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PDF file - 1469K, Different ABL TKIs demonstrate potency differences in relative cellular and biochemical target inhibition (S1); Incomplete restoration of BCR-ABL signaling activity following washout of dasatinib or ponatinib tracks with intracellular drug retention and commitment to apoptosis in LAMA cells (S2); Incomplete restoration of BCR-ABL signaling activity following washout of nilotinib, imatinib, or DCC-2036 tracks with intracellular drug retention and commitment to apoptosis in LAMA cells (S3); BCR-ABL signaling is partially attenuated in conditions of continuous low concentrations of ABL TKIs that induce apoptosis (S4); Commitment to apoptosis in primary CML cells tracks with intracellular retention of ABL TKIs (S5); Intracellular retention of dasatinib, nilotinib, and imatinib is reduced following washout in primary CML cells harboring the resistant BCR-ABLT315I mutant (S6).

PDF file - 337K, Summary of mean fluorescence intensity values for levels of CrkL and STAT5 phosphorylation in K562 cells following acute ABL TKI exposure followed by standard and expanded washout (S1); Kinase:inhibitor dissociation off-rates for ABL TKIs (S2).

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AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

Her3 (also known as ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be 'undruggable' using ATP-competitive small molecules because it lacks appreciable kinase activity. Here we report what is to our knowledge the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3-dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor® 647-labeled HDAC inhibitor or "tracer," which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.

Deubiquitinating enzymes (DUBs) proteolytically cleave ubiquitin from ubiquitinated proteins, and inhibition of DUBs that rescue oncogenic proteins from proteasomal degradation is of emerging therapeutic interest. Recently, USP2 and UCH37 have been shown to deubiquitinate tumor-growth-promoting proteins, and other DUBs have been shown to be overexpressed in cancer cells. Therefore inhibition of DUBs is of interest as a potential therapeutic strategy for treating cancer. DUBs require the presence of properly folded ubiquitin protein in the substrate for efficient proteolysis, which precludes the use of synthetic peptide substrates in DUB activity assays. Because of the requirement for full-length ubiquitin, substrates suitable for use in fluorescent assays to identify or study DUB inhibitors have been difficult to prepare. We describe the development of a time-resolved fluorescence resonance energy transfer (FRET)-based DUB substrate that incorporates full-length ubiquitin that is site-specifically labeled using genetically encoded yellow fluorescent protein (YFP) and a chemically attached terbium donor. The intact substrate shows a high degree of FRET between terbium and YFP, whereas DUB-dependent cleavage leads to a decrease in FRET.

The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Interaction of wild-type AR LBD with the random phage display peptide D11FxxLF was observed with dihydrotestosterone (DHT), testosterone, R1881, estradiol, spironolactone, progesterone, and cortisol resulting in distinct dose dependency (EC50) values for each ligand and correlating well with the reported rank order potency of these agonists. Increasing concentrations of cyproterone acetate and mifepristone resulted in more complete disruption of the DHT-mediated AR-D11FxxLF peptide interaction, while flutamide, hydroxyflutamide, and bicalutamide caused only partial disruption of the complex. The mutant AR T877A LBD exhibited increased binding affinities for all ligands tested except for bicalutamide, mifepristone, DHT, and R1881 in a competitive binding assay as compared to wild-type AR LBD. This mutation was also characterized by increased ligand potency for agonist-induced peptide recruitment. Although usually an antagonist, hydroxyflutamide was more potent in the recruitment of D11FxxLF or an SRC3-1 LXXLL motif to AR T877A LBD than AR LBD. The antagonist cyproterone acetate behaved as a full antagonist of D11FxxLF recruitment to AR LBD and AR T877A LBD but as a more potent agonist in the recruitment of SRC3-1 to AR T877A LBD. These results suggest that the AR T877A mutation affects both ligand affinity and ligand dose dependency for peptide recruitment and may explain in part the altered responses of antagonists and increased transcriptional activation reported in androgen-independent prostate cancers.

Fluorescence-based kinase assays using peptide substrates are an established format for high-throughput screening and profiling of kinases. Among fluorescence-based formats, time-resolved fluorescence resonance energy transfer (TR-FRET) using a lanthanide donor species has advantages over other fluorescent formats in being resistant to many types of optical interference such as autofluorescent compounds, scattered light from precipitated compounds, or colored compounds that absorb excitation or emission radiation ("color quenchers"). By taking advantage of the fact that acceptors such as fluorescein or green fluorescent protein (GFP) can be paired with a terbium donor in a TR-FRET assay, we have developed TR-FRET kinase assays that use physiologically relevant native protein substrates, either labeled with fluorescein or expressed as GFP fusions. Phosphorylation of the labeled protein substrate results in an increase in TR-FRET when incubated with a terbium-labeled antibody that specifically recognizes the phosphorylated product. Thus, a strategy of using terbium-based TR-FRET can be applied to develop kinase assays, and the unique properties of terbium lead to a high degree of flexibility with regard to specific assay design.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes. We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate. Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies. Cancer Res; 73(11); 3356–70. ©2013 AACR.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a frequent cause of late-onset autosomal dominant Parkinson's disease (PD). Some disease-associated mutations directly affect LRRK2 kinase activity and inhibition of LRRK2 is viewed as a potential therapeutic treatment for PD. We demonstrate by both binding and enzymatic assays that alterations in the kinase activity of the PD-associated mutants I2020T and G2019S are due in part to altered ATP affinity. In binding assays, G2019S and I2020T have approximately 2-fold lower and 6-fold higher ATP affinity, respectively, than wild-type LRRK2. Furthermore, using an in vitro kinase activity assay, we demonstrate that at ATP concentrations close to cellular levels (1 mM) I2020T is approximately 10-fold more resistant to ATP-competitive kinase inhibitors than wild-type whereas G2019S is 1.6-fold more sensitive. These results predict that LRRK2 status may impact kinase inhibitor potencies in vivo or in cellular models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (Lantha-ScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRETassays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation,G2019S, results in a robust enhancement of kinase activity with LRRKtideas the substrate. This mutation also affects binding of ATP to LRRK2,with wild-type binding being tighter (Km,app of 57 lm) than with theG2019S mutant (Km,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in nutrient sensing and cell growth and is a validated target for oncology and immunosuppression. Two modes of direct small-molecule inhibition of mTOR activity are known: targeting of the kinase active site and a unique mode in which the small molecule rapamycin, in complex with FKBP12 (the 12-kDa FK506 binding protein), binds to the FRB (FKBP12/rapamycin binding) domain of mTOR and inhibits kinase activity through a poorly defined mechanism. To facilitate the study of these processes, the authors have expressed and purified a truncated version of mTOR that contains the FRB and kinase domains and have developed homogeneous fluorescence-based assays to study mTOR activity. They demonstrate the utility of these assays in studies of active sitedirected and FRB domain-directed mTOR inhibition. The results suggest that these assays can replace traditional radiometric or Western blot–based assays. (Journal of Biomolecular Screening 2008:238-244)

