Hsp90 has become the target of intensive investigation as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. Hsp90 inhibitors also exhibit and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds seven were previously characterized as Hsp90 inhibitors. Four compounds anthothecol garcinol piplartine and rottlerin were further characterized and the ability of these compounds to inhibit the refolding of luciferase and reduce the rate of growth of MCF7 breast cancer cells correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase and deplete cultured cells of Hsp90-dependent client proteins. Thus this screen has identified an Anacardic Acid additional 44 compounds with known beneficial pharmacological properties but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine. normal cells [11 12 and (4) protection of cells from toxicity induced by the accumulation of protein aggregates [13 14 15 ([25]. A number of high-throughput screening (HTS) assays Anacardic Acid based on a variety of techniques have been developed to screen large chemical libraries to identify new Hsp90 inhibitors (reviewed in [26]). Screens have been developed based on the ability of compounds to: (1) inhibit Hsp90 catalyzed ATP hydrolysis; (2) competitively inhibit the binding of ligand to Hsp90’s N-terminal ATP binding domain; (3) inhibit Hsp90-mediated refolding of denatured protein (e.g. luciferase); and (4) deplete cultured cells of Hsp90 client proteins. These assays have identified a large number of potential Hsp90 inhibitors. However only a limited number of follow-up studies have been carried out to verify the mechanism of action of these compounds and to optimize their Hsp90-inhibitory activity. As Anacardic Acid noted above most HTS have been carried out Anacardic Acid on large chemical libraries. Here we focus on the use of a high-throughput assay to screen natural product libraries for novel inhibitors of the Hsp90 chaperone machine. The screening is based on the ability of Hsp90 inhibitors to block the refolding of thermally denatured firefly luciferase which is catalyzed by the Hsp90 chaperone machinery present in rabbit reticulocyte lysate [27 28 29 It was reasoned that natural products would be a fertile territory for identification of additional Hsp90 inhibitors as it would be reasonable to expect that evolutionary pressure would give plant or other species which have acquired pathways leading to the synthesis of secondary metabolites that inhibit Hsp90 a competitive advantage because such compounds would be expected to inhibit the growth and development of insect and pathological pests. Furthermore as noted in a recent review the majority of drugs approved for use by the FDA during the past 30 to 50 years are natural products or derivatives thereof [30 31 In addition there is vast literature on active Anacardic Acid compounds that have been isolated from traditional folk medicines that allowed us to mine the literature for compounds identified in our screen that have properties of Hsp90 inhibitors that were discussed above. 2 Experimental Section 2.1 High-Throughput Screen of Natural Product Libraries 2.1 Rabbit Reticulocyte Lysate Rabbit reticulocyte lysate prepared by lysing one volume of packed reticulocytes in two volumes of deionized water followed by centrifugation for twenty minutes at 15 0 × g was purchased from Green Hectares (Oregon WI USA). 2.1 Denatured Luciferase Recombinant luciferase from Promega was diluted to 0.5 mg/mL in buffer consisting of 25 mM Tricine-HCl (pH 7.8) 8 Ntn2l mM MgSO4 0.1 mM EDTA and 10 mg/mL acetylated BSA. Next the solution was adjusted to include 10% glycerol and 1% Triton X-100. Finally the luciferase solution was heated to ~41°. Once the activity of the luciferase reached ~1% of its initial value the mixture was placed on ice or flash frozen in liquid nitrogen and placed at ?80° for storage. To prepare the denatured luciferase for use in re-folding assays 125 μL of the 0.5 mg/mL mixture was added into a 10 mL mixture containing 80 mM Tris HCl pH 7.7 8 mM Mg(OAc)2 300 mM KCl 12 mM ATP.