Genetic Engineering & Biotechnology News

JAN15 2018

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Genetic Engineering & Biotechnology News | GENengnews.com | JANUARY 15, 2018 | 13 NanoBRET TE Intracellular Kinase Assays, K-4 or K-5. Additionally, Promega has de- veloped more than 120 kinase-NanoLuc fu- sion DNA vectors. The pairing of a kinase- NanoLuc fusion vector with the appropriate NanoBRET TE Intracellular Kinase Assay (K-4 or K-5) results in a kinase-specific live- cell TE assay. The Table lists some of the kinases, asso- ciated kinase-NanoLuc fusion vectors, and NanoBRET TE assays; all commercially available vectors and NanoBRET TE kinase assays are listed on the Promega website. For each kinase, the company has created a technical data sheet (downloadable PDF) with kinase specific assay data, recommend- ed tracer concentration, and relevant techni- cal notes. Additional NanoBRET TE Kinase Assays and kinase-NanoLuc fusion DNA vectors are in development and are available through Promega's Custom Assay Services. All of these assays are compatible with 96-well plates, many scalable to 384-well or higher densities, making them suitable for multiple phases of drug discovery. Recently, Promega has shown that when compound binding is measured against multiple kinases, com- pound selectivity within live cells can also be determined. 1 Multiple Types of Kinase Inhibitors Detected To demonstrate the broad utility of the NanoBRET TE kinase assays for quantify- ing binding of inhibitors utilizing different mechanisms, Promega performed competi- tive binding analysis for ABL kinase with type I and type II inhibitors, as well as al- losteric kinase inhibitors (Figure 2C). As ex- pected for ABL kinase, increasing concentra- tions of type I (dasatinib) and type II (ima- tinib and ponatinib) inhibitors displaced the tracer, resulting in a decrease in BRET signal. The allosteric inhibitor GNF-2, which binds the myristoyl binding pocket of ABL (and not the ATP pocket), was also able to par- tially displace tracer from ABL. GNF-2 is functionally competitive with ATP as previously demonstrated in the lit- erature. These assays have also been used in studies on reversible covalent inhibitor de- velopment (data not shown). Residence Time Compound residence time is an ad- ditional parameter that NanoBRET TE ki- nase assays are able to monitor. Residence time is the lifetime that a ligand remains bound to its target protein under nonequi- librium, open conditions. With NanoBRET, the faster a compound dissociates from its target, the sooner the tracer can bind, re- sulting in an increase in BRET signal as a function of time. Drugs are designed to be used in living systems where they are subject to nonequi- librium conditions associated with absorp- tion, distribution, metabolism, and excretion. Monitoring compound residence time can be important during inhibitor development. Summary The need for quantitative, specific, and scalable cellular kinase assays is growing as our understanding of intracellular kinase bi- ology deepens. The NanoBRET TE method allows direct quantitative measurement of compound affinity for full-length kinases under physiological cellular conditions. Each NanoBRET TE kinase assay is specific for the full-length kinase-NanoLuc fusion pro- tein expressed in cells. Because NanoLuc is so bright, kinase- NanoLuc fusion proteins can be expressed at low levels. These assays are easily scalable and therefore suitable for multiwell-plate formats. We have demonstrated the binding of multiple types of kinase inhibitors (types I and II, as well as allosteric and covalent) using these assays. Finally, we recently intro- duced more than 120 live-cell NanoBRET TE kinase assays with representative data, and we have additional cell-based kinase as- says in development. Reference 1. Vasta et al., "Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement," Cell Chem. Biol. (2017), https://doi.org/10.1016/j.chembiol.2017.10.010. We have four BMG LABTECH plate readers and they are all used heavily. They are smart, reliable and help my company to achieve its strategic goals. More than this, the support team and servicing engineers are brilliant! Thank you BMG! Hayley M Saunders, MRC Technology, London Visit us at SLAS in San Diego at booth #1129 from February 3 rd - 7 th ! www.bmglabtech.com * Source: SelectScience customer reviews © 2018 All rights reserved. All logos and trademarks are the property of BMG LABTECH. The BMG LABTECH All Stars Drug Discovery Tutorial Figure 2. Quantitative analysis of kinase target engagement determined using NanoBRET in live cells.¹ (A) Competitive binding experiments were used with varying tracer concentrations to determine a recommended tracer concentration for each kinase. (B) Linear regression analysis of test compound IC 50 vs. tracer concentration allows for determination of apparent intracellular K D for the test compound. (C) Compound competitive binding analysis using NanoBRET TE measured type I inhibitor, type II inhibitor, and allosteric kinase inhibitor binding to ABL kinase. B A C

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