Design and Synthesis of Class-Selective Activity Probes for Protein Tyrosine Phosphatases

Lo, Lee‐Chiang; Pang, Te-Ling; Kuo, Chih‐Yu; Chiang, Ying-Ling; Wang, Hsin‐Yi; Lin, Jing‐Jer

doi:10.1021/pr015506a

Cited by 112 publications

(59 citation statements)

References 27 publications

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“…However, until now, classselective probes for PTPs were not available. The suicide substrate, 4-fluromethylaryl phosphate, was recently explored as a PTP probe (12), because hydrolysis of 4-fluoromethylaryl phosphate generates a highly reactive quinone methide intermediate, which can alkylate nucleophilic side chains at, or near, the phosphatase active site (13). Unfortunately, 4-fluromethylaryl phosphate is not specific for PTPs; it also forms a covalent adduct with other classes of phosphatases such as nonspecific prostatic acid phosphatase and protein Ser͞Thr phosphatase calcineurin (13)(14)(15).…”

Section: Resultsmentioning

confidence: 99%

Activity-based probes for protein tyrosine phosphatases

Kumar

Zhou

Liang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

152

143

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Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival. Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome. Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for functional characterization of these enzymes. To facilitate the global analysis of PTPs, we designed and synthesized two activity-based probes that consist of ␣-bromobenzylphosphonate as a PTP-specific trapping device and a linker that connects the trapping device with a biotin tag for visualization and purification. We showed that these probes are active site-directed irreversible inactivators of PTPs and form a covalent adduct with PTPs involving the active site Cys residue. Additionally, we demonstrated that the probes are extremely specific toward PTPs while remaining inert to other proteins, including the whole proteome from Escherichia coli. Consequently, these activity-based PTP probes can be used to profile PTP activity in complex proteomes. The ability to interrogate the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets. P rotein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes (Ͼ100 in humans) that are important for the regulation of cell proliferation, differentiation, metabolism, migration, and survival (1, 2). Dysfunction in PTPs results in aberrant Tyr phosphorylation, which has been linked to the etiology of several human diseases, including cancer and diabetes (3, 4). Unlike protein kinases, of which Tyr-and Ser͞Thr-specific kinases share sequence identity, the PTPs show no sequence similarity with Ser͞Thr phosphatases or the broad-specificity phosphatases, such as acid or alkaline phosphatases. The hallmark that defines the PTP superfamily is the active site amino acid sequence C(X) 5 R, also called the PTP signature motif, in the catalytic domain. The PTPs can be broadly divided into two groups based on active site substrate specificity: the Tyr-specific and the dual-specificity phosphatases, which hydrolyze pSer͞Thr as well as pTyr. Despite variations in primary structure and differences in substrate specificity, key structural features in the active site and the mechanism of catalysis are conserved among all members of the PTP superfamily (5, 6).Although PTPs share a common catalytic mechanism, they have distinct (and often unique) biological functions in vivo. One of the major challenges in the field is to rapidly establish functional roles for PTPs, in both normal physiology and pathogenic conditions. Gene knockout analysis is useful in assessing the role of a number of PTPs in cellular signaling. However, this process is often tedious, and gene a...

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Section: Resultsmentioning

confidence: 99%

Activity-based probes for protein tyrosine phosphatases

Kumar

Zhou

Liang

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

152

143

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“…19,20 The activity-based probes (ABPs) were targeted to distinct enzyme classes, which include serine hydrolases, [21][22][23] cysteine proteases, 24 and tyrosine phosphatases. 25 However, to the best of our knowledge, such probes have not yet been described for protein kinases.…”

Section: Fsba As An Activity-based Probe For Protein Kinasesmentioning

confidence: 99%

A Liquid Chromatography/Mass Spectrometry-Based Method for the Selection of ATP Competitive Kinase Inhibitors

Khandekar

Feng²,

et al. 2005

SLAS Discovery

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The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5′-fluorosulfonylbenzoyl 5′-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases. (Journal of Biomolecular Screening 2005:447-455)

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“…10,11 In addition to profiling the functional state of enzymes, mechanism-based probes are also seeing application in drug discovery, target identification, and discovery of previously uncharacterized enzyme activity. 9 To develop class-selective PTP probes, 4-fluromethylaryl phosphate was initially examined 12 as its hydrolysis generates a highly reactive quinone methide intermediate, which alkylate nucleophiles at, or near, the phosphatase active site. 13 Unfortunately, 4-fluromethylaryl phosphate is not specific for the PTPs, as the diffusible, unmasked quinone methide electrophile will nonspecifically label any nearby proteins.…”

Section: Introductionmentioning

confidence: 99%

Aryl Vinyl Sulfonates and Sulfones as Active Site-Directed and Mechanism-Based Probes for Protein Tyrosine Phosphatases

et al. 2008

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Protein tyrosine phosphatases (PTPs) play key roles in the regulation of normal and pathological processes ranging from cell proliferation, differentiation, metabolism, and survival to many human diseases including cancer and diabetes. Functional studies of PTP can be greatly facilitated by small molecule probes that covalently label the active site of a PTP through an activity-dependent chemical reaction. In this communication, we characterize phenyl vinyl sulfonate (PVSN) and phenyl vinyl sulfone (PVS) as a new class of mechanism-based PTP probes. PVSN and PVS inactivate a broad range of PTPs in a time-and concentration-dependent fashion. The PVSN and PVS-mediated PTP inactivation is active site-directed and irreversible, resulting from a Michael addition of the active site Cys Sγ onto the terminal carbon of the vinyl group. Structural and mechanistic analyses reveal the molecular basis for the preference of PVSN/PVS toward the PTPs, which lies in the ability of PVSN and PVS to engage the conserved structural and catalytic machinery of the PTP active site. In contrast to early α-bromobenzyl phosphonate based probes, PVSN and PVS are resistant to solvolysis and are cell permeable, and thus hold promise for in vivo applications. Collectively, these properties bode well for the development of aryl vinyl sulfonates/sulfones based PTP probes to interrogate PTP activity in complex proteomes.

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Design and Synthesis of Class-Selective Activity Probes for Protein Tyrosine Phosphatases

Cited by 112 publications

References 27 publications

Activity-based probes for protein tyrosine phosphatases

Activity-based probes for protein tyrosine phosphatases

A Liquid Chromatography/Mass Spectrometry-Based Method for the Selection of ATP Competitive Kinase Inhibitors

Aryl Vinyl Sulfonates and Sulfones as Active Site-Directed and Mechanism-Based Probes for Protein Tyrosine Phosphatases

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