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Tumor Selectivity of Hsp90 Inhibitors: The Explanation Remains Elusive Gabriela Chiosis†* and Len Neckers‡
† Department of Medicine and Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, and ‡Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892-1107
T
he molecular chaperone heat-shock protein 90 (Hsp90) plays an important role in maintaining the functional stability and viability of cells under a transforming pressure. It allows cancer cells to tolerate the deregulation of components of the signaling pathway that occur during cellular transformation. Hsp90 protects the cell by interacting with and stabilizing several client substrates, including kinases, hormone receptors, and transcription factors, which are directly involved in driving multistep malignancy, and also with mutated oncogenic proteins that drive the transformed phenotype (see reviews 1–5). Association of Hsp90 with these client proteins maintains their ability to function in the deregulated state and appears to be essential for their transforming, aberrant activity. Enhanced Hsp90 affinity for mutated or functionally deregulated client proteins has been observed, and several examples of this behavior have been documented. Historically, v-src was the first oncogene shown to display unusually stable interaction with the chaperone (6, 7). In fact, the first Hsp90 inhibitors, geldanamycin (GM) and radicicol (RD), were identified in a screen for compounds that could reverse the phenotype of cells transformed by v-src (8). In contrast, the non-oncogenic c-src requires only limited assistance from the Hsp90 machinery for its maturation and cellular function. Examples may be extended to other transformed phenotypes, and in this regard, almost every protein www.acschemicalbiology.org
involved in cell-specific oncogenic processes has been shown to be regulated by Hsp90 (2, 5). In addition to its role as a chaperone of oncoproteins, Hsp90 is involved in protein folding processes that occur in normal cells. Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations, a process requiring the concerted action of multiple molecular chaperones. From yeast to mammals, Hsp90 functions together with Hsp70 in the folding of a diverse set of proteins, including transcription factors, regulatory kinases, and numerous other proteins that appear to lack common structural or functional features (9). Even under nonstressed conditions, Hsp90 accounts for as much as 1–2% of total cellular protein. Conceiving that Hsp90 is a viable target in cancer therapy is hard because constitutive Hsp90 genetic knockout in eukaryotes is lethal (3, 5). That it is such a target has become apparent only after the discovery of pharmacological agents that selectively inhibit its function (10). These agents have been useful in probing the biological functions of Hsp90 at the molecular level and in validating it as a novel target for anticancer drugs. The N-terminal region ATP pocket binders are the first identified inhibitors of Hsp90 activity (Figure 1) . Some of these are natural products, such as ansamycins (GM, 17allylamino-17-demethoxygeldanamycin [17-AAG] and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin
A B S T R A C T Two recent papers attempt to solve both the tumor selectivity and the in vivo tumor accumulation profiles seen with some Hsp90 inhibitors. They spotlight the higher affinity of ansamycins’ hydroquinone over the quinone form for Hsp90 and further discuss its possible contribution to ansamycins’ tumor selectivity.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online June 16, 2006 10.1021/cb600224w CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. Representative Hsp90 inhibitors. Ansamycins (GM, 17-AAG, 17-AG), RD, and the PUscaffold derivatives (PU3, PU24FCl, and PUH64).
