Mechanism-Based Profiling of Enzyme Families

Evans, Michael J.; Cravatt, Benjamin F.

doi:10.1021/cr050288g

Cited by 530 publications

(449 citation statements)

References 243 publications

(427 reference statements)

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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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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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“…Several ABPs have been developed to target enzymes implicated in cancer progression and tumorigenesis, including metalloproteases, cysteine cathepsins, and esterases [11,14,16]. These ABPs have been used to profile human tumors and tumor cell lines and identify novel enzyme activities for the diagnosis and treatment of cancer (Table 1).…”

Section: Profiling and Discovery Of Enzymes Involved In Cancermentioning

confidence: 99%

“…However, in vitro assays provide only limited information regarding the in vivo potency and selectivity of an inhibitor for a related series of enzymes. Since ABPs bind to the active sites of their enzyme targets, these probes have been used to develop small molecule inhibitor screens that resolve many of the shortcomings that plague standard in vitro inhibitor assays [11,14,33]. In an ABP-based screen, whole cells, cell lysates, or even whole organisms are treated with a range of concentrations of a potential inhibitor (Figure 3).…”

Section: Enzyme Inhibitor Discovery and Verificationmentioning

confidence: 99%

“…Modification of protein targets by these probes thus provides an indirect measure of enzyme activity and also allows for their purification and identification by mass spectrometry (MS). Several recent reviews have outlined the design of ABPs and their biological applications [10][11][12][13][14][15][16]. In this review, we will focus on the use of small molecule probes in cancer biology.…”

Section: Introductionmentioning

confidence: 99%

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Application of activity-based probes to the study of enzymes involved in cancer progression

Paulick

Bogyo

2008

Current Opinion in Genetics & Development

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SummaryMany tumor cells have elevated levels of hydrolytic and proteolytic enzymes, presumably to aid in key processes such as angiogenesis, cancer cell invasion, and metastasis. Since the activities of these enzymes are often regulated by post-translational mechanisms, their functional roles in cancer progression are difficult to study using traditional genomic and proteomic methods. Thus, methods that allow for the direct monitoring of enzyme activity in a physiologically relevant environment are required to better understand the roles of specific players in the complex process of tumorigenesis. This review highlights advances in the field of activity-based proteomics, which uses small molecules known as activity-based probes (ABPs) that covalently bind to the catalytic site of target enzymes. We discuss the application of ABPs to cancer biology, specifically to the discovery of tumor biomarkers, the screening of enzyme inhibitors, and the imaging of enzymes implicated in cancer.

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“…This reactive group is often referred to as a 'warhead' since it leads to permanent inactivation of the target by covalent modification of a primary catalytic nucleophile (for reviews, see Berger et al, 1 Speers and Cravatt 2 and Verhelst and Bogyo 3 ). ABPs that target a number of diverse protease families have been described, 4 including a number of probes employing the acyloxymethyl ketone reactive warhead that label active caspases. 5,6 The caspases are clan CD proteases whose concerted action is responsible for dismantling the cell during apoptotic cell death.…”

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confidence: 99%

Specificity of aza-peptide electrophile activity-based probes of caspases

et al. 2006

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Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.

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Mechanism-Based Profiling of Enzyme Families

Cited by 530 publications

References 243 publications

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

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

Application of activity-based probes to the study of enzymes involved in cancer progression

Specificity of aza-peptide electrophile activity-based probes of caspases

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