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Protein kinases are important regulators of signal-transduction pathways. Dysregulated kinase activity is observed in a variety of human diseases such as cancer, making them targets for the development of molecular therapies. Identification of new drugs is greatly aided by molecular imaging tools which enable real time, non-invasive, dynamic and quantitative imaging of kinase activity in vivo. We have recently described a new reporter platform based on conformation dependent complementation of firefly luciferase to monitor serine/threonine kinase (Akt) activity. The reporter system provides unique insights into the pharmacokinetics and pharmacodynamics of drugs that modulate kinase activity in living subjects and also provide a platform for cell based high-throughput drug screening for modulators of kinase activity.
Protein kinases are enzymes that covalently attach a phosphate group donated by ATP to a specific amino acid residue on protein substrates resulting in changes in target protein activity, cellular location and interaction with other proteins. These functional changes play an important role in mediating intracellular signal-transduction pathways in a range of processes from embryogenesis to cell death.1 Dysregulated kinase activity has been implicated in more than 400 human diseases such as cancer, rheumatoid arthritis, cardiovascular and neurological disorders, asthma and psoriasis.1–6 Therefore, these enzymes make a very attractive target for therapeutic interventions. It is no surprise that the first molecularly targeted drug, Gleevec (imatinib), used in treatment of chronic myelogenous leukemia (CML) and gastrointestinal stromal tumors (GISTs) is a tyrosine kinase inhibitor.7 For the treatment of human maligancies, a number of kinase inhibitors are being developed and are under various stages of preclinical or clinical investigation.8
The serine/threonine kinase Akt/PKB is one of the best characterized and is involved in tumor initiation and progression as well as in resistance to cancer treatment.9 Akt is a central signaling hub wherein many upstream oncogenic stimuli (e.g., growth factor signaling/cytokine cascades) converge (Fig. 1).10 The integration of these intracellular signals at the level of Akt and its kinase activity results in downstream signaling cascades, such as NFkappa B, mTOR, Forkhead, Bad, GSK-3 and MDM-2. These downstream modulators in turn mediate the effects of Akt on cell growth, proliferation and protection from pro-apoptotic stimuli and stimulation of neo-angiogenesis. The activation of Akt is regulated by both translocation to the plasma membrane and phosphorylation at Thr 308 and Ser473. Constitutive phosphorylation of Akt at these sites is frequently observed in a wide range of solid tumors and hematologic malignancies. Although, there have been major developments in our understanding of the biology of Akt and its role in human malignancies, the development of molecular imaging technologies to monitor and quantify Akt activity has been inadequate. Existing modalities for monitoring Akt activity utilize techniques such as western blotting or immunocytochemistry using a phospho-specific target antibody, and in vitro kinase assays. However, these methods are invasive, cumbersome and only provide a “snap-shot” view of the kinase activity at a specific time point. Molecular imaging is defined as a non-invasive visual representation of biological processes at the cellular and molecular level in the whole organism while also encompassing the modalities and instrumentation to support the visualization and measurement of these processes.11 This is an attempt to bridge the gap between discovery of important disease biomarkers and their use in clinical. In the last decade, at least three different molecular imaging technologies have been utilized for the understanding of disease biomarkers, drug development or monitoring therapeutic outcome, which are (1) optical imaging (bioluminescence and fluorescence imaging) (2) Magentic resonance imaging (MRI) and (3) Nuclear imaging [e.g., single photon emission computed tomography (SPECT) and positron emission tomography (PET)]. They have emerged as powerful tools that enable real time and repetitive visualization of gene expression, signal transduction, protein-protein interaction and cell trafficking in intact cells and living animals. This emerging field of molecular imaging promises to bring in novel tools for the dynamic measurement of signaling cascades and its players thereby eliminating the needs for time-consuming dissection and histological methods for tissue analysis and longitudinal studies of biological processes. Molecular imaging tools provide non-invasive real time, dynamic imaging and quantification of kinase activity in living cells and subjects. These tools can aid in preclinical determination of drug dosage, schedule and combination. Further, since an increase in Akt activity is synonymous with cancer cell proliferation and tumor growth while the reduction in Akt kinase activity is associated with decreased tumorgenicity, molecular imaging of Akt activity could be exploited as a surrogate marker for monitoring real time response of cancer cells to therapeutic regimens. The applications of molecular imaging assays in vitro also provide an excellent platform for cell based high throughput screening. Inhibitors identified by such assays typically pass the barriers of solubility, membrane permeability and toxicity, thus enhancing their chance of being successful in experimental and clinical investigation. In summary, the development of molecular imaging tools for kinases would significantly enhance our understanding of the biology of cancer and assist in the development and evaluation of novel therapeutic agents.
