Anticancer Effect And Mechanism Study Of Novel Imidazopyridine Derivatives As Potent CDC25Inhibitors

Backgrounds:Different types of cancers may have different pathogenesis and show different clinical symptoms, but one of the most prominent features shared in cancer cells is the deregulated growth. The uncontrolled proliferation is mostly attributed to cell cycle deregulation. Cell cycle is a tightly controlled procedure, which needs a delicate signaling network to determine the proper progression. Cyclin-dependent kinases (CDKs) are foremost cell cycle regulators that phosphorylate and activate the downstream players like retinoblastoma (Rb) protein to promote cell cycle progression. The cell division cycle25(CDC25) family of proteins are highly conserved dual specificity phosphatases that could activate CDKs, which in turn regulate progression through the cell division cycle.CDC25dual phosphatase family can be found in all eukaryotic organisms except plants. In mammalian cells, it has three members:CDC25A, CDC25B, and CDC25C. CDC25A acts on the control of Gl-to-S and G2-to-S transitions in cell cycle whereas CDC25B and CDC25C are mainly responsible for regulating the progression at the G2-to-M transition. CDC25B is proposed to be responsible for the initial activation of CDK1-Cyclin B at the centrosome during the G2-M transition, which is then followed by a complete activation of CDK1-Cyclin B complexes by CDC25C in the nucleus at the onset of mitosis. Owing to such an important contribution to the cell cycle regulation, CDC25phosphatases have been considered to be involved in oncogenic transformations and human cancers. CDC25A and CDC25B overexpression are frequently found in many cancers, such as breast cancer, colorectal cancer, hepatocellular carcinoma, non-hodgkin lymphoma etc., and are often associated with high-grade tumors and poor prognosis. Therefore, the inhibition of CDC25phosphatases may represent a novel approach for the development of anticancer therapeutics, although more details about the involvement of CDC25A and CDC25B overexpression in tumorigenesis remain to be clarified.Many compounds with CDC25phosphatase inhibitory activities are currently being developed. The existing structural types include quinonoids, phosphate surrogates, or electrophilic derivatives. Starting from the structure of vitamin K published in1993as a potent CDC25inhibitor, numerous compounds were derived from a para-quinonoid structure. And many other quinonoid derivatives were able to affect CDC25activity and tumor cell proliferation. The most potent quinonoid-based compounds identified so far are active on xenografted tumour models. To date, there is no suitable inhibitor for further drug development, and thus innovative compounds are required.Nitrogen heterocycles are central structures for the chemistry of life, and continue to play an increasingly important role in lead discovery and biological activities in the pharmaceutical industry and academic research. In the area of anticancer drug discovery, design and synthesis of pharmacologically important heterocyclic structures are the main objective of medicinal chemists (Song Y. et al,Curr Pharm Des.2013;19(8):1528-48.).In recent years, heterocycle-thioacetic acid derivatives, a group of molecules containing a heterocycle core linked with a thioacetic acid-derived fragment, represent an important type of "privileged scaffold" possessing a wide spectrum of biological properties (Song Y. et al,Med. Chem. Commun.2013;4:810-816; Song Y. et al,Curr Pharm Des.2013;19(40):7141-54.).As a versatile and rigid-flexible joint scaffold for designing potential bioactive agents, heterocycle-thioacetic acid may offer the following advantages:1) They serve as versatile building blocks to position dependent functional groups for optimal engagement withthe binding pocket;2) as critical pharmacophore elements to make aromaticity interaction or hydrogen bonds for facilitating the spatial filling at the binding site of the drug-specific targets. Moreover, the relative flexibility of the "S-CH2-CO-" linker might allow heterocycle-thioacetic acid derivatives to adapt to a drug pocket more readily than the rigid ring structure. These versatile properties of heterocycle-thioacetic acids endow their derivatives with interesting