A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time

Thongwichian, Rossukon; Kosten, Jonas; Benary, Uwe; Rose, Honor May; Stuiver, Marchel; Theillet, François‐Xavier; Dose, Alexander; Koch, Birgit; Yokoyama, Hideki; Schwarzer, Dirk; Wolf, Jana; Selenko, Philipp

doi:10.1021/jacs.5b02987

Cited by 14 publications

(18 citation statements)

References 20 publications

(30 reference statements)

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“…'In-cell' and 'in-lysate' NMR spectroscopy (Danielsson et al 2013(Danielsson et al , 2015Serber et al 2005Serber et al , 2007Smith et al 2013) have developed to a point where new aspects of protein function can be revealed (Banci et al 2013). For instance in a recent study Selenko and co-workers developed an NMR-based methodology that enables quantification of the temporal phosphorylation and de-phosphorylation of short protein segments in cell lysates (Thongwichian et al 2015). With this approach the efficacy of kinase drugs can be studied for many kinase targets simultaneously and also under near-cellular conditions.…”

Section: Discussionmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

143

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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Section: Discussionmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

143

122

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“…High-resolution NMR is an ideal technique for characterization of chemical modifications of proteins at the residue level, and has been shown to be particularly suited for monitoring protein phosphorylation events. [40][41][42] Using time-resolved in vitro NMR, we unambiguously identified four serine phosphorylation sites, which react with CK2 at different rates, in NS5A-D3. [38,39] Moreover,f ast NMR experimentsa cquiredw ith sensitivee quipment allow protein phosphorylation eventst ob em onitored in real time, including in intact cells and extracts.…”

Section: Introductionmentioning

confidence: 99%

“…[38,39] Moreover,f ast NMR experimentsa cquiredw ith sensitivee quipment allow protein phosphorylation eventst ob em onitored in real time, including in intact cells and extracts. [40][41][42] Using time-resolved in vitro NMR, we unambiguously identified four serine phosphorylation sites, which react with CK2 at different rates, in NS5A-D3. Interestingly, some sites do not have a" canonical" CK2 recognition motif, and had not been predicted previously.…”

Section: Introductionmentioning

confidence: 99%

The Casein Kinase 2‐Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time‐Resolved NMR Spectroscopy

2016

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Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level.

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“…For instance, Dose et al [ 425 ] used acetylation- and deacetylation-based assays to monitor the activity of histone deacetylase and acetyl-transferase. Thongwichian et al [ 426 ] used peptide-based reporters to identify active kinases and phosphatases in cellular conditions. Lastly, Doura et al [ 427 ] designed a 19 F probe that operates in biological conditions in order to study the adherence and dynamics of proteins found in human blood.…”

Section: In-cell Nmr Approachesmentioning

confidence: 99%

NMR as a “Gold Standard” Method in Drug Design and Discovery

et al. 2020

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Studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. Microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. Cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. The pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. Viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of SARS-CoV-2, which originated in Wuhan, China, illustrates the critical and immediate need to improve drug design and development techniques. Modern day drug discovery is a time-consuming, expensive process. Each new drug takes in excess of 10 years to develop and costs on average more than a billion US dollars. This demonstrates the need of a complete redesign or novel strategies. Nuclear Magnetic Resonance (NMR) has played a critical role in drug discovery ever since its introduction several decades ago. In just three decades, NMR has become a “gold standard” platform technology in medical and pharmacology studies. In this review, we present the major applications of NMR spectroscopy in medical drug discovery and development. The basic concepts, theories, and applications of the most commonly used NMR techniques are presented. We also summarize the advantages and limitations of the primary NMR methods in drug development.

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A Multiplexed NMR-Reporter Approach to Measure Cellular Kinase and Phosphatase Activities in Real-Time

Cited by 14 publications

References 20 publications

Protein dynamics and function from solution state NMR spectroscopy

Protein dynamics and function from solution state NMR spectroscopy

The Casein Kinase 2‐Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time‐Resolved NMR Spectroscopy

NMR as a “Gold Standard” Method in Drug Design and Discovery

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