Discovery and Synthesis of a Pyrimidine-Based Aurora Kinase Inhibitor to Reduce Levels of MYC Oncoproteins.

The A-type Aurora kinase is upregulated in many human cancers, and it stabilizes MYC-family oncoproteins, which have long been considered an undruggable target. Here, we describe the design and synthesis of a series of pyrimidine-based derivatives able to inhibit Aurora A kinase activity and reduce levels of cMYC and MYCN. Through structure-based drug design of a small molecule that induces the DFG-out conformation of Aurora A kinase, lead compound 13 was identified, which potently (IC50 < 200 nM) inhibited the proliferation of high-MYC expressing small-cell lung cancer (SCLC) cell lines. Pharmacokinetic optimization of 13 by prodrug strategies resulted in orally bioavailable 25, which demonstrated an 8-fold higher oral AUC (F = 62.3%). Pharmacodynamic studies of 25 showed it to effectively reduce cMYC protein levels, leading to >80% tumor regression of NCI-H446 SCLC xenograft tumors in mice. These results support the potential of 25 for the treatment of MYC-amplified cancers including SCLC.

Discovery of novel 2,4-disubstituted pyrimidines as Aurora kinase inhibitors.

In order to explore novel Aurora kinase inhibitors, a series of novel 2,4-disubstituted pyrimidines were designed, synthesized and evaluated their in vitro anti-proliferative activities against a panel of cancerous cell lines (A549, HCT-116 and MCF-7). Among them, compound 12a showed the moderate to high anti-proliferative activities against A549 (IC50 = 12.05 ± 0.45 µM), HCT-116 (IC50 = 1.31 ± 0.41 µM) and MCF-7 (IC50 = 20.53 ± 6.13 µM) cells, as well as the Aurora A and Aurora B inhibitory activities with the IC50 values of 309 nM and 293 nM, respectively. Furthermore, compound 12a induced apoptosis by upregulated the pro-apoptotic proteins Bax and decreased the anti-apoptotic protein Bcl-xl in HCT-116 cells. Moreover, the molecular docking study showed that compound 12a had good binding modes with Aurora A and Aurora B and the bioinformatics prediction discovered that compound 12a exhibited good drug likeness using SwissADME. Taken together, these results indicated that 12a may be a potential anticancer compound that was worthy of further development as Aurora kinase inhibitor.

Identification and evaluation of novel drug combinations of Aurora kinase inhibitor CCT137690 for enhanced efficacy in oral cancer cells.

Oral cancer is the most prevalent subtype of head and neck cancers and arises mainly from squamous cells of the oral cavity. Patients with advanced metastatic disease have poor overall survival resulting primarily from limited treatment options. Recent advances in the understanding of molecular basis of oral tumorigenesis provide an opportunity for identification and validation of new drug targets. The deregulated expression of the Aurora family of mitotic kinases, for example, has been associated with pathogenesis and poor prognosis in oral cancer. Here, we have evaluated the efficacy of the pan-Aurora inhibitor (CCT137690) alone and in combination with different chemotherapeutic and targeted drugs to identify its synergistic partners in oral cancer cell lines (ORL-48 and ORL-115). CCT137690 effectively inhibits Aurora kinases in both the cell lines and displays potent antiproliferative activity towards them. Prolonged treatment of these cells with CCT137690 results in abrogated mitotic spindle formation, misaligned chromosome attachment and polyploidy that ultimately leads to apoptotic cell death. We further identified that inhibitors of EGFR (gefitinib) and PI3-kinase (pictilisib) synergize with CCT137690 to inhibit the proliferation of the oral cancer cell lines. Moreover, we demonstrate that polyethylene glycol-based nanocapsules harboring combinations of CCT137690 with gefitinib or pictilisib inhibit the growth of oral cancer cell lines in 3D spheroid cultures and induce apoptosis that is comparable to free drug combinations. In conclusion, we have demonstrated the in vitro efficacy of CCT137690 in oral cancer cell lines, identified novel drug combinations with CCT137690 and synthesized nanocapsules containing these drug combinations for co-administration.

Induction of Mitotic Catastrophe via Inhibition of Aurora B by Ionizing Radiation With Additive of Mulberry Water Extract in Human Bladder Cancer Cells.

Mulberry fruit water extract (MWE) has been reported to synergistically enhance the cytotoxic effect of paclitaxel by promoting mitotic catastrophe to induce apoptosis in bladder cancer cells in our previous work. The aim of this study was to evaluate and to mechanistically explore the effects of MWE on bladder cancer responses to ionizing radiation (IR) by treating TSGH 8301 bladder carcinoma cells with MWE after exposing to IR. The results of MTT assay showed a synergistic cytotoxicity of IR with the co-treatment of MWE (IR/MWE) by inducing G2/M phase arrest as demonstrated by flow cytometry analysis in TSGH 8301, HT1367 and HT1197 bladder carcinoma cells lines. The IR/MWE-treated cells expressed increased levels of the G2/M phase arrest-related proteins cdc2/cyclin B1 and displayed giant multinucleated morphology, a typical characteristic of mitotic catastrophe. Immunofluorescent confocal microscopy revealed that the combined strategy inhibited Aurora B phosphorylation through Ras/Raf/MEK/ERK signaling cascade as demonstrated by Western blotting analysis. IR/MWE also caused an inhibitory effect on Plk1 and the subsequent downstream regulator RhoA repression and Cep55 induction, which would influence cell cycle progression in the early steps of cytokinesis. A profound tumor growth suppression and inactivation of Aurora B activity in the tumor tissues by IR/MWE treatment were confirmed in the TSGH 8301 xenograft model in vivo. These data demonstrated that MWE could be an effective auxiliary to synergize with radiation on the anticancer efficacy by promoting mitotic catastrophe through inhibition of Aurora B, providing a novel and effective therapeutic option for bladder cancer management.

