A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Fouda, Ahmed E.; Pflum, Mary Kay H.

doi:10.1002/anie.201503041

Cited by 18 publications

(17 citation statements)

References 46 publications

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“…19 Among these analogs, ATP-biotin (Figure 1b) is an attractive probe for characterizing phosphorylation by affixing a biotin handle onto phosphoproteins, which can be used for subsequent visualization. 20, 21 In addition, kinase-catalyzed biotinylation coupled with avidin affinity purification offers a powerful phosphoprotein enrichment strategy. 22 …”

Section: Introductionmentioning

confidence: 99%

The generality of kinase-catalyzed biotinylation

Senevirathne

Embogama

Anthony

et al. 2016

Bioorganic & Medicinal Chemistry

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Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signalling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.

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Section: Introductionmentioning

confidence: 99%

The generality of kinase-catalyzed biotinylation

Senevirathne

Embogama

Anthony

et al. 2016

Bioorganic & Medicinal Chemistry

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“…Therefore, it is expected that triphosphate modifications, which gave rise to the 8‐halogenated‐7‐deazadGTP derivatives, could result in candidates for antitumor activity by targeting hMTH1. In future work, 8‐halogenated‐7‐deazadGTP modifications, including nucleotide diphosphate or triphosphate prodrug approach, will be completed to allow the compounds to penetrate cells . Evaluation of nucleotide kinase and DNA polymerase activities are also ongoing.…”

Section: Methodsmentioning

confidence: 99%

Inhibitory Effect of 8‐Halogenated 7‐Deaza‐2′‐deoxyguanosine Triphosphates on Human 8‐Oxo‐2′‐deoxyguanosine Triphosphatase, hMTH1, Activities

2016

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hMTH1 (8-oxo-2'-deoxyguanine triphosphatase) hydrolyzes oxidized nucleoside triphosphates; its presence is non-essential for survival of normal cells but is required for survival of cancer cells. In this study, 8-halogenated-7-deaza-2'-deoxyguanosine triphosphate (8-halogenated-7-deazadGTP) derivatives were synthesized. Interestingly, these triphosphates were poor substrates for hMTH1, but exhibited strong competitive inhibition against hMTH1 at nanomolar levels. This inhibitory effect is attributed to slower rate of hydrolysis, possibly arising from enzyme structural changes, specifically different stacking interactions with 8-halogenated-7-deazadGTP. This is the first example of using nucleotide derivatives to inhibit hMTH1, thus demonstrating their potential as antitumor agents.

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“…A fully orthogonal system-in which the engineered enzyme utilizes the bumped substrate analog with high efficiency, while no longer recognizing the native substrate-may also benefit from a high signal-to-noise ratio. The bump-hole strategy has been successful with other enzyme families, including kinases, 30,31 acetyltransferases, 32 methyltransferases, 33 and ADPribosyltransferases. 34 Previous studies have demonstrated that glycosyltransferases are amenable to enzyme-substrate engineering, wherein the active site is modified to accept a non-native substrate.…”

Section: Introductionmentioning

confidence: 99%

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Choi¹,

Wagner²,

Timmermans³

et al. 2019

Preprint

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<div><div><div><p>O-GalNAc glycans constitute a major part of the human glycome. They are difficult to study becauseof the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form ofglycosylation, the polypeptide GalNAc transferases (GalNAc-Ts). Despite proven disease relevance,correlating the activity of individual GalNAc-Ts with biological function remains challenging due to alack of tools to probe their substrate specificity in a complex biological environment. Here, wedevelop a “bump–hole” chemical reporter system for studying GalNAc-T activity in vitro. IndividualGalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with anewly-synthesized collection of 20 (bumped) UDP-GalNAc analogs to identify enzyme–substratepairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme–substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer non-glycosylated peptide substrates and GalNAcT-10 that prefers a pre-glycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way forstudying substrate specificities in living systems.</p></div></div></div>

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A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Cited by 18 publications

References 46 publications

The generality of kinase-catalyzed biotinylation

The generality of kinase-catalyzed biotinylation

Inhibitory Effect of 8‐Halogenated 7‐Deaza‐2′‐deoxyguanosine Triphosphates on Human 8‐Oxo‐2′‐deoxyguanosine Triphosphatase, hMTH1, Activities

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

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