29. New Way to Interrogate PTPs

A New Way to Interrogate PTPs

A review of literature describing an assay that could help researchers use protein tyrosine phosphatases in drug development.

Protein tyrosine phosphatases (PTPs) control posttranslational tyrosine phosphorylation, and thus play a critical role in the regulation of many signaling molecules. Despite the disease relevance of PTPs (for example, PTP1B is considered a target for treatment of diabetes type 2; DEP1 and PTPγ are cancer targets) and several PTP inhibitors entering clinical trials (Hardy et al., Curr Oncol 2008;15:5–8), this class of enzymes has been known as one of the “undruggable” targets. This is largely due to the promiscuity of the targets and challenges associated with obtaining potent and specific PTP inhibitors. A need for PTP assays, preferentially with the ability to interrogate both substrate specificity and kinetic parameters in real time, is still highly desirable. Traditional phosphatase assays utilize artificial chromogenic or fluorogenic substrates, which may skew data interpretation. The Malachite green assay, on the other hand, quantifies inorganic phosphate production in an end-point mode, limiting itself for kinetic time course application.

In this article highlighted herein, the authors* present a PTP assay that utilizes a physiologically relevant peptidic substrate that contains 3-nitrophosphotyrosine moiety. The assay is based on the observation that 3-nitrophosphotyrosine is widely accepted by different PTPs, and upon enzyme turnover, the resulting 3-nitrotyrosine can be monitored spectrophotometrically at 415 nm.

After assay validation using two known phosphatase inhibitors, vanadate and NSC87877 (see Figure 2, panel C), the authors further demonstrated several distinct features of the assay. PTP activity profiling and substrate specificity could be conveniently achieved using the assay in plate format (see for example Figure 2, panel B). For instance, between two model cancer targets, DEP1 and PTPγ, their substrate specificity was differentiated among eight different peptidic substrates.

Another advantage of the assay lies in its insensitivity to the presence of background phosphate, such as elements from assay buffer or cell lysates. The same assay was subsequently applied to HEK293 cell lysates that had been treated under four different conditions, including wild type, transfected with PTP1B or inactive PTP1B, nontransfected but with 250 μM vanadate. PTP activity was readily discriminated as expected based on their respective initial reaction velocities, with cell lysates transfected with PTP1B showing the highest activity, while non-transfected lysates treated with vanadate showed the lowest

Figure 2. Sample data for the spectrophotometric PTP assay. All data points are averages of three experiments, errors expressed as SE. (A) Absorption spectra at different concentrations of 3-nitrotyrosine showing the maximum at 415 nm. (B) Initial velocities (v_ini) at different substrate concentrations for PTP1B with a 3-nitrophosphotyrosine peptide substrate derived from SIGLEC2. (C) Inhibition of PTPγ by different concentrations of sodium ortho-vanadate using a 3-nitrophosphotyrosine peptide substrate derived from ZAP70. (D) Initial velocities (v_ini) for HEK293 lysates (WT, nontransfected; PTP1B, transfected with PTP1B; C215S, transfected with inactive PTP1B; VO4, nontransfected in the presence of 250 µM vanadate) using a 3-nitrophosphotyrosine peptide substrate derived from INSR.

Overall, the method reported by van Ameijde et al. represents a welcomed addition to the existing toolbox and drug development programs for protein phosphatases. Potential improvements could include further enhancement in assay sensitivity (such as using fluorescence as a readout and reduce enzyme/substrate consumption).