[17-DMAG] (11–13)) and RDs (RD, RD oxime-derivatives, (14); cycloproparadicicol, (15); and pochonin D, (16)). Others are synthetic small molecules discovered either by design or by high-throughput screening. These can be subclassified into purinescaffold derivatives (PU-class), discovered by our laboratory (17, 18), and their further re-scaffolding products, such as triazolopyrimidines, pyrazolopyrimidines, and pyrrolopyrimidines (19), and other scaffolds, such as pyrazole derivatives (20). Compounds that interfere with chaperone cycling by binding to important domains in the C-terminus have also been identified (21, 22). Recently, a peptidomimetic modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator survivin was reported (23). This peptidomimetic, termed shepherdin, mimics the survivin sequence I74–L87, the minimal peptide span that retains Hsp90 inhibitory activity. Shepherdin was shown to make extensive contacts with the ATP pocket of Hsp90, resulting in destabilization of chaperone client proteins. Probing Hsp90 function with these agents in cellular and animal models of cancer has led to some surprising yet rewarding findings. First, several of these inhibitor classes have shown selective binding to Hsp90 in tumor cells (23–28). Second, cancer cells have proven to be significantly more sensitive to Hsp90 inhibition than are nontransformed cells (23–30). 280
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Third, Hsp90 inhibitors at nontoxic doses have demonstrated anticancer activity in multiple animal models (23, 27, 28, 31, 32). Moreover, drug accumulation in tumors, coupled with rapid clearance from blood and normal tissue, has been observed for some Hsp90 inhibitor classes in these animal models (26, 27, 33–35). Although no comprehensive explanation has yet been presented for the remarkable selectivity of Hsp90 inhibitors toward cancer cells, many hypotheses have emerged. Both in vitro and in vivo approaches have been used to implicate co-chaperones, tumorspecific drug modification, oncogene addiction, tumor-specific post-translational modification (PTM), and so forth, as the basis of this selectivity. In Vitro Explanations Using Biochemical Approaches. Several groups have analyzed the Hsp90 inhibitor tumor selectivity at a biochemical level. First, Kamal et al. (24) have measured a 100-fold difference for 17-AAG (Figure 1) in affinity between transformed and normal-cell Hsp90. Further, studies with the PU-scaffold Hsp90 inhibitors (Figure 1) have reported similar selectivity with several members of this class of molecules (25–28). Recently, Altieri et al. (23) have demonstrated that immobilized shepherdin pulled down Hsp90 only from transformed cells, not from normal ones. Several attempts to shed light on the higher affinity of these agents for tumor Hsp90 have focused on ansamycins. CHIOSIS AND NECKERS
Initially, the affinity of these drugs for the chaperone in solution was determined by several biochemical methods to be ⬃1 M. This is contrary to their cellular potency seen at low-nanomolar concentrations. Multiple explanations of this apparent discrepancy have been proposed. In a recent paper, Maroney et al. (36) suggested that the dihydroquinone form of 17-AAG, 17-AAGH2 (Figure 1), which may form in cells, has a better affinity for Hsp90 than does the quinone 17-AAG. First, an assay that monitors protein unfolding as a function of temperature by using the environmentally sensitive dye bis-1-anilino-8-naphthalene sulfonate was employed to test Hsp90 thermal stability in the presence and absence of GM, 17-AAG, RD, and nucleotides, with or without added reducing agents. For both GM and 17-AAG, a 40-fold increase in protein stabilization was seen in the presence of reducing agents such as DTT and tris-(2-carboxyethyl)phosphine hydrochloride (TCEP). Further, a filter binding assay that measures drug binding, under equilibrium conditions (48 h), of [3H]-17AAG to a truncated Hsp90 ␣ containing the N-terminal region determined Kd of 1.1 M and 2.4 nM in the absence or presence of TCEP, respectively. The same assay was used to determine an extended koff for 17-AAGH2 (Figure 1) as compared to 17-AAG (⬎⬎4.5 h vs several minutes). In a different approach, using a fluorescence polarization assay, Llauger et al. (37) reported that two fluorescently labeled GMs, GM-BODIPY and GM-FITC, bound tightly to Hsp90 ␣ with Kd of 33.8 ⫾ 1.2 and 23.3 ⫾ 0.9 nM, respectively. These same authors further demonstrated that binding was affected by DTT, a higher affinity with Kd of 6.6 ⫾ 1.3 nM being favored by more DTT in the assay buffer and longer incubation times (38, 39). These authors also reported that equilibration to the high-affinity state was slow at low-DTT concentrations but accelerated as the DTT content was increased. Gooljarsingh et al. (40) reprowww.acschemicalbiology.org
Point of
