Research paper
Discovery of pyrazole derivatives as cellular active inhibitors of histone lysine specific demethylase 5B (KDM5B/JARID1B)

https://doi.org/10.1016/j.ejmech.2020.112161Get rights and content

Highlights

  • Pyrazole derivatives were discovered as novel KDM5B inhibitors.

  • 35 compounds were synthesized and studied on their KDM5B inhibitory activities for SARs.

  • Compound 27 ab is a potent and cellular active KDM5B inhibitor that can inhibit MKN45 cell proliferation and migration.

Abstract

KDM5B (also known as PLU-1 and JARID1B) is 2-oxoglutarate and Fe2+ dependent oxygenase that acts as a histone H3K4 demethylase, which is a key participant in inhibiting the expression of tumor suppressors as a drug target. Here, we present the discovery of pyrazole derivatives compound 5 by structure-based virtual screening and biochemical screening with IC50 of 9.320 μM against KDM5B, and its subsequent optimization to give 1-(4-methoxyphenyl)-N-(2-methyl-2-morpholinopropyl)-3-phenyl-1H-pyrazole-4-carboxamide (27 ab), a potent KDM5B inhibitor with IC50 of 0.0244 μM. In MKN45 cells, compound 27 ab can bind and stabilize KDM5B and induce the accumulation of H3K4me2/3, bona fide substrates of KDM5B, while keep the amount of H3K4me1, H3K9me2/3 and H3K27me2 without change. Further biological study also indicated that compound 27 ab is a potent cellular active KDM5B inhibitor that can inhibit MKN45 cell proliferation, wound healing and migration. In sum, our finding gives a novel structure for the discovery of KDM5B inhibitor and targeting KDM5B may be a new therapeutic strategy for gastric cancer treatment.

Introduction

To data, some studies indicate that the occurrence and development of cancer are closely related to epigenetics, including DNA and RNA modification, histone modification and non-coding RNAs [[1], [2], [3], [4], [5]]. Among the diverse epigenetic mechanisms that modify chromatin structure to regulate gene expression, histone modification plays a significant role in gene transcription, genomic integrity and chromosome stability [[6], [7], [8], [9]]. Until now, two classes of lysine demethylases can remove methyl groups from lysine residues: one is Histone lysine demethylase 1 (KDM1) subfamily, the other is Histone lysine demethylase 2–7 (KDM2-KDM7) subfamily which contain JmjC domain. KDM1 subfamily is flavin adenine dinucleotide (FAD) dependent oxygenase and KDM2-7 subfamily is α-ketoglutarate (2-OG)/Fe2+ - dependent oxygenase [[10], [11], [12]]. Of the JmjC containing KDMs, lysine demethylase 5B (KDM5B) is a lysine demethylase that can erase methyl group from H3K4me1/2/3 [13], and it is overexpressed and plays a vital role in diverse cancers [14,15], such as breast cancer [[16], [17], [18]], gastric cancer [[19], [20], [21], [22]], hepatocellular carcinoma [23], and lung cancer [24,25]. Hence, targeting KDM5B is a new therapeutic strategy for cancer treatment [26,27], and a range of small-molecule inhibitors of KDM5B has been reported, the majority of which are 2-OG competitive and coordinate to Fe2+ in the catalytic site, for example compounds 14 [[28], [29], [30], [31]] (Fig. 1). Although several KDM5B inhibitors have been developed, there is still no KDM5B inhibitors in clinic trials due to their cell permeability, specificity and potency and so on. Hence, it is still a challenging subject to develop potent KDM5B inhibitors to abrogate the abnormal overexpression and activation of KDM5B.

High-throughput virtual screening (HTVS) is a powerful and efficient tool for drug discovery [[32], [33], [34]]. In order to discover novel KDM5B inhibitor with new scaffold, HTVS was performed to identify hit compound using known receptor structure (PDB code: 5FYZ, resolution: 1.75 Å). Over 2 million compounds from the database Enamine and our in-house compound library (approximate 500 compounds) were screened, and hit compound 5 (IC50 = 9.320 μM) was found. Moreover, through subsequent structural based optimization, compound 27 ab was developed as a potent KDM5B inhibitor with IC50 = 0.0244 μM, which improves the potency for more than 382-fold, comparing to the hit compound 5.