We have recently described the development of a bioluminescent Akt reporter which can be used to measure Akt phosphorylation in cultured cells and mice (Fig. 2).12 This reporter was based on conformation dependent complementation of firefly luciferase. Protein complementation assays have their roots in protein engineering strategies wherein a monomeric reporter is split into two separate inactive components in such a way that when these components are brought into close proximity they reconstitute the original reporter activity. Protein complementation has been extensively exploited to understand protein-protein interaction. Historically, the yeast two hybrid system first described by Stan Fields13 is based on protein complementation of GAL4, a transcriptional activator protein. This discovery revolutionized the understanding of signaling cascades in eukaryotic organisms. Although, a plethora of interacting partners in a variety of signaling cascades was discovered, this system has limited utility in dissecting mammalian signaling pathways. A number of other reporters routinely utilized in understanding mammalian biology were engineered for complementation studies. These include fluorescent proteins (GFP and YFP), bioluminescent enzymes (Firefly luciferase and renilla luciferase), β-galactosidase, dihydrofolate reductase (DHFR) and TME1 β-lactamase.14
Of the different complementation assays, the bioluminescence reporter has emerged as a useful technique for small animal imaging. Luciferase is a photoprotein which modifies the substrate (luciferin) releasing photons in the presence of oxygen and ATP. The light emitted by Firefly luciferase appears blue to yellow green in color with an emission spectra that peaks at wavelength between 490 nm to 620 nm.15 There are more than 30 luciferase-luciferin systems of independent origin but the most utilized luciferase for in vivo molecular imaging is the ATP-dependent firefly (Photinus pyalis) luciferase.16 This is chiefly due to the fact that 30% of the light generated by firefly luciferase has an emission spectra above 600 nm, a region where the signal attenuation by the absorption and scattering properties of mammalian tissue is minimal.16,17 This is a major advantage compared to other optical imaging systems such as fluorescence imaging wherein the excitation light can also excite other naturally occurring fluorescent molecules in the body which may result in a high level of background autofluorescence.
An optimized firefly luciferase protein fragment complementation was developed by screening incremental truncation libraries of N- and C-terminal fragments of luciferase.18 The N-terminal and C-terminal luciferase fragments were fused with FRB of the mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively. The optimized pair of FRB-NLuc/CLuc-FKBP reconstituted luciferase activity upon single-site binding of rapamycin in an FK506-competitive manner. By employing this strategy, the investigators monitored the lower affinity protein-protein interaction, such as the phosphorylation-dependent interaction between human Cdc25C with 14-3-3ε in vivo. Paulmerugan et al., designed a complementation-based assay using renilla luciferase by exploiting the strong interaction of MyoD and Id.19 Recently, a non-ATP dependent Gaussia princeps luciferase enzyme was engineered for protein complementation assay and demonstrates cross talk between insulin and TGFβ signaling pathways.20
A number of groups have reported development of tools to monitor kinase activity such as serine/threonine kinase PKA, PKB, PKC, PKD and non-receptor tyrosine kinase Src in live mammalian cells by fluorescence resonance energy transfer (FRET).21–25 FRET is a mechanism by which energy is transferred from an excited donor fluorohore to an acceptor fluorophore when the two are in close proximity. The typical strategy for kinase imaging is based on conformational change induced by phosphorylation such that the phosphorylation brings the acceptor and donor fluorphores in close proximity resulting in FRET. Using this strategy, Sasaki26 described kinase reporter wherein the phosphorylation dependent intermolecular binding results in changes in fluorescence. The reporters contain two green fluorescent protein mutants, an Akt substrate, and a phosphorylation recognition domain. Phosphorylation of the Akt substrate in the reporter causes a change in FRET, allowing the detection of phosphorylation catalyzed by Akt. They used the endogenous Akt substrate nitric-oxide synthase and Bad, which were fused with the Golgi targeting domain and mitochondria targeting domain. By using these reporters, they suggested that activated Akt is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing into the cytosol, thereby resulting in efficient phosphorylation of its substrate proteins. A similar strategy was also used by Kunkel et al to monitor Akt activity in the cytosol and nucleus.21 They found that Akt signaling in the cytosol is more rapid and transient when compared to Akt signaling in the nucleus which indicates the possibility of differentially regulated phosphatase activity between these two compartments. Additionally, targeting the reporter to the plasma membrane, where Akt is activated, resulted in an accelerated and a prolonged response compared to the cytosol, suggesting that release of Akt or its substrates from the membrane is required for desensitization of Akt signaling. In summary, the novel biology of kinases has been deciphered through the use of FRET based kinase reporters in vitro.
