A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

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

doi:10.1002/ange.201503041

Cited by 11 publications

(5 citation statements)

References 44 publications

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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

Choi

Wagner²,

Timmermans

et al. 2019

J. Am. Chem. Soc.

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O-Linked -N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide Nacetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bumphole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly-synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs 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

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Section: Introductionmentioning

confidence: 99%

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Choi

Wagner²,

Timmermans

et al. 2019

J. Am. Chem. Soc.

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“…To improve the cell permeability of the ATP probe, Pflum and co-workers developed an analog 6. 26 The improved cell permeability of this probe was ascribed to the partial neutralization of the triphosphate's negative charge with a cationic polyamine linker, methylated spermine. The difference between the identity of proteins biotinylated in the cell vs lysate demonstrates the significance of the environment on the phosphorylation process, indicating the influence of compartmentalization inside the cell.…”

Section: Journal Of Medicinal Chemistrymentioning

confidence: 99%

“…The difference between the identity of proteins biotinylated in the cell vs lysate demonstrates the significance of the environment on the phosphorylation process, indicating the influence of compartmentalization inside the cell. 26 Ferrocene-modified bioconjugates are popular redox reporters thanks to their electrochemical properties and stability in aqueous solution. 32 Kraatz and co-workers have developed γferrocene-modified ATP derivatives 7−10 for monitoring protein kinase activity and for inhibitor screening.…”

Section: Journal Of Medicinal Chemistrymentioning

confidence: 99%

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications

Joachimiak

Błażewska

2018

J. Med. Chem.

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Studies on the human proteome have engaged diverse techniques; however, none of them represent a predominant approach. Chemical biology has made a major contribution to our understanding of human biology, stimulating the generation of biological hypotheses. Tools such as functional probes have advanced studies on biological mechanisms and helped in elucidating off-target reactivity and potential toxicities of drugs and drug candidates. Here, we accentuate the recent developments in the design and applications of phosph(on)ate-containing probes. Phosphate esters and anhydrides are present in a number of vital cell constituents, and their significance can be reflected by a number of biological processes that involve phosphorus-bearing molecules. We discuss the use of phosph(on)ate-derived probes for (1) the identification of phosphate-requiring enzymes, their substrates, interacting partners; (2) developing screening assays; and (3) their potential as diagnostics. Limitations that as yet need to be overcome and possible measures to be undertaken will also be addressed.

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“…ABPP probes covalently target the active site of enzymes for their detection or purification. Substrate ID describes a set of tools for the capture of the downstream substrates of a given enzyme . While ABPP and substrate ID are powerful tools for assessing the functional status and output of enzymes, we sought a way to not only identify active kinases but also to capture the kinases as they bind to a specific substrate, a method we term substrate‐based protein profiling (SBPP).…”

Section: Introductionmentioning

confidence: 99%

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

et al. 2019

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The proteomic mapping of enzyme–substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide‐based substrate analog capable of reprogramming the wild‐type phosphoryl‐transfer reaction to produce a kinase‐acrylamide‐based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP‐based methacrylate crosslinker (ATP‐MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP‐MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP‐based methacrylate (ADP‐MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP‐MA over ATP‐MA is due to reduced inhibition of the second step of the kinase–substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP‐MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site‐specific manner. The kinase–substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

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A Cell‐Permeable ATP Analogue for Kinase‐Catalyzed Biotinylation

Cited by 11 publications

References 44 publications

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

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