Yeast is selectively hypersensitised to heat shock protein 90 (Hsp90)-targetting drugs with heterologous expression of the human Hsp90β, a property that can be exploited in screens for new Hsp90 chaperone inhibitors

Abstract

Heat shock protein 90 (Hsp90) is essential for activation of many of the most important regulatory proteins of eukaryotic cells. It is an extremely conserved protein, such that heterologous expressions of either human Hsp90β or Caenorhabditis elegans Hsp90 will provide the essential Hsp90 function in yeast. The ability of these metazoan Hsp90s to provide this Hsp90 function to yeast cells requires Sti, a Hsp90 system cochaperone. Yeast that is expressing human Hsp90β in place of the normal native yeast Hsp90 is selectively hypersensitised to Hsp90 inhibitor drugs. Hsp90 drugs are promising anticancer agents, their administration simultaneously destabilizing a number of the proteins critical to multistep carcinogenesis. Though one of these drugs (17-allylaminogeldanamycin, 17-AAG) is now progressing to Phase 2 clinical trials, there is a pressing need to identify selective Hsp90 inhibitors that are more soluble than 17-AAG. High-throughput screening for chemical agents that exert greater inhibitory effects against yeast expressing the human Hsp90β relative to yeast expressing its native Hsp90 should therefore facilitate the search for new Hsp90 inhibitors.

Introduction

Anticancer drug development strategies involve identifying drug targets that are crucial for tumourigenesis. One such target is heat shock protein 90 (Hsp90), an essential chaperone that dynamically interacts with a diverse set of metastable ‘client’ proteins (Smith et al., 1998, Piper, 2001, Maloney and Workman, 2002, Neckers, 2002). Typically, Hsp90 keeps these clients in a state where they are ‘poised’ for activation by subtle conformational changes, such as those triggered by the events of signal transduction. Hsp90 is the target of certain antibiotics and it is possible to selectively inhibit the Hsp90 function in vivo using the inhibitor compounds radicicol (RD; a macrolactone produced by certain fungi) and geldanamycin (GA; a benzoquinone ansamycin produced by Streptomyces hygroscopicus). These bind within the Hsp90 ADP/ATP binding site, thereby inhibiting the ATPase step of the Hsp90 chaperone cycle (Prodromou et al., 1997, Stebbins et al., 1997, Roe et al., 1999, Schulte et al., 1999).

Interest in the use of these Hsp90-targetting antibiotics as anticancer drugs was initially triggered by the identification of RD and GA as agents that reversed the phenotype of p60^v-src-transformed cells in culture (Uehara et al., 1986, Kwon et al., 1992). These compounds inhibit the Hsp90 whose action is essential for p60^v-src to become an active tyrosine kinase. As a result of this inhibition p60^v-src protein is rapidly destabilised (Schneider et al., 1996). The administration of these antibiotics also causes marked destabilisation of several other oncologically-relevant proteins in vivo, for example p53, Erb-b, Raf-1 and steroid receptors (Schulte et al., 1995, Schneider et al., 1996, Whitesell et al., 1997, Bagatell et al., 2001). It is probable that this drug-induced destabilisation of Hsp90 ‘client’ proteins is a result of their inability to progress through the chaperone cycle. Cells in which the Hsp90 function is inhibited tend to undergo either cell cycle arrest or apoptosis, depending on the nature of the cell culture system that is under investigation (Maloney and Workman, 2002).

The unique pharmacological profile of Hsp90 inhibitors may stem from their ability to block simultaneously the actions of several proteins critical to multistep oncogenesis (Maloney and Workman, 2002). Hsp90 inhibition may also circumvent certain acquired resistances to other cancer drugs. It will for example prevent the formation of the late-stage Hsp90/progesterone receptor (PR) multiprotein complex, the form of PR that is competent for the high affinity binding of steroid. As the Hsp90 drug is binding not to the receptor but to the Hsp90 in this complex, the receptor mutations that often cause resistance to antiestrogen (e.g. tamoxifen) therapy might not also be associated with resistance to Hsp90 inhibitors (Smith et al., 1998, Bagatell et al., 2001).

The existing Hsp90 inhibitor drugs are all derivatives of RD and GA (Piper, 2001). Using several animal model systems, appreciable antitumour effects of these agents have now been demonstrated, the 17-allylamino derivative of GA (17-AAG) being more effective and less hepatotoxic in vivo than the parent GA (Supko et al., 1995, Maloney and Workman, 2002). Although this 17-AAG is now progressing to Phase 2 clinical trials, its insolubility causes problems in administration. Also it is potentially a redox-cycling drug. There is therefore the need to identify or develop newer Hsp90 inhibitors, compounds that are more soluble and more selective in their ability to solely inhibit Hsp90 in vivo (Maloney and Workman, 2002). Especially needed are improved methods for rapidly assessing the abilities of compounds to act as selective Hsp90 inhibitors. Here we describe the chance observation that yeast expressing the human Hsp90β is selectively hypersensitised to Hsp90 inhibitor compounds. This can be exploited to rapidly screen large numbers of compounds for any potential Hsp90 inhibitory activity.