Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.
Bibliographical noteFunding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work conducted at the University of Minnesota was funded by National Institutes of Health (NIH) NICHD U01 HD076542 and HHSN275201300017C. This work was carried out in part using computing resources at the University of Minnesota Supercomputing Institute. Support for effort at the University of Florida was provided by NIH NICHD U01 HD060474. This work was partly supported by Otsuka Pharmaceuticals, Inc.
- ADP/ATP exchange
- Lactococcus lactis
- adenine nucleotide translocase
- high throughput screening
- yeast mitochondria