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A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer

Acta Pharmacologica Sinica volume 45, pages 422–435 (2024)Cite this article

Abstract

Extracellular regulated protein kinases 1/2 (ERK1/2) are key members of multiple signaling pathways, including the ErbB axis. Ectopic ERK1/2 activation contributes to various types of cancer, especially drug resistance to inhibitors of RTK, RAF and MEK, and specific ERK1/2 inhibitors are scarce. In this study, we identified a potential novel covalent ERK inhibitor, Laxiflorin B, which is a herbal compound with anticancer activity. However, Laxiflorin B is present at low levels in herbs; therefore, we adopted a semi-synthetic process for the efficient production of Laxiflorin B to improve the yield. Laxiflorin B induced mitochondria-mediated apoptosis via BAD activation in non-small-cell lung cancer (NSCLC) cells, especially in EGFR mutant subtypes. Transcriptomic analysis suggested that Laxiflorin B inhibits amphiregulin (AREG) and epiregulin (EREG) expression through ERK inhibition, and suppressed the activation of their receptors, ErbBs, via a positive feedback loop. Moreover, mass spectrometry analysis combined with computer simulation revealed that Laxiflorin B binds covalently to Cys-183 in the ATP-binding pocket of ERK1 via the D-ring, and Cys-178 of ERK1 through non-inhibitory binding of the A-ring. In a NSCLC tumor xenograft model in nude mice, Laxiflorin B also exhibited strong tumor suppressive effects with low toxicity and AREG and EREG were identified as biomarkers of Laxiflorin B efficacy. Finally, Laxiflorin B-4, a C-6 analog of Laxiflorin B, exhibited higher binding affinity for ERK1/2 and stronger tumor suppression. These findings provide a new approach to tumor inhibition using natural anticancer compounds.

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Fig. 1: Semi-synthetic production of Laxiflorin B from Eriocalyxin B.
Fig. 2: Inhibitory effects of Laxiflorin B on non-small cellular lung cancer cells.
Fig. 3: ERK1/2 as a potential novel target of Laxiflorin B.
Fig. 4: Laxiflorin B-induced apoptosis was mediated by BAD via ERK1/2 inhibition.
Fig. 5: Autocrine production of ERK1/2-downstream growth factors was abolished by Laxiflorin B.
Fig. 6: Anticancer effect of Laxiflorin B in vivo.
Fig. 7: The modifiable capacity of Laxiflorin B.
Fig. 8: Laxiflorin B inhibits the EGFR-related pathway in non-small-cell lung cancer by BAD activation and blockage of EREG/AREG-EGFR axis via a positive feedback loop.

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Acknowledgements

The authors would like to thank Dr. Jessica Tamanini of ETediting for editing the manuscript prior to submission and the Instrumental Analysis Center of Shenzhen University for the support. This work was supported by the Natural Science Foundation of Guangdong Province (No. 2021A1515011046, 2021A1515010996), the Guangdong Provincial Science and Technology Program (No. 2017B030301016), the Regional Joint Fund of Guangdong Province (Grant No. 2019B1515120080), the Shenzhen Municipal Government of China (No. JCYJ20210324093408024), the Shenzhen Key Medical Discipline Construction Fund (No. SZXK060).

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Author notes

  1. These authors contributed equally: Cheng-Yao Chiang, Min Zhang, Junrong Huang, Juan Zeng

Authors and Affiliations

  1. Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, International Cancer Center, Department of Cell Biology and Genetics, Shenzhen University Medical School; College of Life Sciences and Oceanography, Shenzhen University; Department of Pharmacy, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital (Shenzhen Institute of Translational Medicine), Shenzhen, 518055, China

    Cheng-Yao Chiang, Min Zhang, Junrong Huang, Chunlan Chen, Dongmei Pan, Heng Yang, Tiantian Zhang, Min Yang, Tian Xiao, Yongdong Zou, Lizhi Zhu & Duo Zheng

  2. School of Biomedical Engineering, Guangdong Medical University, Dongguan, 523808, China

    Juan Zeng

  3. SpecAlly Life Technology Co., Ltd, Wuhan, 430075, China

    Qiangqiang Han

  4. Wuhan Biobank Co., Ltd, Wuhan, 430074, China

    Qiangqiang Han & Zou Wang

  5. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

    Yangchao Chen

  6. Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen University Town, Xili, Shenzhen, 518055, China

    Feng Yin & Zigang Li

  7. Guangdong Provincial Key Laboratory of Systems Biology and Synthetic Biology for Urogenital Tumors, Shenzhen, 518035, China

    Lizhi Zhu

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Contributions

Conceived and designed the experiments: DZ, LZZ and CYC. Chemical compound synthesis: JRH and MY. Conducted most of the experiments and analyzed the data: MZ, CYC, JRH, CLC, DMP, TTZ, HY and TX. Construction of plasmids and lentivirus packaging: MZ, CYC. and CLC. Animal studies: CYC and FY. Performed the LC–MS/MS and proteomic analysis: QQH, ZW and YDZ. Performed the computer simulation of Laxiflorin B and ERK1/2 crystal structures: JZ. Planning and discussion of the project: DZ and LZZ. Supervised the entire project: YCC, DZ and LZZ. Wrote the manuscript, designed the layout of figures and tables: CYC, DZ, ZGL, and JZ.

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Correspondence to Lizhi Zhu or Duo Zheng.

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Chiang, CY., Zhang, M., Huang, J. et al. A novel selective ERK1/2 inhibitor, Laxiflorin B, targets EGFR mutation subtypes in non-small-cell lung cancer. Acta Pharmacol Sin 45, 422–435 (2024). https://doi.org/10.1038/s41401-023-01164-w

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