With the goal of monitoring kinase activity in vivo, we have developed a split firefly luciferase based reporter wherein Akt activity can be measured by bioluminescence imaging.12 A hybrid polypeptide, BAR, was constructed in which an Akt consensus substrate peptide and phosphoamino acid binding domain (FHA2) are flanked by the amino- (N-Luc) and carboxyl- (C-Luc) terminal domains of the firefly luciferase reporter molecule (Fig. 2). In the presence of Akt kinase activity, phosphorylation of the Akt consensus substrate sequences within the reporter results in its interaction with the FHA2 domain, thus stearically preventing reconstitution of a functional luciferase reporter molecule. In the absence of Akt kinase activity, release of this stearic constraint allows reconstitution of the luciferase reporter molecule whose activity can be detected non-invasively by BLI. In contrast to the FRET-based reporters, which allow reporter activity to be monitored in single cells, the split luciferase reporter allows imaging in live cells and animals in a quantitative, dynamic and non-invasive manner. The inhibition of Akt activity using an Akt inhibitor (API-2) and a PI-3K inhibitor (perifosine) resulted in an increase of bioluminescence activity in a time- and dose-dependent manner, which indicates that BAR provides a surrogate for Akt activity in terms of quantity and dynamics. BAR was also used to study upstream signaling events of Akt. For example, stimulation of EGFR can be evaluated using Akt activity as a surrogate and monitored by bioluminescent imaging. The use of an EGFR inhibitor, erlotinib with an erlotinib-sensitive and an erlotinib-resistant cell lines resulted in differential activation of the BAR reporter.
The ability to non-invasively and quantitatively image Akt activity within a specific tissue in live animals significantly enhanced our understanding of pharmacokinetics and bioavailability of specific drugs. For example, at 40 mg/kg API-2 treatment, inhibitory levels of the compound were detected for up to 24 hours which decreased thereafter. In contrast, when 20 mg/kg was delivered, peak inhibition was detected at 12 hours and by 24 hours little inhibitory activity was detected (Fig. 3). In contrast to API-2 for which published pharmacokinetics data are not available, the pharmacokinetics of perifosine has been extensively studied. Published data demonstrated that high plasma concentrations of the drug could be detected for as long as seven days post treatment. Results obtained from the BAR reporter studies in live animals confirm this observation since high levels of Akt inhibitory activity was detected within two hours of treatment which remained elevated for seven days.
Molecular imaging of Akt in cells and live animals will greatly facilitate the process of target validation, dose and schedule optimization as well as for rapid identification of a lead compounds from a library using cell based high throughput screening. This technology will facilitate the determination of pharmacokinetics and pharmacodynamics of drugs in mice which may or may not be of relevance in humans. It provides a unique opportunity to design the optimal dosing and schedule regiments for maximal tumor control (Akt inhibition) and minimal normal tissue toxicity.
Integration of genetically encoded imaging reporters into cells and animals has provided a unique opportunity to monitor molecular, biochemical and cellular pathways in vivo. It will greatly facilitate the process of target validation and dose and schedule optimization, as well as providing a way to identify lead compounds from a library using cell-based, high-throughput screening.
We thank Steven Kronenberg for generation of Figure 1, and Christin Hamilton for critical reading of the manuscript. This work was supported by US National Institutes of Health grants P01CA85878, P50CA01014 and R24CA83099.