VIEW duced these findings in their recent PNAS publication. They reported that GM-BODIPY induced a time-dependent conformational change in both Hsp90 ␣ and  isoforms leading to a high-affinity state. A similar profile was observed for Hsp90 assayed in cell lysates obtained from ovarian epithelial cancer (transformed) and human umbilical vein endothelial (HUVEC, normal) cells. These data are in agreement with previous reports by Llauger et al. (25, 26) on Hsp90 from breast-cancer cells. The dissociation rate of this tightly bound GM was measured to be very slow, with a t1/2 of 4.5 h. All highaffinity binding constants for ansamycins were observed in the presence of 2 mM DTT in the assay buffer. These finding indicated that reducing agents affected affinity, so we further analyzed buffer conditions for several other assays used previously to determine the affinity of ansamycins for Hsp90. Roe et al. (41) used isothermal titration calorimetry to calculate a dissociation constant of 1.22 M for GM binding to intact yeast Hsp90 and 0.78 M for GM binding to the Hsp90 N-terminal domain. These values were obtained in 20 mM Tris-HCl, pH 7.5, and 1 mM EDTA. Chiosis et al. (17, 42) used immobilized GM to obtain an apparent relative affinity of 17-AAG for Hsp90 ␣ of 1 M. This assay used buffer conditions comparable to Roe et al. (50 mM Tris-HCl, pH 7.4, and 1 mM EDTA with added 1% NP40 to reduce background interference from the solid support). Carreras et al. (43) reported that in 10 mM Tris-HCl and 5 mM MgCl2, pH 7.0, the binding of [3H]-17-AAG to Hsp90 reached equilibrium in a few minutes with a Kd of 0.4 ⫾ 0.1 M. Using immobilized biotinylated GM, Le Brazidec et al. (44) reported a relative affinity of 17-AAG for Hsp90 in PBS of 800 nM. Collectively, these data support the hypothesis that reducing agents can influence the affinity of ansamycins for Hsp90, suggesting that the hydroquinone is a better Hsp90 binder than the quinone itself. Howwww.acschemicalbiology.org
ever, they do not support a role for this phenomenon in explaining the higher tumor affinity of Hsp90 inhibitors, because neither PU-scaffold derivatives nor shepherdin are DTT-sensitive. In addition, these findings do not preclude an additional effect of DTT on the structural conformation of Hsp90. Further, the concept of an “encounter complex” between GM and Hsp90 with a relatively weak Ki of 450 nM, which then equilibrates to a tight complex with Ki of 10 nM (e.g., “time-dependent” binding of ansamycins), presented by Gooljarsingh et al. (40) can be explained by either an induced fit model or the active ansamycin isomerization model described by Lee et al. (45). In any case, such a binding schema is ansamycinspecific. Another biochemical explanation for tumor selectivity of Hsp90 inhibitors comes from work by Kamal et al. (24), who reported that Hsp90 from tumor cells is present entirely in multichaperone complexes with high ATPase activity and also high affinity for ligands, whereas in normal tissues, Hsp90 exists in a latent uncomplexed state. Maroney et al. (36) confirmed this finding by demonstrating that the amount of Hsp90 complexed to co-chaperones is higher in tumor cells than in resting ones. Gooljarsingh et al. (40), however, contradicted the finding and argued that Hsp90 co-chaperones such as Hsp70, Hsp40, Hop, and p23 do not alter the GM-Hsp90 binding profile, even though they are sufficient to refold a denatured Hsp90 client protein. Interpretation of such in vitro data has its limitations, however, because these cochaperones are not bound to the same conformation of Hsp90. Further, there are many more co-chaperones whose interaction with Hsp90 could significantly impact (at least theoretically) access to or the shape of the nucleotide pocket (or surrounding regions). Putting the Cell to Work. Another finding for pharmacological Hsp90 inhibition is that cancer cells are significantly more sensitive
to these drugs than are nontransformed cells. Such selectivity in inhibiting the growth of cancer cells has been reported for ansamycins, RD derivatives, and PU-scaffold derivatives (23–30). Such sensitivity was first believed to be class-specific and due to an intracellular reduction of 17-AAG. Kelland et al. (46) observed that ansamycins were substrates for purified human NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase, EC 1.6.99.2). This flavoenzyme can use either NADH or NADPH as reducing cofactors to catalyze the direct two-electron reduction of quinones to hydroquinones. Elevated cellular activity of this enzyme sensitized cells to 17-AAG, but surprisingly, not to GM or 17-amino, 17-demethoxygeldanamycin (17-AG). Although 17-AAG was a reasonable substrate for human DT-diaphorase, it was not an appreciably better substrate than GM or 17-AG. Ross et al. (47) confirmed the 17-AAG findings and further demonstrated that the metabolism of 17-AAG by recombinant human NQO1 led to the appearance of 17-AAGH2. The formation of 17-AAGH2 was NQO1-dependent and could be inhibited by the addition of a mechanism-based (suicide) inhibitor of NQO1. Maroney et al. (36) further demonstrated that 17-AAGH2 does not spontaneously forms in aqueous media. They do, however, find that cells possess enough reductive potential to conduct this transformation, and a 68% transformation of 17AAG to 17-AAGH2 was observed in MCF7 breast-cancer cells. However, quiescent HUVEC cells reduced 17-AAG to the same extent. Nonetheless, the inhibitory activity of 17-AAG is 100 nM in MCF7 but ⬎10 M in quiescent HUVECs; thus, it is unlikely that inhibitor selectivity for tumor Hsp90 is explained by tumor-specific conversion of 17-AAG to 17-AAGH2. Chiosis et al. (48) reported another peculiar behavior of ansamycins in cells. In tissue-culture experiments, when the molar amount of ansamycins was kept constant but the volume of the medium was inVOL.1 NO.5 • 279–284 • 2006
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Figure 2. Hsp90 may exist in an equilibrium between an “activated” state prevalent in cancer cells and a “latent” state predominant in normal cells. The activation state of the chaperone may be regulated by co-chaperones and perhaps PTMs. This is a schematic representation of Hsp90 states and does not represent actual individual complexes.
