A simple yeast-based system for analyzing inhibitor resistance in the human cancer drug targets Hsp90a/b Stefan H. Millson a , Chrisostomos Prodromou b , Peter W. Piper a, * a Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK b Section for Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK 1. Introduction Heat shock protein 90 (Hsp90) orchestrates a multi-stage chaperone cycle, essential for the final maturation, stabilisation and localization events of a diverse set of important proteins in eukaryotic cells. It is increasingly attracting attention as a promising target for cancer drug development, since many of the proteins responsible for the oncogenic phenotype of cancer cells are highly dependent upon Hsp90 for their activity. In cancer cells treated with highly-selective inhibitors of this chaperone, several oncogenic activities are inactivated and destabilized simultaneously, enabling such drugs to cause the combinatorial depletion of many cancer-causing pathways and a modulation of all of the hallmark traits of malignancy [1–3]. Fortuitously, Hsp90 inhibitors also show a high selectivity for cancer versus normal cells [4,5] and a therapeutic activity at doses that are well tolerated in cancer patients [6]. Inhibitors of Hsp90 include the natural antibiotics geldanamy- cin (GdA), monocillin 1 and radicicol/monorden (RAD). These, together with the most promising synthetic Hsp90 inhibitor drugs [7,8], bind within the highly-conserved ATP binding site of the Hsp90 N-terminal domain [9–13]. Cancer clinic trials of both derivatives of GdA [14], as well as of purine and 4,5-diaryisoxazole resorcinol Hsp90 inhibitors based on the interactions of RAD, are now well advanced [6,15,16]. Cancer chemotherapy is often compromised by the development of drug resistance. In cell cultures, a partial resistance to one of the GdA derivatives now in clinical trials, 17-allylamino-demethox- ygeldanamycin (17-AAG), is able to develop through an altered expression of NAD(P)H:quinone oxidoreductase 1 [17,18]. However, whether any appreciable resistance to Hsp90 inhibitors could arise by mutation to tumour Hsp90 is still unknown. Part of the strong case for Hsp90 drug development is the prediction that the probability of such an occurrence might be relatively low. The amino acid residues that facilitate drug interactions within the nucleotide binding site of Hsp90 are generally highly conserved in Hsp90-family proteins from bacteria to man [19], such that mutational changes that compromise drug binding would mostly be expected to inactivate this essential chaperone. However, since Biochemical Pharmacology 79 (2010) 1581–1588 ARTICLE INFO Article history: Received 5 December 2009 Accepted 26 January 2010 Keywords: Hsp90 Molecular chaperone Cancer Drug target Drug resistance Yeast ABSTRACT Heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, is one of the most promising targets for cancer drug development. Whether any resistance to these Hsp90 inhibitor drugs could arise by Hsp90 mutation is still unknown. Yeast is readily engineered so that its essential Hsp90 function is provided by either isoform of the human cytosolic Hsp90, Hsp90a or Hsp90b. However, its high intrinsic resistance to most drugs poses a major obstacle to the use of such Hsp90a- or Hsp90b-expressing yeast cells as a model system to analyse whether drug resistance might arise by Hsp90 mutation. In order to overcome this problem, we have generated a strain that is both hypersensitive to Hsp90 inhibitors as it lacks multiple drug resistance genes, and in which different heterologous and mutant Hsp90s can be expressed by plasmid exchange. It is not rendered appreciably stress sensitive when made to express Hsp90a or Hsp90b as its sole form of Hsp90. Should there be any development of resistance to the Hsp90 drugs now in cancer clinic trials, this system can provide a rapid initial test of whether any single nucleotide polymorphism appearing within the coding regions of Hsp90a or Hsp90b could be a contributory factor in this resistance. We have used this strain to demonstrate that significant levels of resistance to the Hsp90 inhibitors radicicol and 17-allylamino-demethoxygeldanamycin (17-AAG) are generated as a result of the same single point mutation within the native Hsp90 of yeast (A107N), the human Hsp90a (A121N) and the human Hsp90b (A116N). ß 2010 Elsevier Inc. All rights reserved. Abbreviations: RAD, radicicol; GdA, geldanamycin; 17-AAG, 17-allylamino- demethoxygeldanamycin; DMSO, dimethylsulphoxide; 5-FOA, 5-fluoroorotic acid. * Corresponding author. Tel.: +44 114 222 2851; fax: +44 114 222 2800. E-mail address: peter.piper@sheffield.ac.uk (P.W. Piper). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2010.01.031