Abstract 3504 The imatinib paradigm established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). However, once-daily dasatinib (serum half-life: 3–5 h) is clinically effective despite only transient BCR-ABL inhibition, opening an opportunity for in-depth study of the mechanistic requirements for ABL TKI-induced CML cell death. Apoptosis commitment after potent, transient target inhibition is observed with ABL TKIs in vitro (Blood, 114, 2009, 3459–63), although variations in required TKI concentrations relative to their activity against BCR-ABL kinase suggest involvement of previously unrecognized factors. The “oncogenic shock” concept holds that temporary disruption of BCR-ABL-mediated prosurvival and proapoptotic signaling sets up a kinetic imbalance in favor of apoptosis. We have undertaken a comprehensive mechanistic exploration of this issue, wherein CML cells were transiently exposed to the ABL TKIs imatinib (50 and 500 nM), nilotinib (50 and 500 nM), dasatinib (10 and 100 nM), and ponatinib (AP24534; 10 and 100 nM) and then investigated with respect to pathways critical to drug efficacy and intracellular residence time. Cellular studies utilized multi-parameter intracellular FACS and immunoblot analysis, liquid chromatography-mass spectrometry, and high-throughput real-time qPCR expression analysis. Corresponding biochemical studies to determine ABL kinase/inhibitor dissociation parameters were also performed. All four ABL TKIs tested were capable of triggering apoptosis following transient exposure, although nilotinib and imatinib (which feature much narrower kinase target profiles than dasatinib and ponatinib) did so only at high concentrations. In contrast to potent, transient inhibition of BCR-ABL being necessary and sufficient for commitment of CML cells to apoptosis, we found that apoptosis could be reversed under conditions involving extensive additional TKI washout protocols. Consistent with the best indicator of apoptosis induction in our experiments being incomplete restoration of BCR-ABL signaling activity to pre-treatment levels, in all cases for which apoptosis commitment was irreversible, we identified a small, functionally important pool of intracellular TKI after washout of drug from the media. This property correlated with results of ABL kinase/inhibitor dissociation studies. For example, we found that ponatinib is a tight-binding inhibitor with a remarkably slow off-rate (t1/2 >95 h). Transient exposure to ponatinib followed by thorough washout committed CML cells to apoptosis despite very low intracellular concentrations that did not completely inhibit BCR-ABL. Based on these findings, we explored the possibility that effective TKIs are inhibiting additional targets involved in committing cells to an apoptotic fate, and used high-throughput qPCR assays to identify a preliminary profile of 30 apoptosis-related genes differentially expressed in TKI-treatment conditions that do and do not irrevocably commit CML cells to apoptosis. Taken together, our findings reveal that even slightly attenuated restoration of BCR-ABL signaling correlates with apoptosis commitment and that cryptic cellular retention of ABL TKIs is important in mediating this effect, potentially via sustained low-level inhibition of auxiliary targets. By extension, monitoring intracellular drug levels by LC-MS may be informative, especially for short serum half-life kinase inhibitors such as dasatinib. These studies further establish and refine the guiding principles of commitment of CML cells to apoptosis and improve our ability to design kinase inhibitors for CML and other malignancies. Disclosures: Riddle: Life Technologies Corporation: Employment, Equity Ownership. Apgar:BD Biosciences: Employment. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.

Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for the well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET-based binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified or do not demonstrate measurable activity against putative or potential substrates. This assay addresses kinase assay requirements early in the drug discovery process, and should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Imatinib, are sometimes preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C95.

Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations to be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified, completely lack activity, or do not have a known substrate. These new assay options for addressing kinase assay requirements in early drug discovery should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Gleevec®, are generally preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3688.

There is a great clinical need to develop selective, high affinity kinase inhibitors. While many high throughput kinase activity assays have become readily available, easy-to-use and cost-effective, activity-based assays have significant limitations in terms of both the extent of target coverage and the type of information they can provide about compounds. We have developed a competitive TR-FRET binding assay platform for characterization of protein kinase inhibitors. This assay platform has several advantages over traditional activity-based assays, in part due to simplicity of the assays as well as the ability to measure binding events in real time. To extend the breadth of target coverage of this platform, we have developed novel tracers for Class I PI3-kinases and the related protein kinase mTOR. We present a comparison of inhibitor potencies measured in binding assays relative to kinase activity assays for a diverse set of commonly used PI3 kinase family inhibitors. These novel assays enable simple, rapid measurements of IC50 values for PI3 kinases and mTOR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 362.