Kate Byth

AZD1152, a Selective Inhibitor of Aurora B Kinase, Inhibits Human Tumor Xenograft Growth by Inducing Apoptosis

Purpose: In the current study, we examined the in vivo effects of AZD1152, a novel and specific inhibitor of Aurora kinase activity (with selectivity for Aurora B). Experimental Design: The pharmacodynamic effects and efficacy of AZD1152 were determined in a panel of human tumor xenograft models. AZD1152 was dosed via several parenteral (s.c. osmotic mini-pump, i.p., and i.v.) routes. Results: AZD1152 potently inhibited the growth of human colon, lung, and hematologic tumor xenografts (mean tumor growth inhibition range, 55% to ≥100%; P < 0.05) in immunodeficient mice. Detailed pharmacodynamic analysis in colorectal SW620 tumor-bearing athymic rats treated i.v. with AZD1152 revealed a temporal sequence of phenotypic events in tumors: transient suppression of histone H3 phosphorylation followed by accumulation of 4N DNA in cells (2.4-fold higher compared with controls) and then an increased proportion of polyploid cells (>4N DNA, 2.3-fold higher compared with controls). Histologic analysis showed aberrant cell division that was concurrent with an increase in apoptosis in AZD1152-treated tumors. Bone marrow analyses revealed transient myelosuppression with the drug that was fully reversible following cessation of AZD1152 treatment. Conclusions: These data suggest that selective targeting of Aurora B kinase may be a promising therapeutic approach for the treatment of a range of malignancies. In addition to the suppression of histone H3 phosphorylation, determination of tumor cell polyploidy and apoptosis may be useful biomarkers for this class of therapeutic agent. AZD1152 is currently in phase I trials.

Imidazo[1,2-A]Pyridines: A Potent and Selective Class of Cyclin-Dependent Kinase Inhibitors Identified Through Structure-Based Hybridisation

High-throughput screening identified the imidazo[1,2-a]pyridine and bisanilinopyrimidine series as inhibitors of the cyclin-dependent kinase CDK4. Comparison of their experimentally-determined binding modes and emerging structure-activity trends led to the development of potent and selective imidazo[1,2-a]pyridine inhibitors for CDK4 and in particular CDK2.

Mechanism and in vitro pharmacology of TAK1 inhibition by (5Z)-7-Oxozeaenol.

Transforming growth factor-β activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.

AZ703, an Imidazo[1,2-a]Pyridine Inhibitor of Cyclin-Dependent Kinases 1 and 2, Induces E2F-1-Dependent Apoptosis Enhanced by Depletion of Cyclin-Dependent Kinase 9

Preclinical studies were performed of a novel selective imidazopyridine cyclin-dependent kinase (cdk) inhibitor, AZ703. In vitro kinase assays showed that IC50 values for AZ703 against purified cyclin E/cdk2 and cyclin B/cdk1 were 34 and 29 nmol/L, respectively. In contrast, the IC50 against cdk4 was 10 micromol/L. AZ703 also inhibited cdk7 and cdk9 with IC50 values of 2.1 micromol/L and 521 nmol/L, respectively. Treatment of U2OS, NCI-H1299, and A549 cells for 24 hours resulted in growth arrest involving multiple cell cycle phases. At low drug concentrations (< 2 micromol/L), G2 arrest predominated, whereas at higher concentrations (> or = 2 micromol/L), S-G2 arrest was observed. When cells were synchronized in G1 by starvation and released into AZ703, a block in G1 occurred that was not evident in exponentially growing cells. Cell cycle arrest was associated with reduced phosphorylation of the retinoblastoma protein and p27(Kip1) at cdk2 phospho-sites. Following longer exposures, apoptosis was evident. Cells were further sensitized to AZ703 following recruitment to S phase by synchronization. Consistent with the inhibition of cdks during S and G2 that modulate the activity and stability of E2F-1, AZ703 treatment induced E2F-1 expression. In U2OS and NCI-H1299 cells engineered to inducibly express the dominant-negative mutant E2F-1 (1-374), expression of the mutant decreased AZ703-mediated apoptosis, indicating dependence on E2F-1 transcriptional targets. AZ703-induced apoptosis in NCI-H1299 cells was enhanced by small interfering RNA-mediated depletion of cdk9, which caused reduced levels of Mcl-1 and XIAP, suggesting that cdk2, cdk1, and cdk9 represent a rational subset of family members for drug targeting.

AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts

Deregulation of the cell cycle has long been recognized as an essential driver of tumorigenesis, and agents that selectively target key cell cycle components continue to hold promise as potential therapeutics. We have developed AZD5438, a 4-(1-isopropyl-2-methylimidazol-5-yl)-2-(4-methylsulphonylanilino) pyrimidine, as a potent inhibitor of cyclin-dependent kinase (cdk) 1, 2, and 9 (IC50, 16, 6, and 20 nmol/L, respectively). In vitro, AZD5438 showed significant antiproliferative activity in human tumor cell lines (IC50 range, 0.2–1.7 μmol/L), causing inhibition of the phosphorylation of cdk substrates pRb, nucleolin, protein phosphatase 1a, and RNA polymerase II COOH-terminal domain and blocking cell cycling at G2-M, S, and G1 phases. In vivo, when orally administered at either 50 mg/kg twice daily or 75 mg/kg once daily, AZD5438 inhibited human tumor xenograft growth (maximum percentage tumor growth inhibition, range, 38–153; P < 0.05). In vivo, AZD5438 reduced the proportion of actively cycling cells. Further pharmacodynamic analysis of AZD5438-treated SW620 xenografts showed that efficacious doses of AZD5438 (>40% tumor growth inhibition) maintained suppression of biomarkers, such as phospho-pRbSer249/Thr252, for up to 16 hours following a single oral dose. A comparison of different schedules indicated that chronic daily oral dosing provided optimal cover to ensure antitumor efficacy. These data indicate that broad cdk inhibition may provide an effective method to impair the dysregulated cell cycle that drives tumorigenesis and AZD5438 has the pharmacologic profile that provides an ideal probe to test this premise. [Mol Cancer Ther 2009;8(7):1856–66]