bioactivities, thereby rapidly expanding the therapeutic arsenal.In view of its predominance and to seek new type of CDC25phosphatase inhibitor from this structure motif, in the present work, on the basis of our research interest involved in this scaffold, a focused in-house compound library containing more than60heterocycle-thioacetic acidderivatives was constructed and assayed for their inhibitory activity against CDC25phosphatase, which led to the identification of a set of novel imidazopyridine derivatives as potent CDC25inhibitors. Further in vitro and in vivo investigations led to the identification of compound CHEQ-2as a promising anticancer drug candidate with remarkable antiproliferative effects.Objective:The present study was designed to screen theanticancer effects of six novel imidazopyridine derivativesas potent CDC25inhibitors. Among them, the most potent derivative was CHEQ-2. Therefore, further in vitro and in vivo investigations were conducted to evaluate the potential of CHEQ-2as an anticancer drug candidate. These findings make it a good starting point for further investigation and structure modification.Methods:The enzyme inhibition activity of the novel imidazopyridine derivatives was measured by using OMFP as fluorescent substrate. The effects of CHEQ-1～CHEQ-6on cellular viability were assessed by using the MTT assay in human hepatocellular carcinoma HepG2cells and human colon cancer HT-29cells. The antiproliferation effect of CHEQ-2was assayed and compared with VK3and XDW-1in more different types of tumor cell lines. The ratio of MCF-7, HepG2and HT-29cells in the G0/G1, S and G2/M phases of cell cycle was determined by their DNA content using PI fluorescent dye. And western blotting were used to assay the expression of CDC25A, CDC25B, CDK2, Cyclin A, Cyclin B and Cyclin E. In addition, the apoptosis induction of CHEQ-2was estimated with Hoechst33342staining and Annexin V/PI staining analysis by using flow cytometry. The expression of apoptosis-related proteins such as Caspase-3, PARP, Bax and Bcl-2were evaluated by western blotting. Fluorescent probe DCFH-DA and JC-1were employed to determine the ROS production and mitochondrial membrane potential in MCF-7, HepG2and HT-29cells. The antioxidant NAC was employed to determine the contribution of ROS in the anti-cancer effects of CHEQ-2. In acute toxicity experiment, mortality and clinical signs were continually observed for6h after oral administration of abdominal injection of a single dose (2000mg/kg) on day0and at one time per day for2weeks. The animals (10mice per group) were weighed before dosing (day0, justified as100%) and after dosing (day1to day7, once a day) using an electronic balance. In vivo experiments, the anti-tumor effect of CHEQ-2was examined by HepG2xenografts bearing in mice. And the tumors were harvested, weighed, and prepared for further examination forthe expression levels of CDC25A, CDC25B,CDK2, Cyclin A, Cyclin B and Cyclin E.Results:The six imidazopyridine derivatives showed notable activity against CDC25A/B with IC50valuesranged from0.3to30μM. And compared with VK3and XDW-1, most of the compounds (except for CHEQ-4and CHEQ-6) displayed equivalent or more potent antiproliferative activities in HT-29and HepG2cell lines.CHEQ-2was the most potent inhibitor of CDC25A/B activity as well as cell proliferation. Further, the inhibitory effects of CHEQ-2on a mass variety of different tumor cells were investigated. CHEQ-2led to a decreased viability in these different tumor cells and IC50values were lower than VK3and XDW-1.According to the flow cytometry results, cells treated with different doses of CHEQ-2(7.5,15,30μM) for12,24and48h could trigger significant reduction of G0/G1phase cellsin MCF-7, HepG2and HT-29cell lines. And this reduction was accompanied by an apparent increase in the proportion of cells in the S phase in concentration-dependent manner. And the maximum increases of S-phase cell fraction reached40.0%(MCF-7),38.1%(HepG2) and27.7%(HT-29) respectively. According to western blotting results, CHEQ-2not only suppressed the expression of CDC25A and B, but also inhibited the expression of CDK2, Cyclin A, B and E, which mediated the S-phase cell cycle arrest.The induction of apoptosis by CHEQ-2was first proved by Hoechst staining under fluorescence microscopy. After