A Chemical Genetic Screen for Modulators of Asymmetrical 2,2′-Dimeric Naphthoquinones Cytotoxicity in Yeast

Ashkan Emadi,Kathleen M Cowan,Ashley E Ross,Milena Vuica-Ross,Yolanda M Fortenberry

doi:10.1371/journal.pone.0010846

Abstract

BackgroundDimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones.Methodology/Principal findingsExposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC50s in the µM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ.Conclusions/SignificanceIn yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Highlights

Multimeric naphthoquinones are unique molecules, which possess a diverse array of biologic activities including antineoplastic, antiprotozoal and antiviral effects [1]
Binaphthoquinones inhibit yeast growth To establish whether yeast could be used as a model system for binaphthoquinone cytotoxicity, we first aimed to determine the ability of binaphthoquinones to suppress yeast growth
Our analysis showed that wild type yeast is sensitive to Binaphthoquinone 7 (BiQ7) with an IC50 of 0.960.2 mM while other tested binaphthoquinones showed IC50 in the 762 mM range (Fig. 1B)

Summary

Introduction

Multimeric naphthoquinones are unique molecules, which possess a diverse array of biologic activities including antineoplastic, antiprotozoal and antiviral effects [1] Their structures are based on two or more naphthoquinone units linked together in different positions. Elucidation of the cellular components necessary to protect cells or sensitize them to binaphthoquinones might allow for the enhanced use of these drugs as antiretroviral, antiparasitic or antineoplastic agents To this end, we carried out genome wide screens for binaphthoquinone sensitivity and resistance in yeast. We utilized several different binaphthoquinone analogues and developed a genetic model of their cytotoxicity by performing a systematic high-throughput screen of a yeast isogenic deletion mutant array for drug enhanced or suppressed growth.

Results

Discussion

Materials and Methods