creased, no change was observed in MCF7 cells’ growth-inhibitory potency. In contrast, when such an experiment was repeated with the PU-scaffold derivatives PU3 and PU24FCl (Figure 1), activity diminished with drug concentration. Upon addition to aqueous tissue culture media, ansamycins rapidly accumulated in cells and were mostly depleted from the media, producing higher intracellular concentrations than expected. This behavior was also observed in cultured normal epithelial cells. The nonspecific accumulation of GM and 17-AAG into cells in tissue culture conflicted with their specific tumor accumulation observed in vivo. It is thus important to differentiate between nonselective drug accumulation into cells in tissue culture, which may be due to physicochemical peculiarities of a compound in those settings, and selective accumulation into cancer (vs normal) cells due to different biological characteristics of these cells. These data warn us that any peculiarities seen in tissue culture may not explain the in vivo tumor accumulation of Hsp90 inhibitors. Whole-Animal Case Study: No Plastic to Blame. Hsp90 inhibitors have anticancer activity in multiple-animal xenograft models at nontoxic doses. Such effects have been observed with ansamycins such as 17-AAG, IPI-504, and 17-DMAG (32–35) and also with RD derivatives (31), PU-scaffold inhibitors (27, 28), and shepherdin (23). In addition, drug accumulation in tumors coupled to rapid clearance from normal tissue has been observed for multiple Hsp90 inhibitor classes in these models. The first agent reported to be retained in tumors was our PU-scaffold derivative PU24FCl (Figure 1) (27). While this agent was rapidly cleared from blood, pharmacologically relevant 282
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concentrations were recorded in MCF7 xenografts at ⬎24 h post-administration. Second-generation, water-soluble PU-class agents such as PUH64 (Figure 1) were similarly shown to be retained in MDA-MB-468 xenografts, suggesting that tumor retention is not influenced by drug lipophilicity (26). Similar results were later reported by Eiseman et al. (33) for 17-DMAG in MDA-MB-231 breast-cancer xenografts and by Workman et al. (34) for 17-AAG in human-ovariancancer xenografts. Recently, IPI-504, the reduced form of 17-AAG, was shown to be rapidly cleared from plasma while selectively retained in tumor tissue. These results were reported in a multiple-myeloma xenograft model (35). Further, Serenex has claimed a tumor retention profile for their structurally novel SNX-2112 Hsp90 inhibitor in an HT29 human colon adenocarcinoma model (www.serenex.com). Thus, the tumorspecific accumulation of such diverse chemical classes of Hsp90 inhibitors clearly cannot be entirely explained by the biochemical and cellular hypotheses presented above (which focus only on the peculiarities of ansamycins). The Answer May Lie in Hsp90 Itself. In tumors, Hsp90 comprises as much as 4–6% of total cellular protein, and this translates into an intracellular concentration in the ⬃500 M range. It is thus noteworthy that administration of 50–200 mg kg⫺1 of diverse Hsp90 inhibitors led to only 0.5–5.0% of drug being retained in tumors. Eiseman et al. (33) reported an ⬃5 g mL⫺1 level of 17DMAG retained in MDA-MB-231 tumors at ⬃12 h post-administration when drug was injected intraveneously at 75 mg kg⫺1. Banerji et al. (34) observed in A2780 and CH1 human-ovarian-cancer xenografts treated with a single dose of 17-AAG CHIOSIS AND NECKERS
