Research paper
Identification of highly potent and selective Cdc25 protein phosphatases inhibitors from miniaturization click-chemistry-based combinatorial libraries

https://doi.org/10.1016/j.ejmech.2019.111696Get rights and content

Highlights

  • 96 quinone derivatives were synthesized via miniaturized click-chemistry reaction.

  • M2N12 is a potent and selective Cdc25C inhibitor.

  • M2N12 showed significant anti-tumor activity against cancer cells.

  • The selectivity of M2N12 for Cdc25C was rationalized by MD simulation.

Abstract

Cell division cycle 25 (Cdc25) protein phosphatases play key roles in the transition between the cell cycle phases and their association with various cancers has been widely proven, which makes them ideal targets for anti-cancer treatment. Though several Cdc25 inhibitors have been developed, most of them displayed low activity and poor subtype selectivity. Therefore, it is extremely important to discover novel small molecule inhibitors with potent activities and significant selectivity for Cdc25 subtypes, not only served as drugs to treat cancer but also to probe its mechanism in transitions. In this study, miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening were used to discover selective Cdc25 inhibitors. The bioassay results showed that compound M2N12 proved to be the most potent Cdc25 inhibitor, which also act as a highly selective Cdc25C inhibitor and was about 9-fold potent than that of NSC 663284. Moreover, M2N12 showed remarkable anti-growth activity against the KB-VIN cell line, equivalent to that of PXL and NSC 663284. An all-atom molecular dynamics (MD) simulation approach was further employed to probe the significant selectivity of M2N12 for Cdc25C relative to its structural homologs Cdc25A and Cdc25B. Overall, above results make M2N12 a promising lead compound for further investigation and structural modification.

Introduction

Although there are different types of cancers, with each cancer type having diverse causes and various clinical symptoms, one of the most common features they all share is a disordered cell cycle. Therefore, a major strategy in anti-cancer treatment is to target molecules which regulate the cell cycle. The Cdc25 phosphatases are highly conserved dual specificity phosphatases that play important roles in cell cycle under normal conditions and in response to DNA damage [1]. They could activate cyclin-dependent kinases (CDKs) through the dephosphorylation of pThr14 and pTyr15 [2]. Cyclin-dependent kinases, the central regulators of the eukaryotic cell cycle, could phosphorylate and activate downstream players like retinoblastoma (Rb) protein to promote cell cycle progression [3]. Several studies have shown that the Cdc25 phosphatases are overexpressed in a wide variety of cancers [4]. Therefore, Cdc25 phosphatases have been demonstrated as promising targets for the discovery of novel small molecule antitumor drugs [[5], [6], [7]].

Cdc25 phosphatases are present in all eukaryotes except plants [8]. There are three subtypes of Cdc25 phosphatases including Cdc25A, Cdc25B and Cdc25C. Cdc25A is thought to be regulators of G1/S and G2/M transition, and it could dephosphorylate and activate the CDK2/cyclin A and CDK2/cyclin E complexes which is necessary for mitosis [9]. Cdc25B and Cdc25C are regulators of G2/M transition through their ability to dephosphorylate and activate Cdk1/cyclin A, Cdck2/cyclin A and Cdk1/cyclin B [1]. Cdc25 phosphatases also play a vital role in the checkpoint control in response to DNA damage induced by ionizing radiation (IR) or ultraviolet light (UV). Especially, Cdc25C is a crucial downstream effector of Chk1 response on account of its phosphorylation on Ser216 which can generate 14-3-3-mediated separation of the phosphatases away from their substrates and a consequential balance of Cdk1/cyclin B within the phosphorylated and inhibited state [10]. Previous research about FPD/AML (familial platelet disorder/acute myeloid leukemia) has illustrated that mutated Cdc25C could disrupt the G2/M checkpoint and promote malignant transformation [11]. It is also found that Cdc25C and phospho-Cdc25C (Ser216) play a key role in the development and progression of vulvar carcinomas [12]. Moreover, overexpression of Cdc25C and an alternatively spliced variant were found in prostate cancer, but the role of Cdc25C and its spliced variant is not clear [13]. Taken together, these results could present Cdc25C as a novel target for the treatment of cancer while potent and selective Cdc25C inhibitors could help disrupt the pathogenesis of these cancers.