Small Molecule Supplements Improve Cultured Megakaryocyte Polyploidization by Modulating Multiple Cell Cycle Regulators.

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.

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.

Bioactive compounds of Eriocaulon sieboldianum blocking proliferation and inducing apoptosis of HepG2 cells might be involved in Aurora kinase inhibition.

Eriocaulon sieboldianum (Sieb. & Zucc. ex Steud.) is an edible and medicinal plant used in traditional Chinese medicine. Often in combination with other herbs, it is processed into healthcare beverages for expelling wind-heat, protecting eyes, and reducing blood lipids. Besides, its water decoction together with other herbs has been utilized to treat cancer in China. However, the active ingredients and the precise cellular mechanisms of E. sieboldianum remain to be elucidated. The Aurora kinase family plays critical roles in the regulation of cell division and has attracted great attention to the identification of small-molecule Aurora kinase inhibitors for potential treatment of cancer. A molecular docking study was employed for docking of the most bioactive compounds. Hispidulin (HPDL) and quercetin-3-O-(6''-O-galloyl)-ß-D-galactopyranoside (QGGP) were singled out as potent inhibitors of Aurora kinase. Their inhibitory activity towards Aurora kinase was further confirmed by the obvious decrease in autophosphorylation of Aurora-A (Thr288) and Aurora-B (Thr232). Moreover, the induction of cell cycle arrest in HepG2 cells and the suppressed phosphorylation of histone H3 were also consistent with the inhibition of Aurora kinase. The data indicate that the E. sieboldianum extract and its two active compounds, HPDL and QGGP, could effectively induce apoptosis via p53, MAPKs and the mitochondrial apoptotic pathways. These findings could improve the understanding and enhance the development of drugs based on E. sieboldianum and raise its application value in anticancer therapy or prevention. In addition, our results indicated that Aurora kinase might be a novel target of HPDL and QGGP.

Identification of 3,5,6-substituted indolin-2-one's inhibitors of Aurora B by development of a luminescent kinase assay.

Aurora B kinase plays an important role in the cell normal mitosis and overexpresses in a variety of tumors. Inhibition of Aurora B kinase resulted in an apoptosis of cancer cells, which prevented tumor growth in xenograft models. In this Letter, we developed a luminescent kinase assay to perform high-throughput screening for identification of small molecule Aurora B inhibitors. Two 3,5,6-substituted indolin-2-one derivatives were identified within an in-house compound library. Their new derivatives were then designed and synthesized that resulting two new inhibitors of Aurora B kinase with improved potency. Docking simulation further demonstrated the proposed binding modes between indolin-2-one inhibitor and Aurora B.

Discovery of 4-aminoquinazoline--urea derivatives as Aurora kinase inhibitors with antiproliferative activity.

Two series of 20 novel 4-aminoquinazoline-urea derivatives have been designed and synthesized. The entire target compounds were investigated for their in vitro antiproliferative activity against six human cancer cell lines (K562, U937, A549, NCI-H661, HT29 and LoVo) using the MTT-based assay. Most compounds showed significant antiproliferative activities against four solid tumor cell lines, but no or poor activities against two leukemia cell lines. Furthermore, the target compounds were screened for Aurora A/B kinases inhibitory activity. Among them, 7c, 7d, 8c, and 8d are more potent against Aurora A kinase than ZM447439. Docking study of compounds 7d and ZM447439 revealed that they bound strongly to the ATP-binding sites of Aurora A and B. Thus, they may be promising lead compounds for the development of novel anti-tumor drug potentially via inhibiting Aurora kinases.

A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors.

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

Discovery of 7-aryl-substituted (1,5-naphthyridin-4-yl)ureas as aurora kinase inhibitors.

As part of our research projects to identify new chemical entities of biological interest, we developed a synthetic approach and the biological evaluation of (7-aryl-1,5-naphthyridin-4-yl)ureas as a novel class of Aurora kinase inhibitors for the treatment of malignant diseases based on pathological cell proliferation. 1,5-Naphthyridine derivatives showed excellent inhibitory activities toward Aurora kinases A and B, and the most active compound, 1-cyclopropyl-3-[7-(1-methyl-1H-pyrazol-4-yl)-1,5-naphthyridin-4-yl]urea (49), displayed IC50 values of 13 and 107 nM against Aurora kinases A and B, respectively. In addition, the selectivity toward a panel of seven cancer-related protein kinases was highlighted. In vitro ADME properties were also determined in order to rationalize the difficulties in correlating antiproliferative activity with Aurora kinase inhibition. Finally, the good safety profile of these compounds imparts promising potential for their further development as anticancer agents.