treatment with CHEQ-2for48hours, cells of all three cell lines showed bright fluorescent nuclei blebbing and DNA fragmentation, comparing with non-treated control. CHEQ-2-induced apoptosis was also confirmed by flow cytometric analysis of MCF-7, HepG2and HT-29cells stained with annexin V and PI. The results showed that CHEQ-2caused a dose-dependent increase in both early and late stage of apoptosis in all three cell lines, and the apoptosis rate increased to49.5%(MCF-7),45.7%(HepG2), and52.8%(HT-29). Evaluation of apoptosis protein showedthat CHEQ-2dramatically induced activation of caspase3and PARP. Ourwork also found that CHEQ-2treatment caused downregulation of Bcl-2expression, as well as elevated Bax protein levelsin MCF-7, HepG2and HT-29cells.We performed DCFH-DA-based fluorescence detection by both fluorescence microscopy and flow cytometry in MCF-7, HepG2and HT-29cells. The results showed that CHEQ-2treatment elevated ROS levels in all three cell lines in a dose-dependent manner. Moreover, CHEQ-2treatment resulted in a dose-dependent loss of mitochondrial potential in all three cell lines, as evidenced by the shift of fluorescence. And30μM of CHEQ-2caused an increase (31.1%(MCF-7),67.6% (HepG2) and29.1%(HT-29)) of cells with green fluorescence, indicating that CHEQ-2treatments led to reduction of mitochondrial membrane potential and dysfunction of mitochondria.Treatment of5mM NAC significantly suppressed CHEQ-2-induced ROS generation in all three cell lines. Moreover, NAC could attenuat CHEQ-2induced elevation of percentages of cells with green fluorescence in all three cell lines which indicated NAC could abrogate the loss of mitochondrial membrane potential in these cells. Meanwhile, it was noteworthy that CHEQ-2-mediated inhibition on MCF-7, HepG2and HT-29cell viability was also compromised by the pre-incubation of5mM NAC.In acute toxicity experiment, there were no animal deaths in oral administration groups and only1death in abdominal injection CHEQ-2-treated group. Accordingly, the approximate lethal doses of CHEQ-2in female mice are higher than2000mg/kg. Observed clinical signs included loss of appetite and energy in3/4animals after dosing for1or2days in CHEQ-22000mg/kg group. The symptoms were found more serious in the abdominal injection groups, and there was also a temporary body weight drop on the second day of dosing (day1). Other than that, normal body weight gains were observed, no significant differences were observed between the vehicle control and CHEQ-2treatment groups. No abnormal gross findings were observed in any animals.In vivo experiments, oral administration of CHEQ-2(10mg/kg) significantly inhibited xenografted human liver tumor growth in nude mice. The tumor weight was reduced by43.0%in CHEQ-2-treated mice compared to that of the vehicle group. And the body weight of mice was consistent within14days of treatment with and without CHEQ-2. All animals showed no signs of weight loss or severe adverse action and none of the mice died during this study.Conclusions: In summary, the present study described the discovery and characterization of a novel inhibitor of CDC25A/B, CHEQ-2. We provide evidence that CHEQ-2improved the inhibitory potential compared to positive compound VK3as well as XDW-1, and good inhibition on the proliferation of tumor cells. Moreover, CHEQ-2could cause dramatic S-phase cell cycle arrest by suppressing CDC25A/B expression, eventually leads to activation of apoptosis. The CHEQ-2-treated cells also exhibited much higher ROS level and apparent decline in membrane potential, which may attribute to the cell apoptosis and could be abolished by NAC treatment. The in vivo approach further proved CHEQ-2could inhibit the tumor growth in nude mice with low toxicity. Over all, with robust anticancer potential and lower toxicity, CHEQ-2is more effective on multiple cancer cell death than positive compound VK3and XDW-1. The expression of CDC25A, B, CDK2, and Cyclin A, B, E in the tumor tissues was further studied by western blotting. Compared with the vehicle, the CDC25A, CDC25B and Cyclin B expression were significantly reducedby51.3%,61.1%and64.1%respectively in the tissue from the CHEQ-2treated mice. Therefore, this compound deserves further investigation in the hope of providing new alternatives to treat cancer patients.