Improving colon cancer therapy with a new promising small-molecule activator of the p53-pathway through disruption of p53-MDM2/MDMX interactions

Abstract

Impairment of the tumour suppressor p53 pathway is a major event in human cancers, making p53 activation one of the most attractive therapeutic strategies [1]. This work describes the synthesis and biological evaluation of the (R)-tryptophanol-derived bicyclic lactam SYNAP as a selective p53 activator with potent anticancer activity against colon cancer [2]. The anticancer activity and mechanism of action of SYNAP was studied in both 2D and 3D models of human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53+/+) and the corresponding p53-null isogenic derivative cells (HCT116 p53-/-), alone and in combination with conventional chemotherapeutic agents. The anti-proliferative activity of SYNAP was analysed by sulforhodamine B assay and by clonogenic assays. The compound presented an anti-proliferative effect in human cancer cells dependent on p53 status. In HCT116 p53+/+cells, SYNAP p53-dependent growth inhibition was associated with cell cycle arrest and apoptosis, analysed by flow cytometry, and with anti-migratory activity. Western blot and RT-PCR analysis also showed an upregulation of several p53 transcriptional targets upon treatment with SYNAP. A yeast-based assay and a co-immunoprecipitation assay in human cancer cells were performed to study the disruption of the p53 interaction with its endogenous inhibitors murine double minute (MDM)2 and MDMX by SYNAP. The obtained results indicated that SYNAP potentially targeted p53 by disruption of the p53-MDM2/MDMX interactions. Moreover, SYNAP sensitized colon cancer cells to the cytotoxic effect of known chemotherapeutic agents. In addition, SYNAP did not induce acquired or cross-resistance and re-sensitized doxorubicin-resistant colon cancer cells to the therapy. Importantly, SYNAP was non-genotoxic and presented low cytotoxic effects against normal cells. Collectively, this work reports a new selective dual inhibitor of p53-MDM2/MDMX interactions with promising application in colon cancer therapy, both as monotherapy and in combination with known chemotherapeutic agents. Additionally, SYNAP represents a starting point for improved p53 activators, particularly inhibitors of the p53 interaction with MDM2 and MDMX. 1 – Graves B et al. PNAS 2012; 109: 11788-11793; 2 – Raimundo L et al. Br. J. Pharmacol. 2018; 175: 3947-3962. This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through grant UID/QUI/50006/2019. This work received financial support from the European Union (FEDER funds through the Operational Competitiveness Program (COMPETE) POCI-01-0145-FEDER-006684/POCI-01-0145-FEDER-007440 and (3599-PPCDT) PTDC/DTP-FTO/1981/2014 – POCI-01-0145-FEDER-016581) and the FCT grants PTDC/QUIQOR/29664/2017, UID/DTP/04138/2013 (iMed.ULisboa), IF/00732/2013 (M.M.M. Santos). We thank FCT and ESF (European Social Fund) through POCH (Programa Operacional Capital Humano) for: L. Raimundo PhD grant ref. SFRH/BD/117949/2016; J. Loureiro PhD grant ref SFRH/BD/128673/2017; M. Espadinha PhD grant ref SFRH/BD/117931/2016. J. Calheiro thanks ICETA for her grant ref. ICETA2019-71. We thank (POCH), specifically the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences; PD/00016/2012).