A quantitative LumiFluo assay to test inhibitory compounds blocking p53 degradation induced by human papillomavirus oncoprotein E6 in living cells

Lorenzo Messa,Chiara Bertagnin,Giorgio Palù,Giulio Nannetti,Marta Celegato,Arianna Loregian,Beatrice Mercorelli

doi:10.1038/s41598-018-24470-4

Lorenzo Messa, Chiara Bertagnin + Show 5 more

Open Access

https://doi.org/10.1038/s41598-018-24470-4

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Journal: Scientific Reports	Publication Date: Apr 16, 2018
Citations: 11	License type: open-access

Affiliation: University of Padua

Abstract

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.

Highlights

Papillomaviruses (PVs) are small DNA viruses that can infect a wide range of mammals, including humans, and cause distinctive hyperproliferative lesions of the skin[1]
Full-length high-risk E6 proteins can efficiently induce p53 degradation through the direct association with both p53 and the cellular ubiquitin ligase E6AP, to form a trimeric complex that leads to p53 ubiquitination and degradation[19]
The intimate addiction of cancer cells to the sustained activity of E6 represents an advantage for the development of anti-cancer drugs, since perturbing E6 activities can restore the intracellular levels of active p53 and reactivate p53-mediated pathways, leading to oncogene-induced senescence and eventually apoptosis of cancer cells[20]

Summary

Introduction

Papillomaviruses (PVs) are small DNA viruses that can infect a wide range of mammals, including humans, and cause distinctive hyperproliferative lesions of the skin[1]. Blocking the formation of the trimeric complex among E6, E6AP and p53 through a small-molecule compound represents a novel intriguing strategy to inhibit the E6-mediated degradation of p53 and to counteract the progression of HPV-associated cancers. A major limit was represented by the lack of simple biological assays able to and quantitatively evaluate the inhibitory activity of test compounds against the E6-mediated p53 degradation in a cellular context. To overcome this drawback, we developed a luminescence/fluorescence-based (LumiFluo) assay to quantitatively monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule compound testing

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