A high-throughput screen to identify novel calcineurin inhibitors Lekha Menon Margassery a , Jonathan Kennedy a , Fergal O'Gara a, b , Alan D. Dobson a , John P. Morrissey a, ⁎ a Marine Biotechnology Centre, Environmental Research Institute, and Microbiology Department, University College Cork, Ireland b BIOMERIT Research Centre, University College Cork, Ireland abstract article info Article history: Received 29 August 2011 Received in revised form 19 October 2011 Accepted 19 October 2011 Available online 26 October 2011 Keywords: Calcineurin inhibitor High-throughput screen β-galactosidase activity Yeast reporter Calcineurin is a eukaryotic protein phosphatase important for many signalling and developmental processes in cells. Inhibitors of this enzyme are used clinically and there is interest in identifying novel inhibitors for therapeu- tic applications. This report describes a high-throughput assay that can be used to screen natural or chemical li- braries of compounds to identify new calcineurin inhibitors. The microtitre plate assay is based on a yeast reporter strain and was validated with known inhibitors and tested in a pilot screen of bacterial extracts. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Calcineurin is a highly conserved, eukaryotic calmodulin-dependent serine/threonine phosphatase type 3CA (formerly PP2B). In mammalian cells, it participates in diverse cellular processes such as signal transduc- tion, cell-cycle regulation, stress response and apoptosis (Rusnak and Mertz, 2000), and has recently been implicated in several important neurodegenerative disorders, including schizophrenia (Kvajo et al., 2010); (Tabarés-Seisdedos and Rubenstein, 2009). In the immune sys- tem, calcineurin is centrally involved in maturation of T cells and, for this reason, most immunosuppressive therapies involve the use of calci- neurin inhibitors (CNI) (Castroagudín et al., 2011). The most widely used CNIs are cyclosporin A and tacrolimus (FK506), both of which are natural microbial metabolites that bind to intracellular receptors known as immunophilins, which then block calcineurin function. The mode of action of and history of these CNIs has been extensively studied and very well-documented (Liu et al., 1991); (Pritchard, 2005). Interest- ingly, the main details of the mechanism of action of both cylcosporin A and FK506 were first determined in the budding yeast, S. cerevisiae, something made possible by the strong conservation of calcium signal- ling pathways and calcineurin across the Eukarya (Fox and Heitman, 2002). Although CNI are extensively used for immunosuppressive ther- apy, most notably in organ transplantation, there are cardiovascular, renal and other side-effects that can occur with long-term use (Rezzani, 2006); (Fung et al., 1991) and thus there is interest in identify- ing novel calcineurin inhibitors. Furthermore, such molecules may also have potential as novel anti-fungal agents (Steinbach et al., 2007) and applications even as cell biology reagents to study calcium signalling and homeostasis are possible. The most prolific reservoir of bioactive molecules has always been the natural world and the majority of molecules used in medicine are derived from microbes or plants. Many antibiotics, as well as important drugs such as CsA, tacrolimus (FK506) and sirolimus (rapamycin), are of microbial origin and in the current era of genome biology there is renewed interest in bioprospecting to identify new microbial metabo- lites with therapeutic applications. The challenge of developing screens to identify specific inhibitory activities remains, however, and modern requirements stipulate that any methods are specific, rapid, high- throughput and automatable in so far as is possible. Current methods to screen for calcineurin inhibitors are limited and do not satisfy these requirements (Fruman et al., 1992); (Enz et al., 1994); (Sellar et al., 2006). The aim of this study was to develop a new screen for calcineurin inhibitors that would be compatible with robotic handling systems for screening large numbers of microbial extracts, metagenomic libraries or combinatorial chemistry libraries. The screen was developed using yeast reporter strains. In yeast, as in mammalian cells, various extracellular signals lead to a transient increase in calcium ions in the cytoplasm. The cytoplasmic protein calmodulin binds calcium and then activates calcineurin. The targets of calcineurin differ among organisms, with the transcription factor Crz1p, the main target in yeast. Following dephosphorylation by cal- cineurin, Crz1p translocates to the nucleus where it activates tran- scription of target genes by binding to a promoter sequence known as the CDRE element (Kraus and Heitman, 2003); (Cyert, 2003). The conservation of calcineurin in Eukarya facilitated the develop- ment of this yeast screen. Journal of Microbiological Methods 88 (2012) 63–66 ⁎ Corresponding author. Tel.: + 353 21 4902392; fax: + 353 21 4903101. E-mail address: j.morrissey@ucc.ie (J.P. Morrissey). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.10.012 Contents lists available at SciVerse ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth