ATP-competitive Kinase Inhibitors Research Articles

Jak1 Has a Dominant Role over Jak3 in Signal Transduction through γc-Containing Cytokine Receptors

Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: CHARACTERIZATION OF A SELECTIVE ALLOSTERIC KINASE INHIBITOR

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

Discovery of selective irreversible inhibitors for EGFR-T790M.

Targeting the epidermal growth factor receptor kinase (EGFR) with ATP-competitive kinase inhibitors results in dramatic but short-lived responses in patients with EGFR mutant non small cell lung cancer. A series of novel covalent EGFR kinase inhibitors with selectivity for the clinically relevant T790M 'gatekeeper' resistance mutation relative to wild-type EGFR were discovered by library screening. A representative compound 3i was obtained through a systematic SAR study guided by mutant EGFR-dependent cellular proliferation assays.

A New BCR-ABL1 Mutation (L248R) Is Highly Resistant to Imatinib, Bosutinib, Nilotinib and Dasatinib, but Can Be Inhibited by AP-24534 and DCC-2036.

AbstractAbstract 3398Chronic Myeloid Leukemia (CML) treatment was radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion kinase Bcr-Abl. Second generation ATP-competitive tyrosine kinase inhibitors (TKIs) bosutinib (BOS), nilotinib (NIL) and dasatinib (DAS) further improved CML therapy. However, resistance to TKIs occurs in a variable proportion of patients; it can arise from different mechanisms but in the majority of cases it is due to Bcr-Abl point mutations that alter directly or indirectly the drug-protein binding. Over 70 mutations have been described in patients, with T315I showing resistance to IM, NIL, BOS and DAS. Here we report the discovery of a new P-loop mutant (L248R) that is highly resistant to all currently available inhibitors. L248R was identified in a lymphoid Blast Crisis CML patient. The patient initially presented with an IM-resistant F359I mutation. A cytogenetic and molecular response was obtained with BOS, but after 1 year haematological relapse developed; at this point a F359I/L248R clone was identified. L248R was previously reported only in an in vitro during a mutagenesis screen involving IM (Bradeen et al. 2006) and in a study with the T315I inhibitor SGX393 (O'Hare et al. 2008). L248R was never isolated from a clinical sample. Activity profile of BOS, IM, DAS and NIL against L248R, F359I and F359I/L248R was performed (Table). Stable transfectant Ba/F3 cells were generated and the TKIs anti-proliferative activity was determined. The relative IC50 increase over wild type Bcr-Abl (Relative Resistance, RR) was calculated. We classified RR values in four categories: sensitive (RR≤ 2), moderately resistant (RR between 2.1 and 4), resistant (RR between 4.1 and 10) or highly resistant (RR>10). In all cases a RR >10 was obtained, for L248R. Recently new compounds were developed to overcome resistance generated by the T315I mutant. Among them AP-24534 (AP), a panBcr-Abl inhibitor (O'Hare et al. 2009) and the switch pocket inhibitor DCC-2036 (DCC) were reported as potently active against the T315I mutation. Both compounds showed activity against L248R (RR 6.2 and 0.4), although the activity against the double mutant F359I/L248R was reduced especially for AP (RR 17.7 and 1.0). The activity profile of AP and DCC against L248R and a panel of 26 mutated forms of Bcr/Abl covering the most common mutations is also presented (Table). Activity of BOS, IM, DAS and NIL is also reported for comparison. According to our data, only mutation E255V is classified as highly resistant to AP, in addition to F359I/L248R: 6/26 mutations are considered “resistant” to AP (L248R, G250E, G252H, E255K, F359V, H396R) and 3/26 to DCC (D276G, E279K, F317V). The huge difference in the IC50 values for AP between Bcr/Abl wild type (2.1 nM) and parental Ba/F3 cells (>1000 nM) could render some of the mutants with high RR values to AP still sensitive to this inhibitor. It is also important to note that, according to our data, every mutation analysed shows sensitivity to at least one of the tested TKIs. In conclusion we describe a novel mutation that is highly resistant to the commonly used Bcr-Abl TKIs but which is inhibited by AP and DCC. Modelling data on the L248R mutant in complex with different TKIs will also be presented [Display omitted] IC 50 values are based on tritiated thymidine incorporation assay. Results are an average of at least 3 independent experiments Disclosures:Wise:Deciphera Pharmaceuticals: Employment. Flynn:Deciphera Pharmaceuticals: Employment. Gambacorti-Passerini:Pfizer pharmaceuticals: Research Funding.

Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells

AbstractAbstract 791Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures:Guichard:AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

New Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/ Pk25 design

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on theE. coliprotein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence ofStreptomyces coelicolorPk25 protein kinase. Mutant and wild-typeaphVIII were cloned intoE. coliwith the catalytic domain ofpk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants ofE. colicontaining bothaphVIIIand рк25were more resistant to kanamycin than those carrying onlyaphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of РknB ofMycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.

Knowledge-Based Scoring Functions in Drug Design. 1. Developing a Target-Specific Method for Kinase−Ligand Interactions

Protein kinases are attractive targets for therapeutic interventions in many diseases. Due to their importance in drug discovery, a kinase family-specific potential of mean force (PMF) scoring function, kinase-PMF, was developed to assess the binding of ATP-competitive kinase inhibitors. It is hypothesized that target-specific PMF scoring functions may achieve increased performance in scoring along with the growth of the PDB database. The kinase-PMF inherits the functions and atom types in PMF04 and uses a kinase data set of 872 complexes to derive the potentials. The performance of kinase-PMF was evaluated with an external test set containing 128 kinase crystal structures. We compared it with eight scoring functions commonly used in computer-aided drug design, either in terms of the retrieval rate of retrieving "right" conformations or a virtual screening study. The evaluation results clearly demonstrate that a target-specific scoring function is a promising way to improve prediction power in structure-based drug design compared with other general scoring functions. To provide this rescoring service for researchers, a publicly accessible Web site was established at http://202.127.30.184:8080/scoring/index.jsp .

Abstract A10: Off-target toxicity in white blood cells by AG-012986, a pan-CDK inhibitor, is mediated through inhibition of p38 MAPK signaling pathway

Abstract Cyclin-dependent kinases (CDKs) have been an attractive target for cancer research because these kinases are predominantly active during cell division. AG-012986 is a small molecule pan-CDK inhibitor that has in vitro and in vivo antitumor properties. This compound was stopped during development due in part to rapid bone-marrow independent white blood cell toxicity in non-human primate and rodent species, and the potential for acute and delayed immunosuppression in humans. Because peripheral leukocytes are largely nonproliferating cells, it was hypothesized that the effects of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. Using primary human peripheral blood mononuclear cells (PBMCs) and T-cells, we show that AG-012986 has a toxicity profile with greater specificity and more rapid cell death compared to the prototypical ATP-competitive kinase inhibitor, staurosporine. The cell toxicity mechanism was caspase-dependent and contained the hallmarks of apoptosis as indicated by cells staining double positive for propidium iodide and annexin-V. PBMCs or T-cells treated with AG-012986 and acutely stimulated either pharmacologically or through the T-cell receptor (TCR), exhibited decreased cellular toxicity compared to non-stimulated control cells. Interestingly under activating conditions with the pan-CDK compound, T-cells did not undergo significant cell division, as measured by BrdU incorporation, and were also rescued from apoptosis. This indicates that the pharmacology of AG-012986 was functioning as expected but that the toxicity due to its off-target effect had now been decoupled through cellular activation. Investigating the downstream signaling network showed that phosphorylation of p38 MAP kinase was impaired in the presence of AG-012986. Thus we hypothesize that AG-012986 may target the upstream kinase MKK3 and/or MKK6 for inhibition under basal conditions, leading to cell death in T cells and perhaps other white blood cells. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A10.

Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment

Over the past few years a number of components of the PI3K/mTOR pathway have been the subject of intense drug discovery activities both in pharmaceutical companies and in academia. This review article summarizes progress made in the identification and development of allosteric and ATP-competitive kinase inhibitors of mTOR and their potential therapeutic use in oncology.

Abstract 5753: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

Abstract Activating mutations in KRAS or B-RAF are found in over 30% of all human tumors and targeting this pathway could have broad therapeutic impact. Small molecule ATP-competitive RAF kinase inhibitors have potent anti-tumor effects on B-RAFV600E tumors but, in contrast to MEK inhibitors, are not potent against RAS mutant tumor models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. In this study we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on cellular context. In B-RAFV600E tumors, RAF inhibitors effectively block the MAPK signaling pathway and decrease tumor growth. Surprisingly, in KRAS mutant and RAS/RAF wildtype tumors, RAF inhibitors activate the RAF/MEK/ERK pathway in a RAS-dependent manner, thus enhancing tumor growth in some xenograft models. Inhibitor binding activates wildtype RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, rather, linked to direct conformational effects of inhibitors on the RAF kinase domain. Based on these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signaling pathways, depending upon cellular context. In addition, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors. 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 5753.

Abstract 1533: Sunitinib reverses ABCG2/BCRP-mediated multidrug resistance by inhibiting its efflux activity

Abstract Background and objective: Overexpression of ATP-binding cassette (ABC) transporters, which play a key role in the development of multidrug resistance (MDR), is a significant impediment to successful cancer treatment. Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we investigated in vitro the possible interaction of sunitinib with the major members of the ABC transporters: P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP). Methods: Cytotoxicity tests were performed using the MTT assay; The intracellular accumulation of doxorubicin and rhodamine 123 (Rho123) was analysed by flow cytometry; Membrane vesicles were prepared by the nitrogen cavitation and transport assays were performed using the rapid filtration method to confirm the effect of sunitinib on the transporter activity of ABCG2; Western Blotting was used to determine the protein expression; and reverse transcription-PCR was used to assess the expression of ABCG2 at mRNA level. Results: The IC50 concentration for parental cell line S1 to topotecan or doxorubicin were 0.135 ± 0.056 µM and 0.165 ± 0.009 µM, respectively; However, the concentration of topotecan or doxorubicin required to inhibit ABCG2- overexpressing cell line S1-M1-80 by 50% was about 5.132 ± 2.024 and 6.845 ± 1.035 µM; Coincubation with 2.5 sunitinib, the concentrations required to inhibit the growth of S1-M1-80 cells by 50% for topotecan was 0.091 ± 0.012 µM, and for doxorubicin was 0.181 ± 0.037 µM, respectively. Sunitinib potently reverses ABCG2-mediated resistance to topotecan and doxorubicin in vitro and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1-overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1-overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine-123 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [3H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Conclusions: Sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic. 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 1533.

RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

Beyond Rapalog Therapy: Preclinical Pharmacology and Antitumor Activity of WYE-125132, an ATP-Competitive and Specific Inhibitor of mTORC1 and mTORC2

The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC(50): 0.19 +/- 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy.

SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

Cytostatic Activity of Adenosine Triphosphate-Competitive Kinase Inhibitors inBRAFMutant Thyroid Carcinoma Cells

The V600E mutation accounts for the vast majority of thyroid carcinoma-associated BRAF mutations. The aim was to study the effects of the two BRAF V600E ATP-competitive kinase inhibitors, PLX4032 and PLX4720, in thyroid carcinoma cell lines. We examined the activity of PLX4032 and PLX4720 in thyroid carcinoma cell lines harboring BRAF V600E (8505C, BCPAP, SW1736, BHT101), NRAS Q61R (HTH7), KRAS G12R (CAL62), HRAS G13R (C643), or RET/PTC1 (TPC-1) oncogenes. Normal thyrocytes (PC Cl 3) were used as control. Both compounds inhibited the proliferation of BRAF mutant cell lines, but not normal thyrocytes, with a half maximal effective concentration (EC(50)) ranging from 78-113 nm for PLX4720 and from 29-97 nm for PLX4032. Doses equal to or higher than 500 nm were required to achieve a similar effect in BRAF wild-type cancer cells. Phosphorylation of ERK 1/2 and MAPK kinase (MEK) 1/2 decreased upon PLX4032 and PLX4720 treatment in BRAF mutant thyroid carcinoma cells but not in normal thyroid cells or in cell lines harboring mutations of RAS or RET/PTC1 rearrangements. PLX4032 and PLX4720 treatment induced a G(1) block and altered expression of genes involved in the control of G(1)-S cell-cycle transition. 8505C cell tumor xenografts were smaller in nude mice treated with PLX4032 than in control mice. This inhibition was associated with reduction of phospho-ERK and phospho-MEK levels. This study provides additional evidence of the promising nature of mutant BRAF as a molecular target for thyroid carcinoma cells.