(80 mg kg⫺1 intraperitoneally) ⬃5 M of 17-AAG and 17-AG (an active metabolite of 17-AAG, Figure 1) at 24 h. Similar data were reported for the PU-scaffold Hsp90 inhibitors. Administration of 200 mg kg⫺1 PU24FCl to mice bearing MCF7 xenografts led to 5–10 M tumor drug levels at 24 h (27). PUH64 also accumulated to lowmicromolar concentration in tumors at 24 h when administered intraperitoneally at 50–100 mg kg⫺1 to mice bearing MDAMB-468 xenografts (26). The Serenex derivative SNX-2112 is reported to reach a concentration of 3 M in HT29 colon-cancer xenografts at 24 h when administered at 100 mg kg⫺1 orally (www.serenex.com). At a concentration of 3–10 M, these drugs occupy only a small fraction (0.5–2.0%) of total cellular Hsp90 binding sites. However, in all of the examples described above, the doses of Hsp90 inhibitors used resulted in tumor growth inhibition. One may infer from such observations that these drugs target only a relatively low-abundance but highaffinity conformation of Hsp90, likely found in a multichaperone complex with transformation-specific oncoproteins. This may represent the small fraction of client proteins regulating the transformed phenotype. The latent Hsp90 complexes regulating normal misfolding processes and comprising at any time ⬎95% of total cellular Hsp90 may not be effectively inhibited by these drugs at the relatively nontoxic doses used. Such an interpretation leads to the hypothesis that, under normal conditions, Hsp90 interacts with client proteins in a dynamic, low-affinity manner regulated by low-affinity binding and release of ATP and ADP “latent state”. Upon mutation or deregulation characteristic of the cancer phenotype, many of these client proteins may display (and require) unusually stable association with Hsp90containing chaperone complexes “activated state” (Figure 2). This state also exhibits a high affinity for ATP and ADP or other ligands of this regulatory pocket (i.e., N-terminal Hsp90 inhibitors). The shift in equilibrium www.acschemicalbiology.org
Point of
VIEW from the latent to the activated state may be dictated by the degree of transformation or amount of “stress” on the system (abundance of mutated and deregulated proteins, a hypoxic and/or low-nutrient environment, etc.). The mechanism of this enhanced affinity has not yet been entirely elucidated but is likely due to tumor-specific modification of Hsp90 itself and not to any unique tumorspecific metabolism of its inhibitors. Possible factors responsible for Hsp90 modification include its specific interaction with co-chaperones (24, 36) and/or alterations in the post-translational state of Hsp90, co-chaperones, or both (49). While they confirm previous observations about ansamycins, the two new papers by Maroney et al. (36) and Gooljarsingh et al. (40) do not extend our understanding of the specific biology of tumor Hsp90, particularly its remarkable sensitivity to pharmacological inhibition. Many questions remain unanswered about the uniqueness of Hsp90 in tumors. REFERENCES 1. Workman, P. (2004) Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone, Cancer Lett. 206, 149–157. 2. Zhang, H., and Burrows, F. (2004) Targeting multiple signal transduction pathways through inhibition of Hsp90, J. Mol. Med. 82, 488–499. 3. Whitesell, L., and Lindquist, S. L. (2005) HSP90 and the chaperoning of cancer, Nat. Rev. Cancer 5, 761–772. 4. Neckers, L., and Neckers, K. (2005) Heat-shock protein 90 inhibitors as novel cancer chemotherapeutics—An update, Expert Opin. Emerging Drugs 10, 137–149. 5. Chiosis, G. (2006) Targeting chaperones in transformed systems—A focus on Hsp90 and cancer, Expert Opin. Ther. Targets 10, 37–50. 6. Xu, Y., and Lindquist, S. (1993) Heat-shock protein hsp90 governs the activity of pp60v-src kinase, Proc. Natl. Acad. Sci. U.S.A. 90, 7074–7078. 7. Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and Neckers, L. M. (1994) Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: Essential role for stress proteins in oncogenic transformation, Proc. Natl. Acad. Sci. U.S.A. 91, 8324–8328. 8. Uehara, Y. (2003) Natural product origins of Hsp90 inhibitors, Curr. Cancer Drug Targets 3, 325–330.
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