PTP inhibitor

Extrapolating from well-characterized enzymatic inhibition in test tubes, numerous mechanistic ideas concerning the in vivo effects of vanadium compounds have been advanced. The effects of vanadium compounds as transition-state analogs of certain enzymes with a phosphoprotein intermediate in their reaction scheme is proposed to account for the action of vanadium [11] in many biological systems. Unfortunately, it is often difficult to determine if the inhibition observed in the test tube occurs in vivo. For example, although vanadate is a potent inhibitor of plasma membrane ion pumps (such as the sodium potassium ATPase) in the test tube, it is difficult to determine if these pumps are actually inhibited in animals exposed to vanadium compounds. Currently, the role of vanadium compounds as protein phosphatase (PTP) inhibitors is believed to be related to the metabolic effects of this... 

Protein-tyrosine (PTP) inhibitors are potentially valuable pharmacological tools for studying cellular signal transduction and for therapeutic intervention. Derivatives of 1,1-difluoromethylphosphonic acid are known to be potent PTP inhibitors. An important method for synthesis of these compounds is fluorina-tion of acyl phosphonates by diethylaminosulfur trifluoride (DAST). This method allows synthesis of fcrf-butyl-protected difluoro(aryl)- or (naphthal-enyl)methylphosphonates, which under mildly acidic deprotection conditions afford desirable phosphonic acids [16, 39]. Acyl phosphonate fluorination by DAST has also been utilized for synthesis of a,a-(difluoroprop-2-ynyl)phos-phonates [14]. 

Key words High-throughput screening, HTS, Protein tyrosine phosphatase, PTP, Inhibitors, pNPP,... 

Fig. 3 (a) Buffer optimization for a pNPP colorimetric assay in 384-well format using p-nitrophenol detection. Activity of HePTP at a concentration of 50 nM was tested in the presence of 1.3 mM pNPP, 1 mM DTT, 0.005 % Tween-20, and various concentrations of Bis-Tris, pH 6.0, and NaCI, over a reaction time of 1 h. The optimal buffer under HTS conditions was 20 mM Bis-Tris, pH. 6.0,1 mM DTT, and 0.005 % Tween-20. The value for pNPP was 0.4 mM. The reaction demonstrated linearity of the progress curves over a period of 2 h. Enzymatic activity was proportional to enzyme concentration. Assay performance was confirmed using the general PTP inhibitor orthovanadate, the ICeo value of which was 150 (b) Buffer optimization for an OMFP... 

Validate the assay by testing a positive control, e.g., a known inhibitor of the PTP of interest. If no specific inhibitor is available, use orthovanadate, which is a general, transition state analogue PTP inhibitor. 

PTP Inhibitor ( Hit/Lead Compounds 1—K Cytotoxicity, k (nFAT/AP-1 LudfeiaseT, —v J... 

We recommend performing two different immunoblot experiments First, a dose response experiment to determine an optimal dose of inhibitor, i.e., the lowest concentration that yields maximal response and second, a time course experiment that captures the effects of inhibitors (used at optimal dose) at various time points after TCR stimulation. Efficacious PTP inhibitors not only are expected to augment phosphorylation levels of substrates, but also to induce a sustained effect on these molecules, compared to the vehicle control. For instance, treatment of Jmkat T cells with the chemical probe MLl 19 [21 ] resulted in augmented and sustained phosphorylation of the MAP kinases ERKl/2 and p38 [22]. This effect was due to direct inhibition of HePTP PTPN7), a classical PTP that specifically dephosphorylates the phosphotyrosine residue in the activation loop of ERKl/2 andp38. 

Select PTP inhibitors that dose-dependently augment the phosphorylation level of substrate proteins without affecting global tyrosine phosphorylation for further consideration. Confirm selected inhibitors by repeating this experiment. In order to quantify western blots, scan films in gray scale at a resolution of a least 600 dpi and analyze the scans using the ImageJ software (http // rsbweb.nih.gov/ij/index.html). 

We regard a dose-dependent effect of a FTP inhibitor at sub-and low-micromolar concentration on a direct substrate of the FTP (without affecting global tyrosine phosphorylation) as the gold standard in terms of efficacy of a PTP inhibitor in T cells. To confirm the specificity of the inhibitor, we recommend testing it in T cells where the target PTP has been acutely eliminated via RNA interference as described in the next section. 

To determine whether inhibitors specifically inhibit the PTP of interest in primary human T cells, purified T cells can be used, in which the target PTP has been acutely eliminated (residual expression <20 % of normal expression) by short interfering RNA (siRNA)- or small hairpin RNA (shRNA)-mediated knockdown. Signaling in T cells with severely reduced protein levels of the PTP of interest is expected to be similar to signaling in T cells treated with a specific inhibitor of the PTP. However, this may not always be the case. Inhibition of PTP activity by the inhibitor, or eliminating the protein altogether via RNAi, may have different outcomes due to a more complex function of the PTP that is not based on its phosphatase activity alone [31]. Nonetheless, the combined action of PTP inhibitor and PTP knockdown should not alter signaling beyond what is observed for each alone. 

Count cells and make aliquots of 5 x 10 cells per 1.5 mL sterile microtube. Prepare six tubes for each target gene two tubes for the control non-targeting siRNA, and two tubes for each of the two specific siRNAs (the first tube is for vehicle treated cells the second tube is for PTP inhibitor treated cells). 

Cells are now ready for immunoblot assays. Proceed as described in Subheadings 3.12 and 3.5, respectively. Selective PTP inhibitors are expected to have a similar effect on phosphorylation levels of substrate proteins as observed in cells in which the PTP has been acutely eliminated by RNAi. However, the combination of knockdown and small-molecule inhibition should not result in greater effects than observed for each alone. 

If working with whole blood, 50 mL can be collected in five vacutainer tubes (of 10-mL) containing anticoagulant and EDTA (potassium salt). Employing PBMC collected from healthy volunteers or patients offers the possibility to evaluate the effects of a PTP inhibitor on cells from a donor with a specific genotype. However, preparing PBMC from whole blood results in about seven times fewer cells, compared to preparation from buffy coat. 

Treatment of PBMC with PTP inhibitor should be done in the absence of serum in the culture medium, as serum proteins can deplete the inhibitor through nonspecific binding. Thus, cells... 

I 3 Strategies for Designing Specific PTP Inhibitors and Their Intracellular Activation... 

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