An allosteric kinase inhibitor binds the p21-activated kinase autoregulatory domain covalently

Kinases are important therapeutic targets in oncology due to their frequent deregulation in cancer. Typical ATP-competitive kinase inhibitors, however, also inhibit off-target kinases that could lead to drug toxicity. Allosteric inhibitors represent an alternative approach to achieve greater kinase selectivity, although examples of such compounds are few. Here, we elucidate the mechanism of action of IPA-3, an allosteric inhibitor of Pak kinase activation. We show that IPA-3 binds covalently to the Pak1 regulatory domain and prevents binding to the upstream activator Cdc42. Preactivated Pak1, however, is neither inhibited nor bound significantly by IPA-3, demonstrating exquisite conformational specificity of the interaction. Using radiolabeled IPA-3, we show that inhibitor binding is specific and reversible in reducing environments. Finally, cell experiments using IPA-3 implicate Pak1 in phorbol-ester-stimulated membrane ruffling. This study reveals a novel allosteric mechanism for kinase inhibition through covalent targeting of a regulatory domain.

Reduction of BCR-ABL1 mutant clones after discontinuation of TKI therapy

In this letter we describe two case reports of CML patients with acquired mutations of the BCR-ABL1 kinase domain, in whom the mutant clone regressed and drug sensitivity was restored after temporary interruption of TKI.We believe that temporary interruption of an ATP-competitive tyrosine kinase inhibitor and switching to non-selective therapy can be a valid therapeutic option in CML patients. In addition, we highlight the potential of a flow cytometric CRKL phosphorylation assay to explore TKI sensitivity in CML cells ex vivo, and its correlation with clinical and haematological sensitivity or resistance.

Leucine-rich repeat kinase 2 mutants I2020T and G2019S exhibit altered kinase inhibitor sensitivity

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.

Sensitization of ABCG2-overexpressing cells to conventional chemotherapeutic agent by sunitinib was associated with inhibiting the function of ABCG2

Sunitinib is an ATP-competitive multi-targeted tyrosine kinase inhibitor. In this study, we evaluated the possible interaction of sunitinib with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), breast cancer resistance protein (BCRP, ABCG2) and lung-resistance protein (LRP) in vitro. Our results showed that sunitinib completely reverse drug resistance mediated by ABCG2 at a non-toxic concentration of 2.5 μM and has no significant reversal effect on ABCB1-, ABCC1- and LRP-mediated drug resistance, although a small synergetic effect was observed in combining sunitinib and conventional chemotherapeutic agents in ABCB1 overexpressing MCF-7/adr and parental sensitive MCF-7 cells, ABCC1 overexpressing C-A120 and parental sensitive KB-3-1 cells. Sunitinib significantly increased intracellular accumulation of rhodamine 123 and doxorubicin and remarkably inhibited the efflux of rhodamine 123 and methotrexate by ABCG2 in ABCG2-overexpressing cells, and also profoundly inhibited the transport of [ 3H]-methotrexate by ABCG2. However, sunitinib did not affect the expression of ABCG2 at mRNA or protein levels. In addition, sunitinib did not block the phosphorylation of Akt and Erk1/2 in ABCG2-overexpressing or parental sensitive cells. Overall, we conclude that sunitinib reverses ABCG2-mediated MDR through inhibiting the drug efflux function of ABCG2. These findings may be useful for cancer combinational therapy with sunitinib in the clinic.

573 POSTER Identification of potent, selective JAK2 inhibitors using a fragment-based screening approach

Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the “analog-sensitive” mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.