Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species.

CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies.

Antitumor profiles and cardiac electrophysiological effects of aurora kinase inhibitor ZM447439

Aurora kinases inhibitors, including ZM447439 (ZM), which suppress cell division, have attracted a great deal of attention as potential novel anti-cancer drugs. Several recent studies have confirmed the anti-cancer effects of ZM in various cancer cell lines. However, there have been no studies regarding the cardiac safety of this agent. We performed several cytotoxicity, invasion and migration assays to examine the anti-cancer effects of ZM. To evaluate the potential effects of ZM on cardiac repolarisation, whole-cell patch-clamp experiments were performed with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and cells with heterogeneous cardiac ion channel expression. We also conducted a contractility assay with rat ventricular myocytes to determine the effects of ZM on myocardial contraction and/or relaxation. In tests to determine in vitro efficacy, ZM inhibited the proliferation of A549, H1299 (lung cancer), MCF-7 (breast cancer) and HepG2 (hepatoma) cell lines with IC₅₀ in the submicromolar range, and attenuated the invasive and metastatic capacity of A549 cells. In cardiac toxicity testing, ZM did not significantly affect I(Na), I(Ks) or I(K1), but decreased I(hERG) in a dose-dependent manner (IC₅₀: 6.53 µM). In action potential (AP) assay using hiPSC-CMs, ZM did not induce any changes in AP parameters up to 3 µM, but it at 10 µM induced prolongation of AP duration. In summary, ZM showed potent broad-spectrum anti-tumor activity, but relatively low levels of cardiac side effects compared to the effective doses to tumor. Therefore, ZM has a potential to be a candidate as an anti-cancer with low cardiac toxicity.

MYCN-targeting vaccines and immunotherapeutics.

Amplification and concomitant overexpression of the MYCN oncogene is a frequent event in many malignancies including the childhood tumors, neuroblastoma and medulloblastoma. MYCN is only expressed in a defined time frame during early developmental processes, (1) which is beneficial for approaches combatting tumor-specific MYCN. However, MYCN is a transcription factors that was considered a poor drug target, until recent approaches suggested that down-regulation of MYCN could be possible by indirect targeting using Aurora kinase inhibitors or BET inhibitors. These concepts were proven using preclinical models (2-6) and are now entering clinical trials.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Combining an Aurora Kinase Inhibitor and a Death Receptor Ligand/Agonist Antibody Triggers Apoptosis in Melanoma Cells and Prevents Tumor Growth in Preclinical Mouse Models.

PURPOSE: Preclinical studies show that inhibition of aurora kinases in melanoma tumors induces senescence and reduces tumor growth, but does not cause tumor regression. Additional preclinical models are needed to identify agents that will synergize with aurora kinase inhibitors to induce tumor regression. EXPERIMENTAL DESIGN: We combined treatment with an aurora kinase A inhibitor, MLN8237, with agents that activate death receptors (Apo2L/TRAIL or death receptor 5 agonists) and monitored the ability of this treatment to induce tumor apoptosis and melanoma tumor regression using human cell lines and patient-derived xenograft (PDX) mouse models. RESULTS: We found that this combined treatment led to apoptosis and markedly reduced cell viability. Mechanistic analysis showed that the induction of tumor cell senescence in response to the AURKA inhibitor resulted in a decreased display of Apo2L/TRAIL decoy receptors and increased display of one Apo2L/TRAIL receptor (death receptor 5), resulting in enhanced response to death receptor ligand/agonists. When death receptors were activated in senescent tumor cells, both intrinsic and extrinsic apoptotic pathways were induced independent of BRAF, NRAS, or p53 mutation status. Senescent tumor cells exhibited BID-mediated mitochondrial depolarization in response to Apo2L/TRAIL treatment. In addition, senescent tumor cells had a lower apoptotic threshold due to decreased XIAP and survivin expression. Melanoma tumor xenografts of one human cell line and one PDX displayed total blockage of tumor growth when treated with MLN8237 combined with DR5 agonist antibody. CONCLUSIONS: These findings provide a strong rationale for combining senescence-inducing therapeutics with death receptor agonists for improved cancer treatment.

The discovery of aurora kinase inhibitor by multi-docking-based virtual screening.

We report the discovery of aurora kinase inhibitor using the fragment-based virtual screening by multi-docking strategy. Among a number of fragments collected from eMololecules, we found four fragment molecules showing potent activity (>50% at 100 µM) against aurora kinase. Based on the explored fragment scaffold, we selected two compounds in our synthesized library and validated the biological activity against Aurora kinase.

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma.

BACKGROUND: Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours. METHODS: Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines. RESULTS: Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments. CONCLUSION: Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Comparing immobilized kinase inhibitors and covalent ATP probes for proteomic profiling of kinase expression and drug selectivity.

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification in vivo. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼ 100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets.

A broad activity screen in support of a chemogenomic map for kinase signalling research and drug discovery.

Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

Development of a fluorescence intensity assay for the mitotic serine/threonine protein kinase Aurora-A.

The Aurora kinases are a group of serine/threonine protein kinases that regulate key steps during mitosis, and deregulation of these proteins (e.g., by gene amplification or overexpression) has been linked to a wide variety of tumor types. Thus, Aurora-A and Aurora-B have been intensely studied as targets for anticancer therapy and are now clinically validated targets. Here we report on the development of a novel fluorescence intensity binding assay for Aurora-A kinase inhibitors using a fluorescently labeled probe compound that shows intramolecular quenching when unbound but exhibits a dramatic increase in fluorescence when bound to Aurora-A.

