Original articleIn vitro and in vivo characterization of a benzofuran derivative, a potential anticancer agent, as a novel Aurora B kinase inhibitor
Graphical abstract
Introduction
Aurora is a protein kinase family consisting of Aurora A, Aurora B and Aurora C. Auroras are specifically expressed during mitosis and act as key regulators for multiple signaling networks [1], [2]. Aurora B is a chromosomal protein that is widely expressed in rapidly proliferating tissues and cells. The kinase binds other chromosomal passenger proteins, such as INCENP, survivin, and borealin [3], [4] and phosphorylates histone H3 at Ser 10. It is responsible for cell cycle checkpoint regulation and accurate cell division [5]. The knockdown or inactivation of Aurora B with siRNA or a small-molecule inhibitor impairs cell division, leading to mitotic arrest [6].
Aurora B is overexpressed in many tumors, particularly colon, cervix, liver, kidney, breast and prostate tumors [7]. Breast cancer patients with highly elevated Aurora B expression have the worst prognosis and decreased survival compared to patients with moderate expression of Aurora B [8]. High expression of Aurora B is also correlated with advanced stages of colon cancer [9]. Furthermore, the overexpression of Aurora B induces multi-nucleation and polyploidy in human cells [7]. Studies performed during the last decade suggest a high association of genome instability, cancer development and carcinogenesis with aberrant Aurora signaling [10], [11].
Because these kinases are specific mitotic regulators, Aurora kinases have been validated as drug targets for cancer treatment in several independent studies. In one of the very first studies, Taylor et al. proposed Aurora B as a molecular target for curing many cancers. This led to the development of a series of small-molecule Aurora inhibitors [12], such as ZM447439 (Fig. 1A). ZM447439 was found to inhibit the phosphorylation of histone H3 and impair the cell cycle checkpoint [13], [14]. Another compound Hesperadin (Fig. 1B) was discovered to suppress cancer cell proliferation via inhibition of Aurora B activity [7]. Both ZM447439 and Hesperadin are thought as first generation Aurora kinase inhibitors and were used as molecular tools to study the biology of Auroras and mitotic events. Sun et al. established a high-throughput screening to target Aurora A and obtained several lead compounds [15]. At that same year, VX-680 (Fig. 1C) was developed as an Aurora A inhibitor [16]. The preclinical study also showed it to be a potent inhibitor for Aurora B and C. VX-680 was found to block the phosphorylation of histone H3 on Ser 10 in cancer cells, inhibit cell proliferation and suppress the growth of pancreatic and colon cancer xenografts [15]. However, because of its highly toxic nature, clinical trials were suspended at Phase I. AZD1152-HPQA (Fig. 1D) is the active drug of AZD1152, which is an Aurora B inhibitor [17]. Preclinical studies identified AZD1152 to be very potent in blocking a series of human tumor xenografts with rates of growth inhibition ranging from 55 to 100%. Tumor growth inhibition was found to correlate with blocked phosphorylation of histone H3 at Ser 10 in vivo [18]. PHA-739358 (Fig. 1E) was identified as a pan Aurora inhibitor [19]. MLN8237 (Fig. 1F), an Aurora A inhibitor, improved disease free survival in 80% of solid tumor and 100% in acute lymphoblastic leukemia model [20]. Besides experimental screens, novel alternative compounds for Aurora inhibitors have been discovered by the application of silico approaches [21], [22]. Yan et al. built the classification models to discriminate the selectivity of 512 Aurora inhibitors. This model could be a novel strategy for virtual screen of selective Aurora A or B inhibitor [23]. Moreover, other small-molecule inhibitors, such as AT9283 (Fig. 1G) and SU6668 (Fig. 1H), were found to potently suppress endogenous Aurora B activity [24], [25]. Natural product Fisetin and Luteolin were also reported to inhibit Aurora B kinase activity in cancer cells [26], [27]. In addition to AZD1152, AT9283, PHA-739358, MLN8237 and ENMD2076 (Fig. 1I), which are in Phase II trials, the majority of Aurora inhibitors are either in Phase I trials or the preclinical phase [28], [29]. So far, no Aurora inhibitors are qualified to pass to Phase III or clinical application, so the discovery and investigation of Aurora inhibitors are still needed.
In our previous study, we conducted an in vitro high-throughput screening (HTS) radiometric assay to discover novel Aurora B inhibitors [27], [30], [31] After two rounds of screening a pool of small-molecule compounds, several candidates were selected based on their inhibitory activity and chemical structures. In this study, one of these lead compounds, a small-molecule benzofuran derivative (S6), was identified as an inhibitor that targets Aurora B in the physiological environment. This benzofuran derivative (S6) showed anti-cancer effects in cell culture and human tumor xenografted mouse model. We further classified and evaluated the selectivity of S6 in vitro and in vivo.
Section snippets
Chemicals and reagents
Compound S6, (2Z)-2-(3, 4-dihydroxybenzylidene)-1-benzofuran-3(2H)-one, was synthesized by Medicilon Inc. (≥95%, TLC, Shanghai, China). The chemical synthesis of the compound was previously described [32]. The compound was prepared in DMSO (Sigma Aldrich, St. Louis, MO, USA) to obtain a 10 mM stock solution. Aliquots were stored at −20 °C to avoid freeze–thaw cycles, and a working solution was freshly prepared with culture medium immediately prior to use.
Cell lines and cell culture
Cancer cell lines were purchased from
S6 inhibits Aurora B activity in vitro and binds recombinant Aurora B protein
S6 is a small-molecule benzofuran derivative (Fig. 2A) that was found to inhibit Aurora B kinase activity with an IC50 of 0.735 μM in an in vitro biochemistry assay (Z′-lyte, Invitrogen) (Fig. 2B). Nineteen other kinases were also screened (Table 1). S6 exhibited relatively high activity against Aurora A, with an IC50 of 1.34 μM, which is 1.8-fold higher than the IC50 for Aurora B. Moderate inhibition of GSK3β, FLT-1, PDGFRA, FLT-4 and KDR was also observed at 5∼8-fold higher IC50 values than
Discussion
Aurora kinases are considered as potential oncogenes. In previous reports, the overexpression of Aurora B was found to be highly associated with a poor prognosis or clinical outcomes in patients [35], [36]. Aurora A is a highly conserved homolog of Aurora B, although Aurora A has different functions from those of Aurora B in the cell cycle. The role of Aurora A or B inhibition in cancer treatment remains controversial. Taylor et al. found that colon cancer cells were more sensitive to Aurora B
Conclusion
We report the discovery and characterization of a benzofuran derivative (S6), a potential anticancer agent, as a novel Aurora B inhibitor. We provide sufficient experimental evidence to support the conclusion that Aurora B is the major molecular target of S6 in cancer cells and xenografted tumors. Furthermore, our animal model studies indicate that the drug is safe when administered in vivo. However, further studies to evaluate its off-target effects are warranted.
Acknowledgments
We thank Drs. Reynold C. Ly, Kaustubh N. Bhinge from Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic for critical reading of this manuscript and language modification, Dr. Jian An from Biochemistry and Molecular biology, Mayo Clinic for useful suggestions and Ms. Man Wu for proofreading tables and figures. This work was supported by the National 863 Project of China (2006AA020501, 2008AA02Z311), the National Key Sci-Tech Special Project of China (2008ZX10002-020).
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