Development of a homogenous high-throughput assay for inositol hexakisphosphate kinase 1 activity

Wormald, Michael; Liao, Gangling; Kimos, Martha; Barrow, James C.; Wei, Huijun

doi:10.1371/journal.pone.0188852

Cited by 23 publications

(41 citation statements)

References 26 publications

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“…Although radiolabelled-HPLC or non-radiolabelled sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass-spectrometry-based methods (Azevedo & Saiardi, 2006; Draskovic et al, 2008; Losito et al, 2009) are useful for estimating IP6K activity and cellular IPP levels, they are not suitable for high-throughput screening of thousands of compounds. A recent paper addressed this issue by designing a high-throughput assay for IP6Ks (Wormald et al , 2017). In addition, crystallization of human enzymes will provide an insight into active sites and regulatory regions, which is critical for structure-based drug development.…”

Section: Current and Future Directions Of Ipp Research In Mammalsmentioning

confidence: 99%

The inositol pyrophosphate pathway in health and diseases

Chakraborty

2017

Biological Reviews

123

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Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate-containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet-induced obesity (DIO), type-2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio-metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.

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Section: Current and Future Directions Of Ipp Research In Mammalsmentioning

confidence: 99%

The inositol pyrophosphate pathway in health and diseases

Chakraborty

2017

Biological Reviews

123

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“…It is noteworthy that our IC 50 value for TNP is close to that previously reported using the ADP-Glo Max assay. 9 …”

Section: Resultsmentioning

confidence: 99%

“…At least one pharmacological activity is known for each of these compounds and the main purpose of this library is to examine the performance of a high throughput assay, rather than to identify tractable inhibitors. 9 However, a successful screening exercise depends upon identification of chemically tractable hit molecules. One approach to reaching this goal, in an efficient manner, lies in the curation and application of smaller, focused libraries with functionally and/or chemically related properties.…”

Section: Introductionmentioning

confidence: 99%

Use of Protein Kinase–Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors

et al. 2018

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Inositol hexakisphosphate kinases (IP6Ks) regulate a myriad of cellular processes, not only through their catalytic activity which synthesizes InsP7, a multifunctional inositol pyrophosphate signaling molecule, but also through protein-protein interactions. To further study the enzymatic function and distinguish between these different mechanisms, specific inhibitors that target IP6K catalytic activity are required. Only one IP6K inhibitor is commonly used: N2-(m-(trifluoromethyl)benzyl) N6-(p-nitrobenzyl)purine (TNP). However, TNP is compromised by weak potency, inability to distinguish between IP6K isoenzymes, off-target activities, and poor pharmacokinetic properties. Herein, we describe a new inhibitor discovery strategy, based on the high degree of structural conservation of the nucleotide binding sites of IP6Ks and protein kinases; we screened for novel IP6K2 inhibitors using a focused set of compounds with features known, or computationally predicted, to target nucleotide binding by protein kinases. We developed a time-resolved, fluorescence resonance energy transfer (TR-FRET) assay of ADP formation from ATP. Novel hit compounds for IP6K2 were identified and validated with dose-response curves and an orthogonal assay. None of these inhibitors affected another inositol pyrophosphate kinase, PPIP5K. Our screening strategy offers multiple IP6K2 inhibitors for future development and optimization. This approach will be applicable to inhibitor discovery campaigns for other inositol phosphate kinases.

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“…It is also necessary to determine the therapeutic window for each disease. Several groups are working to screen compound libraries against IP6Ks and to exploit subtle differences in active sites of the isotypes to develop potent and isotype-specific IP6K inhibitors [42,[270][271][272]. Hopefully, IP6K inhibitors, either alone or in combination, will emerge as new drugs to treat metabolic diseases.…”

Section: Can Ip6k Inhibitors Be Used To Treat Metabolic Diseases?mentioning

confidence: 99%

Targeting the Inositol Pyrophosphate Biosynthetic Enzymes in Metabolic Diseases

2020

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In mammals, a family of three inositol hexakisphosphate kinases (IP6Ks) synthesizes the inositol pyrophosphate 5-IP7 from IP6. Genetic deletion of Ip6k1 protects mice from high fat diet induced obesity, insulin resistance and fatty liver. IP6K1 generated 5-IP7 promotes insulin secretion from pancreatic β-cells, whereas it reduces insulin signaling in metabolic tissues by inhibiting the protein kinase Akt. Thus, IP6K1 promotes high fat diet induced hyperinsulinemia and insulin resistance in mice while its deletion has the opposite effects. IP6K1 also promotes fat accumulation in the adipose tissue by inhibiting the protein kinase AMPK mediated energy expenditure. Genetic deletion of Ip6k3 protects mice from age induced fat accumulation and insulin resistance. Accordingly, the pan IP6K inhibitor TNP [N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates obesity, insulin resistance and fatty liver in diet induced obese mice by improving Akt and AMPK mediated insulin sensitivity and energy expenditure. TNP also protects mice from bone loss, myocardial infarction and ischemia reperfusion injury. Thus, the IP6K pathway is a potential target in obesity and other metabolic diseases. Here, we summarize the studies that established IP6Ks as a potential target in metabolic diseases. Further studies will reveal whether inhibition of this pathway has similar pleiotropic benefits on metabolic health of humans.

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Development of a homogenous high-throughput assay for inositol hexakisphosphate kinase 1 activity

Cited by 23 publications

References 26 publications

The inositol pyrophosphate pathway in health and diseases

The inositol pyrophosphate pathway in health and diseases

Use of Protein Kinase–Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors

Targeting the Inositol Pyrophosphate Biosynthetic Enzymes in Metabolic Diseases

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