Recently, many new Cdc25 phosphatase inhibitors were derived from a para-quinonoid structure using VK3 as a lead compound. Besides, small molecule Cdc25 phosphatase inhibitors derived from natural products have also served as research hotspots [[14], [15], [16], [17]]. Currently, although Cdc25 phosphatase inhibitors have been developed, only few of them demonstrate inhibitory activities in vivo [18]. And most of the Cdc25 phosphatase inhibitors demonstrated with low activity and poor selectivity. Moreover, small molecule inhibitors with subtype selectivity can not only serve as probe tools to study the mechanism of the targets, but also have a broad application prospects in clinical drug development [19]. Above all, it is necessary to discover novel Cdc25 phosphatase inhibitors with potent activity and significant subtype selectivity. Previous research indicated that the privileged quinone structure plays an important role to maintain the compounds inhibitory activity against Cdc25 phosphatase. Thus, with the aim to discover promising Cdc25 phosphatase inhibitors with potent inhibitory activity and high subtype selectivity, we chose the representative quinone inhibitors NSC 663284, NSC 668394 and Cpd5 as lead compounds. For the catalytic domains of Cdc25 lack any apparent substrate recognition site and their active site face is surprisingly flat, these lead to difficulties in rational drug design according to structure-activity relationship [20]. Therefore, it is necessary to introduce structural diversity of substituents to modify quinone scaffold.

In order to rapidly generate a large number of diverse molecules, we employed copper(I)-catalyzed azide–alkyne (3 + 2) dipolar cycloaddition (CuAAC) reaction to yield 1,4-disubstituted-1,2,3-triazoles. The reaction is one of the extant click reactions which is the most versatile, enabling exclusive formation of 1,4-disubstituted-1,2,3-triazoles in high yields under mild reaction conditions. Due to its reliability, specificity, and biocompatibility, the 1,2,3-triazole structural motif is regarded as a safe bioequivalent surrogate (nonclassical bioisostere) widely employed in drug design [21]. Besides, previous research demonstrated that “click chemistry” coupled with miniaturization synthesis and in-situ screening is a rapid, reliable and efficient tool to discover novel compounds with different biological activities [22]. Therefore, in this study, we devised a method of parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening. In this method, we introduced the alkyne functional group onto to the quinone skeletons and yielded the privileged alkynyl fragments M1 and M2 (Fig. 1) and obtained a variety of azide substituents. Then each reaction was carried out by incubating an azide substituent with alkynyl fragment in 96-wall plates yielded 96-member quinone derivatives library, and the reaction mixtures were used to test the inhibitory activity against Cdc25s directly without purification. Further in vitro investigations and molecular dynamics (MD) simulation led to the identification of compound M2N12 as a promising lead compound for further investigation.

Section snippets

Building and characterizing the libraries by click chemistry [23,24].

The goal of this work was the synthesis of large libraries of quinone derivatives to identify a suitable lead compound for the search of novel selective Cdc25C inhibitors. Therefore, based on the detailed analysis of the structure-activity relationship (SAR) of substituted quinone compounds demonstrated as Cdc25s inhibitors, we reasoned that generating broad unbiased structural diversity might be more powerful than incremental optimization for developing improved Cdc25s inhibitors. Therefore,

Conclusion

For the Cdc25s were key phosphatases in cell cycle and overexpressed in various types of cancer, Cdc25 inhibitors would be a good target for cancer treatment. In this work, a total of 96 quinone derivatives were synthesized by microscale click chemistry reaction and tested for in vitro biological activities by in situ screening, and interestingly, all the tested compounds showed excellent inhibitory activities toward Cdc25 (A, B and C). Particularly, M2N12, a promising lead compound, with

Chemistry

1H NMR and 13C NMR spectra were obtained via a Bruker Avance-400 NMR spectrometer in Cdcl3 or DMSO using TMS as internal reference. Chemical shifts were expressed in δ units (ppm) and J values were presented in hertz (Hz). All melting points of the compounds were determined on a micro melting point apparatus and were uncorrected. Related mass spectra dates were determined by a LC Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel GF254 for TLC and spots were

Author contributions

All authors contributed to writing the manuscript. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

Acknowledgements

Financial support from the National Natural Science Foundation of China (NSFC Nos. 81603028, 81573347), the Natural Science Foundation of Shandong Province (No. ZR2016HB26), Young Scholars Program of Shandong University (YSPSDU, No. 2016WLJH32), the Fundamental Research Funds of Shandong University (No. 2017JC006), Key research and development project of Shandong Province (No. 2017CXGC1401) and National Institutes of Health (Grant AI033066) are gratefully acknowledged. The authors acknowledge

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