Pharmacological activation of the p53 pathway by a new compound CYZ2017 exerts anti-tumor effects

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Highlights

  • A new p53-MDM2 interaction inhibitor with novel scaffold has been found using p53-MDM2 redistribution assay.

  • CYZ2017 inhibit the proliferation of cancer cells dependent on p53 genotypes.

  • CYZ2017 induce a cell cycle arrest in G0/G1 stage and cell apoptosis in HCT116 cells.

  • CYZ2017 has anti-cancer activity in HCT116 xenograft mice model.

Abstract

Blockage of p53-MDM2 protein-protein interaction has long been a promising strategy of drug development for cancers with wild type p53. In this study, we report a new p53-MDM2 interaction inhibitor, CYZ2017, which could induce p53 nuclear translocation and possess p53-dependent anti-proliferation activity in a range of cancer cells. CYZ2017 treatment led to increase of p53 levels and induced the transactivation of its target genes p21. In addition, CYZ2017 induced G0/G1 cell cycle arrest and apoptosis in HCT116 cells. Besides, CYZ2017 suppressed tumor growth in a HCT116 xenograft model without visible toxicity. These results support that CYZ2017 might be a promising p53-MDM2 interaction inhibitor with good anti-tumor activity. Our finding provides some cues for further investigation of developing anti-tumor drugs based on the blockage of p53-MDM2 interaction.

Introduction

Cancer remains the leading lethal disease of people although much effort has been made. It has been reported that 1,762,450 new cancer cases and 606,880 cancer deaths were projected to occur in the United States in 2019 [1]. The latest data from Chinese National Cancer Center showed that about 3929 thousand cancer cases and 2238 thousand cancer-related death cases were reported in China in 2015 [2]. The situation of cancer prevention and control is still serious although great progresses have been made in the last decades. Therefore, new drugs for cancers are still urgently needed.

p53 gene is a tumor suppressor gene closely related to human tumors. It plays important roles in maintaining genome stability, inhibiting and preventing cell transformation. More than half of all human tumors are related to the mutation of p53 gene. And the function and expression of p53 gene are obviously inhibited in the remaining patients without mutation of p53 gene. MDM2 is the major negative regulation factor of p53. It induces p53 degradation through the ubiquitin-proteasome pathway and inactivates its tumor-suppressing activity [3]. Meanwhile, a negative-feedback loop exists between p53 and MDM2 proteins because p53 can regulate the transcription of mdm2 gene by its transactivation function [3]. MDM2 is frequently overexpressed in many cancers carrying wild-type p53 and can induce the transformation of some normal cells [4]. This pathway has long been an attractive target for cancer therapy and several inhibitors have been advanced into clinical trials.

The first small molecule Nutlin-3a that can selectively inhibit p53-MDM2 interaction through inducing p53 expression, enhancing its transcriptional activity and effectively inhibiting the growth of tumors with wild-type p53 [5]. One Nutlin derivative RG7112 exhibited good dose tolerance and positive effects in the treatment of acute or chronic leukemia in phase I clinical trial [6,7]. Several other agents, such as RG7388, MI-73301, Kevertrin, MK8242 and DS-3032 have been investigated in clinical trials [3,[8], [9], [10], [11]]. However, till now no p53-MDM2 interaction inhibitor has been approved. Hence, new scaffold inhibitors are still pursued.

Here, we screened a series of compounds synthesized by multi-component reactions using a p53-MDM2 screening cell model and discovered that a benzamide compound CYZ2017 had good activity in translocating p53 into the nucleus. Its anti-proliferative activity in several cell lines with different p53 genotypes was determined using Cell Counting Kit-8. The possible binding mode, and effects on p53-MDM2 pathway, cell cycles distribution, apoptosis and in vivo anti-tumor activity were also described in this study.

Section snippets

Materials and methods

Reagent CYZ2017, 2-(benzyloxy)-4-(7-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)imidazo [1,2-a]pyridin-3-yl)benzamide, whose structure is shown in Fig. 1A, with purity >98%, was obtained in our lab (The scheme was shown in supplementary information S1). The compound was dissolved in DMSO as 30 mM stock solution for cell-based assays. RG7112 was purchased from Selleck (USA) and dissolved in DMSO. For animal studies, the compound was dissolved in alcohol and diluted in sterile 0.1 M PBS (phosphate buffer

Screening of p53-MDM2 interaction inhibitor

A series of compounds was synthesized and screened for p53-MDM2 interaction inhibitors using p53-MDM2 redistribution assay. In the CHO cell model, p53 fused to GFP and MDM2 fused to PDE4A4 were both stably expressed. In the untreated cells, p53-GFP fused protein was sequestered in the cytoplasm by MDM2. When the cells were treated with a p53-MDM2 interaction inhibitor, it would dissociate the interaction and induce the nuclear translocation of p53-GFP. Hence, if nuclear green fluorescence was

Discussion

In the present study, we reported a novel p53-MDM2 binding inhibitor CYZ2017, which has good anti-tumor activity and is well-tolerated. CYZ2017 could induce p53 nuclear translocation and have p53-dependent anti-proliferation activity in multicellular models. CYZ2017 treatment resulted in increase of p53 protein levels and induced the transactivation of its targets p21 and MDM2. In addition, CYZ2017 induced G0/G1 cell cycle arrest and apoptosis in HCT116 cells. Besides, CYZ2017 suppressed tumor

Funding

The work was supported by the National Natural Science Foundation of China (21572067, 81501180) and Chinese Medicine Innovation Project of Shanghai Health Committee (ZYKC201601023).

Declaration of competing interest

There is no conflict of interest to be declared.

Acknowledgement

We thanked Prof. Yuanxiang Wang for helping us to revise the manuscript.

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