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January 15, 2018 (Vol. 38, No. 2)

Quantitative Kinase Target Engagement

Live-Cell Platform for Studies Exploring Kinase-Inhibitor Binding

  • Kinases constitute a family of more than 500 enzymes and play critical roles in cellular signaling, both in normal and diseased cells. As a result, kinases continue to be key drug targets, especially for cancer and inflammatory diseases.

    Traditional biochemical assays with recombinant kinases are commonly used to characterize small-molecule effects on kinase activity or affinity. Biochemical-kinase assays, however, can fail to predict how kinase inhibitors might function inside cells. Reasons for failing include: differences in ATP concentration, use of truncated kinase domains, absence of appropriate cellular cofactors, and differences in kinase activation states. Consequently, a biochemical assay often does not recapitulate the physiological complexity that a kinase encounters inside an intact cell.

    There is a growing need for quantitative kinase-inhibitor cellular target engagement (TE) methods. Several methods exist to assess compound TE to kinases expressed in human cells. Some methods use affinity-based chemoproteomics with cell lysates, which disrupt the cell membrane, and cellular cofactors, which affect affinity. Other methods, such as cellular thermal shift assays (CETSAs), do not require cell lysis during compound incubation, but depend on a downstream event such as protein aggregation. CETSAs, therefore, do not provide a quantitative measure of drug affinity. CETSAs are also prone to misreporting on certain TE events, as not all compounds that bind the target kinase will stabilize it.

    Most of these techniques fail to meet the desirable TE assay qualities of quantitating small-molecule inhibitor affinity in live cells via a direct binding event, rather than in lysates or via a downstream event that can be less specific.

  • Live-Cell Quantitative Target Engagement Kinase Assays

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    Figure 1. Schematic of Live-Cell Kinase NanoBRET® Target Engagement Assay. A cell-permeable energy transfer probe (or tracer) reversibly binds the kinase active site, generating a BRET signal in live cells. Unlabeled test compound binding to the selected kinase results in a decrease in BRET signal.1

    Here we describe a novel TE platform, which is the first to quantitatively measure compound binding to kinases in live cells without disrupting the cell membrane. These assays are target kinase specific, use full-length kinases, and are performed in multiwell plates.

    The Promega target engagement assay platform uses a bioluminescence resonance energy transfer technology, NanoBRET™. There are four key components of the assay: 1) cellular-expressed full-length kinase protein fused to the very bright, small NanoLuc® luciferase; 2) cell-permeable energy-transfer probe (or fluorescent tracer) that reversibly binds the active site of the kinase; 3) substrate for NanoLuc luciferase; and (4) extracellular NanoLuc inhibitor to ensure signal originates from live cells.

    Figure 1 is a schematic of the NanoBRET TE kinase assays, which are direct competitive binding assays conducted in live cells. A cell-permeable energy-transfer probe (or tracer) reversibly binds the active site of a kinase-NanoLuc fusion protein expressed in cells.

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    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 IC50 vs. tracer concentration allows for determination of apparent intracellular KD 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.

    Energy transfer from NanoLuc to the fluorescent tracer bound to the kinase occurs due to close proximity and results in a BRET signal. It is this proximity that results in the target kinase specificity of the BRET signal. In the presence of a competitive compound, binding to the kinase-NanoLuc fusion protein results in displacement of the probe and decrease in the BRET signal.

    The NanoBRET-based competitive binding format allows quantitative measures of unmodified compound affinity against the selected target kinase protein in live cells. As shown in Figure 2A, a recommended tracer concentration has been determined for each kinase using competitive binding experiments with varying tracer concentrations. At tracer concentrations ≤KD, this assay should measure apparent occupancy of a compound for the selected kinase in live cells.

    Further confirmation of test compound apparent intracellular KD can be obtained by Cheng-Prousoff analysis of the test compound IC50 vs. tracer concentration (Figure 2B). We have shown that target kinase expression levels do not impact assay results. As NanoLuc is very bright, we have demonstrated that low levels of kinase-NanoLuc fusion protein expression are detectable with NanoBRET, even from kinases expressed from endogenous genetic loci (data not shown).

  • Broad Coverage and Scalable Assays

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    The table shows the top few rows from a list of kinases assayed at Promega using NanoBRET TE Intracellular Kinase Assays. The complete searchable table is available at www.promega.com and includes >120 kinases from all groups (AGC, CAMK, CK1, CMGC, STE, TK, TKL, other, and atypical). Each kinase in the complete table is available as a kinase-NanoLuc fusion vector from the company, as a PDF downloadable technical data sheet for each kinase.

    Broad-coverage cell-permeable NanoBRET kinase tracers have been developed and are commercially available in separate NanoBRET TE Intracellular Kinase Assays, K-4 or K-5. Additionally, Promega has developed more than 120 kinase-NanoLuc fusion 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, associated 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, recommended tracer concentration, and relevant technical 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, compound 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 quantifying binding of inhibitors utilizing different mechanisms, Promega performed competitive binding analysis for ABL kinase with type I and type II inhibitors, as well as allosteric kinase inhibitors (Figure 2C). As expected for ABL kinase, increasing concentrations of type I (dasatinib) and type II (imatinib 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 partially displace tracer from ABL.

    GNF-2 is functionally competitive with ATP as previously demonstrated in the literature. These assays have also been used in studies on reversible covalent inhibitor development (data not shown).

  • Residence Time

    Compound residence time is an additional parameter that NanoBRET TE kinase assays are able to monitor. Residence time is the lifetime that a ligand remains bound to its target protein under nonequilibrium, open conditions. With NanoBRET, the faster a compound dissociates from its target, the sooner the tracer can bind, resulting 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 nonequilibrium conditions associated with absorption, 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 biology 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 protein 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 introduced more than 120 live-cell NanoBRET TE kinase assays with representative data, and we have additional cell-based kinase assays in development. 

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