Essential role of TAK1 in regulating mantle cell lymphoma survival

TGF-β-activated kinase 1 (TAK1), a member of the MAPK kinase family, plays a key role in B-cell growth and development. In the present study, we examined the potential role of TAK1 as a therapeutic target for lymphoma. Here, we show that the active phosphorylated form of TAK1 is abundantly expressed in a panel of lymphoma cell lines, including mantle cell, anaplastic large cell, and Hodgkin lymphoma cell lines. Silencing TAK1 expression via the use of siRNA inhibited the activation of NF-κB and p38 and induced apoptosis in lymphoma cell lines. Moreover, submicromolar concentrations of AZ-TAK1, a novel ATP-competitive small molecule inhibitor of TAK1, dephosphorylated TAK1, p38, and IκB-α in lymphoma cell lines. These molecular events were associated with the release of cytochrome c into the cytosol, down-regulation of X-linked inhibitor of apoptosis, activation of caspase 9, and induction of apoptosis. We also demonstrate that primary lymphoma cells express TAK1 and pTAK1 and were sensitive to AZ-TAK1-mediated cell death. Collectively, our data demonstrate an essential role for TAK1 in regulating critical survival mechanisms in lymphoma and suggest that it may serve as a therapeutic target.

The cellular phenotype of AZ703, a novel selective imidazo(1,2-a)pyridine cyclin-dependent kinase inhibitor

Because the majority of cancers exhibit direct or indirect deregulation of cyclin-dependent kinase (CDK) function, members of the CDK family are attractive targets for the development of anticancer agents. As part of an ongoing program, novel imidazopyridines were identified and developed as potent and selective CDK inhibitors. Here, we describe data on the in vitro biological activities of one of these compounds, AZ703. The selectivity profile of AZ703 was investigated in kinase assays against a range of CDK enzymes as well as a panel of protein kinases in vitro. IC50s were assessed against different tumor cell lines in vitro. The mechanism of action of AZ703 was determined by observing changes in phosphorylation of CDK substrates and cell cycle effects on tumor and normal cells. In vitro studies revealed that AZ703 is a selective inhibitor of CDK1 and CDK2 and displays a mode of action consistent with the induction of G1-, S-, and G2-M-phase arrest. AZ703 also showed potent antiproliferative activity across a wide range of tumor cell lines in vitro. Moreover, AZ703 induced reversible blockade of normal cells while causing tumor cells to undergo apoptosis. We have identified AZ703 as a novel selective imidazo[1,2-a]pyridine CDK inhibitor that shows promising antitumor properties in vitro. [Mol Cancer Ther 2006;5(3):655–64]

Imidazoles: Sar and Development of a Potent Class of Cyclin-Dependent Kinase Inhibitors.

An imidazole series of cyclin-dependent kinase (CDK) inhibitors has been developed. Protein inhibitor structure determination has provided an understanding of the emerging structure activity trends for the imidazole series. The introduction of a methyl sulfone at the aniline terminus led to a more orally bioavailable CDK inhibitor that was progressed into clinical development.

Imidazole pyrimidine amides as potent, orally bioavailable cyclin-dependent kinase inhibitors

The development of a novel series of imidazole pyrimidine amides as cyclin-dependent kinase (CDK) inhibitors is described. The series was found to have much improved CDK2 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. Control of overall lipophilicity was important to achieve good in vitro potency along with acceptable physiochemical properties and margins against inhibition of both CYP isoforms and the hERG potassium ion channel. A compound with an attractive overall balance of properties was profiled in vivo and possessed suitable physiochemical and pharmacokinetic profiles for oral dosing.

Novel approaches in oncology at AstraZeneca

Abstract Advances in the understanding of tumour biology have led to the discovery of new targets that control specific mechanisms essential for tumour spread, growth and survival. In order to fully explore the anticancer potential of these novel approaches, AstraZeneca is developing a broad pipeline of agents targeting a variety of key processes in tumour progression and metastasis. These include two novel antiangiogenic agents, ZD6474 and AZD2171, which are both orally available inhibitors of vascular endothelial growth factor receptor-tyrosine kinase, AZD2171 being a highly potent inhibitor; ZD6474 also has activity against epidermal growth factor receptor tyrosine kinase. Once-daily administration of these agents has been shown to result in effective inhibition of tumour growth in a broad spectrum of human xenograft models. In contrast to this approach, which prevents new vessel formation, the vascular-targeting agent ZD6126 disrupts the microtubular network responsible for maintaining the shape of immature endothelial cells, thereby selectively destroying the existing tumour vasculature and leading to extensive central necrosis. Other agents with a variety of novel antitumour strategies are also in development. These include AZD0530, an orally available Src kinase inhibitor, and AZD3409, an oral prenyl transferase inhibitor, both of which have potential for broad antitumour activity. In addition, an oral, selective, cyclin-dependent kinase inhibitor (AZD5438).