Bioactive tanshinone I inhibits the growth of lung cancer in part via downregulation of Aurora A function.

Lung cancer is the leading cause of cancer death in the world, and the searching for novel efficacious and safe agents for lung cancer prevention remains the top priority of lung cancer research. In the present study, we evaluated the effect of bioactive tanshinones from a Chinese herb Salvia miltiorrhiza, cryptotanshinone (CT), tanshinone I (T1) and tanshinone IIA (T2A), on the proliferation inhibition of lung cancer cell lines. Tanshinones inhibited the lung cancer cell proliferation in vitro, with T1 the most potent, via cell cycle arrest and apoptosis induction. Gene function assay showed that Aurora A knockdown by siRNA dramatically eliminated the T1 activity in vitro, suggesting that Aurora A is an important functional target for T1. We further evaluated the effectiveness of T1 on the growth of H1299 nonsmall lung cancer cell in a mouse model. Tanshinone I inhibited the growth of H1299 lung tumor in a dose-dependent manner. Tanshinone I at 200 mg/kg body weight significantly reduced final tumor weight by 34% (P < 0.05) associated with inhibiting proliferation and inducing apoptosis of lung cancer cells by 54% (P < 0.001) and 193% (P < 0.001), respectively, inhibiting lung tumor angiogenesis by 72% (P < 0.001), and reducing Aurora A expression by 67% (P < 0.001). On the other hand, T1 did not significantly alter food intake or body weight. Our results provided experimental evidence to suggest that T1 may be an efficacious and safe agent for the prevention of lung cancer progression and Aurora A may be an important molecular target for T1 action against lung cancer.

The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle.

Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

A cell-based assay for screening spindle checkpoint inhibitors.

In eukaryotes, the spindle checkpoint acts as a surveillance mechanism that ensures faithful chromosome segregation. The spindle checkpoint prevents premature separation of sister chromatids and the onset of anaphase until every chromosome is properly attached to the mitotic spindle. Tumorigenesis might result from generation of aneuploidy by dysfunction of the spindle checkpoint. Differences of the checkpoint system in normal cells versus tumor cells might provide a new opportunity in cancer drug development; therefore, efforts to identify the spindle checkpoint inhibitors have been fostered. Based on spindle checkpoint inhibitors being able to induce cells to exit mitotic arrest caused by microtubule drug treatment, we developed a cell-based assay to screen compounds that were potential spindle checkpoint inhibitors. This assay was validated with a known spindle checkpoint inhibitor and was easy to adapt to a large-scale screening. It also had the advantages of being high in sensitivity and low in cost.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Ashwagandha derived withanone targets TPX2-Aurora A complex: computational and experimental evidence to its anticancer activity.

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug.

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors.

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.

Adaptation of the plasma inhibitory activity assay to detect Aurora, ABL and FLT3 kinase inhibition by AT9283 in pediatric leukemia.

Non-invasive assessment of biomarker modulation is important for evaluating targeted therapeutics, particularly in pediatrics. The plasma inhibitory activity (PIA) assay is used clinically to assess FLT3 inhibition ex vivo and guide dosing. AT9283 is a novel Aurora kinase inhibitor with secondary activity against FLT3 and ABL. We adapted the PIA assay to simultaneously detect inhibition of Aurora and FLT3 in AML, and Aurora and ABL in CML by AT9283. Furthermore, we optimized the assay for children, where limited blood volumes are available for pharmacodynamic studies. Simultaneously detecting multiple kinase inhibition may identify important mechanisms of action for novel anti-leukemic drugs.

Bioactive tanshinones in Salvia miltiorrhiza inhibit the growth of prostate cancer cells in vitro and in mice.

Searching for efficacious and safe agents for the chemoprevention and therapy of prostate cancer has become the top priority of research. The objective of this study was to determine the effects of a group of tanshinones from a Chinese herb Salvia Miltiorrhiza, cryptotanshinone (CT), tanshinone IIA (T2A) and tanshinone I (T1) on prostate cancer. The in vitro studies showed that these tanshinones inhibited the growth of human prostate cancer cell lines in a dose-dependent manner via cell cycle arrest and apoptosis induction. Among three compounds, T1 had the most potent activity with IC(50) s around 3-6 µM. On the other hand, tanshinones had much less adverse effects on the growth of normal prostate epithelial cells. The epigenetic pathway focused array assay identified Aurora A kinase as a possible target of tanshinone actions. The expression of Aurora A was overexpressed in prostate cancer cell lines. Moreover, knockdown of Aurora A in prostate cancer cells significantly decreased cell growth. Tanshinones significantly downregulated the Aurora A expression, suggesting Aurora A may be a functional target of tanshinones. Tanshinones, especially T1, also showed potent anti-angiogenesis activity in vitro and in vivo. Furthermore, T1 inhibited the growth of DU145 prostate tumor in mice associated with induction of apoptosis, decrease of proliferation, inhibition of angiogenesis and downregulation of Aurora A, whereas it did not alter food intake or body weight. Our results support that T1 may be an efficacious and safe chemopreventive or therapeutic agent against prostate cancer progression.

Bioisosteric approach to the discovery of imidazo[1,2-a]pyrazines as potent Aurora kinase inhibitors.

Our continued effort toward the development of the imidazo[1,2-a]pyrazine scaffold as Aurora kinase inhibitors is described. Bioisosteric approach was applied to optimize the 8-position of the core. Several new potent Aurora A/B dual inhibitors, such as 25k and 25l, were identified.

Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines.

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.