Herein, we described the identification of pyrazole derivatives as KDM5B inhibitors with the aid of HTVS and the subsequent structure-based optimization. Among our synthesized compounds, compound 27 ab (IC50 = 0.0244 μM) is a potent and cellular active KDM5B inhibitor (Fig. 2) that can induce the accumulation of H3K4me2/3 as a chemical tool. Besides, compound 27 ab can also inhibit the proliferation, colony formation and would healing of gastric cancer cell line MKN45. Our finding gives a novel structure for the discovery of KDM5B inhibitor and targeting KDM5B may be a new therapeutic strategy for gastric cancer treatment.

Section snippets

The general routes

The general routes of the designed compounds were depicted in Scheme 1, Scheme 2, Scheme 3, Scheme 4. In this work, the pyrazole scaffold was also replaced with pyridine and triazole. The compounds 13a-b and 14a-b of general routes are presented in Scheme 1. Treatment of ketones 6 with tert-butyl hydrazinecarboxylate 7 in EtOH gave compounds 8a-b, which then reacted with POCl3 in DMF [35,36], affording compounds 9a-b [37]. Oxidization of 9a-b using KMnO4 yielded 10a-b. Compounds 13a-b and 14a-b

Conclusions

In this study, the (R)-N-(2-(2-chlorophenyl)-2-morpholinoethyl)-3-isopropyl-1H-pyrazole-4-carboxamide (compound 5) was firstly discovered as a KDM5B inhibitor based on HTVS and biochemical screening. Then, a series of pyrazole derivatives were designed and synthesized through structural based optimization and obtained compounds were applied to evaluate the inhibitory activities against KDM5B. Among them, 1-(4-methoxyphenyl)-N-(2-methyl-2-morpholinopropyl)-3-phenyl-1H-pyrazole-4-carboxamide

Materials

All the reagents and solvents used in the chemical synthesis were obtained from commercial sources and were used without further purification. 1H NMR and 13C NMR spectra data were obtained on Bruker AVANCE Ⅲ 400 M spectrometer (Bruker Instruments, Inc.), Chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane (TMS) and J values were reported in Hertz. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by

Declaration of competing interest

The authors declare no competing financial interest.

Acknowledgments

This work was supported by National Natural Science Foundation of China (Project No. 81903447, No. 81602961, No. 81430085, No. 21372206, No. 81703328); National Key Research Program (No. 2018YFE0195100, No. 2016YFA0501800 and No. 2017YFD0501401); Science and Technology Innovation Talents of Henan Provincial Education Department (19IRTSTHN001). We thank Xiaoling Zhang (Zhengzhou University) for providing the computing resources for docking analysis.

References (41)

  • M. Esteller

    Cancer epigenomics: DNA methylomes and histone-modification maps

    Nat. Rev. Genet.

    (2007)
  • A. Lujambio et al.

    Genetic unmasking of an epigenetically silenced microRNA in human cancer cells

    Canc. Res.

    (2007)
  • M.F. Fraga et al.

    Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer

    Nat. Genet.

    (2005)
  • A. Roberti et al.

    Epigenetics in cancer therapy and nanomedicine

    Clin. Epigenet.

    (2019)
  • N. Mosammaparast et al.

    Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases

    Annu. Rev. Biochem.

    (2010)
  • S.S. Ng et al.

    Dynamic protein methylation in chromatin biology

    Cell. Mol. Life Sci.

    (2009)
  • C. Martin et al.

    The diverse functions of histone lysine methylation

    Nat. Rev. Mol. Cell Biol.

    (2005)
  • Z. Zhao et al.

    Epigenetic modifications of histones in cancer

    Genome Biol.

    (2019)
  • T.E. McAllister et al.

    Recent progress in histone demethylase inhibitors

    J. Med. Chem.

    (2016)
  • D. Han et al.

    Lysine methylation of transcription factors in cancer

    Cell Death Dis.

    (2019)
  • Cited by (26)

    • Inhibitors of Jumonji-C domain-containing histone demethylases

      2023, Epigenetic Cancer Therapy, Second Edition
    • Identification of the upstream regulators of KDM5B in gastric cancer

      2022, Life Sciences
      Citation Excerpt :

      The present study confirmed that KDM5B is overexpressed at both transcription and protein levels and acts as a biomarker of poor prognosis in GC. Our group, therefore, synthesized some KDM5B inhibitors with novel structures [9]. However, owing to the structural similarity of KDM5B with other JHDMs, obtaining specific KDM5B inhibitors remains a challenge for medicinal chemists.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text