acids and identified 26 YopH inhibitors with IC50 values below 100 mum. The most potent and specific YopH inhibitor is aurintricarboxylic acid (ATA), which exhibits a Ki value of 5 nm for YopH and displays 6-120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in human Jurkat cells transfected with YopH. We show that YopH severely decreases the T-cell receptor-induced cellular tyrosine phosphorylation, ERK1/2 activity, and interleukin-2 transcriptional activity. We demonstrate that ATA can effectively block the inhibitory activity of YopH and restore normal T-cell function. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. In addition, it is expected that potent and selective YopH inhibitors, such as ATA, should be useful reagents to delineate YopH's cellular targets in plague and other pathogenic conditions caused by Yersinia infection.