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Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado
Received April 12, 2006; accepted July 6, 2006
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Abstract |
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; Isaacs et al., 2003); these proteins include ErbB2, Raf-1, Cdk4, Met, mutant p53, telomerase hTERT, Hif-1
, and the estrogen and androgen receptors. The function of Hsp90 has been shown to be dependent on its ability to bind and hydrolyze ATP (Obermann et al., 1998; Panaretou et al., 1998; Pearl and Prodromou, 2001), and competitive inhibition of ATP binding by the natural product geldanamycin (GM), a benzoquinone ansamycin antibiotic isolated from Streptomyces hygroscopicus, leads to the degradation of the client proteins by the ubiquitin-proteosome pathway (Whitesell et al., 1994; Schulte et al., 1995; An et al., 1997), resulting in cell cycle arrest, differentiation, and apoptosis (Hostein et al., 2001; Munster et al., 2001). Therefore, Hsp90 is an attractive cancer drug target in that it has the potential for simultaneous disruption of multiple oncogenic signaling proteins (Csermely et al., 1998; Adams and Elliott, 2000; Richter and Buchner, 2001; Goetz et al., 2003). Furthermore, studies have revealed that the benzoquinone ansamycin Hsp90 inhibitors accumulate in tumor cells more efficiently than in normal tissue, leading to high differential selectivities (Chiosis et al., 2003). In addition, immunoprecipitation studies have shown that Hsp90 from tumor cells has an enhanced affinity for N-terminal ligands, resulting in increased ATPase activity and an increase in sensitivity to benzoquinone ansamycins, than Hsp90 in normal cells (Kamal et al., 2003).
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), and to overcome these undesirable properties, a number of GM analogs of the benzoquinone ansamycin class have been developed, which differ only in their 17-substituent. These include 17-(allylamino)-17-demethoxygeldanamycin (17AAG), which has shown evidence of biological and clinical activity although it has poor solubility and potential toxicity, and the more water-soluble 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin (17DMAG); both are currently in clinical trials (Banerji et al., 2005; Goetz et al., 2005; Smith et al., 2005). The benzoquinone ansamycins are extensively metabolized in vivo, and the 17-position of 17AAG and 17DMAG is prone to dealkylation by cytochrome P450 to generate the benzoquinone ansamycin and Hsp90 inhibitor 17-(amino)-17-demethoxygeldanamycin (17AG) (Egorin et al., 1998). The redox active quinone moiety, of the benzoquinone ansamycins, is susceptible to reduction by flavin-containing reductases to the semiquinone with subsequent generation of superoxide (Dikalov et al., 2002) or hydroquinone species (Kelland et al., 1999; Guo et al., 2005), which is dependent on one- or two-electron reduction. However, these metabolic pathways could introduce further selectivity to the activation of the benzoquinone ansamycins to a more potent species, the hydroquinone ansamycin, within tumor cells, depending on the levels and type of bioreductive enzyme present.
NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, NQO1, DT-diaphorase) is an obligate two electron-reducing flavin-containing enzyme that can use either NADH or NADPH as reducing cofactors and can catalyze the direct two-electron reduction of quinones to hydroquinones (Ernster, 1967). NQO1 is expressed at high levels throughout many human solid tumors, and levels are higher in many human tumor cell lines and cancer tissues (colon, stomach, pancreatic, lung, and breast) compared with the normal equivalents (Siegel et al., 1998; Siegel and Ross, 2000; Cullen et al., 2003). NQO1 has been shown to activate a number of quinone-based bioreductive cytotoxic antitumor agents, including diaziquone, mitomycin C, 3-[5-aziridinyl-3-(hydroxymethyl)-1-methyl-4,7-dioxoindol-2-yl]prop-2-enol, streptonigrin, 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone, and β-lapachone, by reduction to hydroquinone species (Siegel et al., 1990a,b; Walton et al., 1991; Beall et al., 1996; Winski et al., 1998; Pink et al., 2000).
The NQO1-mediated reduction of 17AAG using purified NQO1 and the increased sensitivity of human cancer cell lines expressing NQO1 to 17AAG were originally reported by Kelland et al. (1999). We extended these studies to examine the properties of 17-(allylamino)-17-demethoxygeldanamycin hydroquinone (17AAGH2) formed after reduction of 17AAG by NQO1 (Guo et al., 2005). In addition, we also used the human breast cancer cell line MDA468, deficient in NQO1 because of a genetic polymorphism, and an isogenic paired cell line MDA468/NQ16, a stably transfected clone that expressed high levels of NQO1 protein, in combination with a mechanism-based inhibitor of NQO1 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936)] to determine the effect of NQO1 on the metabolism of 17AAG in cells. Hsp90 ATPase activity assays with purified yeast and human Hsp90 showed that 17AAGH2 was a more potent inhibitor of Hsp90 than the parent quinone (Guo et al., 2005). Molecular modeling studies of 17AAG and 17AAGH2 in the nucleotide-binding pocket of the N-terminal domain of the yeast and human Hsp90 crystal structures displayed a greater number of hydrogen bond interactions with the hydroquinone, resulting in greater interaction energies (Guo et al., 2005).
Here, we have examined the reduction of a series of benzoquinone ansamycins, which include GM, 17DMAG, 17AG, and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin (17AEP-GA), using purified NQO1 to the corresponding hydroquinone ansamycins (Scheme 1) and the inhibition of purified yeast Hsp90 ATPase activity by this series of benzoquinone ansamycins in the presence and absence of NQO1. To confirm the bioreduction of the benzoquinone ansamycins by NQO1 in cells, we used the human breast cancer cell line MDA468 and the isogenic paired cell line MDA468/NQO1 in combination with ES936. We have also extended our molecular modeling study to examine the interactions of both the quinone and hydroquinone forms of this series of benzoquinone ansamycins in the N-terminal domain of the yeast Hsp90 crystal structure. In this article, we describe the data for this series of benzoquinone ansamycins. Where possible, we have illustrated the full data set for the series; otherwise, we have shown the data for 17DMAG and supplemented the remaining data as supporting information.
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Materials and Methods |
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ES936 was supplied by Christopher J. Moody (School of Chemistry, University of Nottingham, Nottingham, UK). Yeast Hsp90 and radicicol were obtained from Alexis (San Diego, CA). Recombinant human NQO1 (rhNQO1) was purified from Escherichia coli as described previously (Beall et al., 1994). The activity of rhNQO1 was 4.5 µmol of DCPIP/min/mg protein.
Cell Lines. The human breast cancer cell line MDA468 and the NQO1 stably transfected cell line MDA468/NQ16 have been described previously (Dehn et al., 2004). Cells were grown in RPMI 1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin, streptomycin, and glutamine. MDA468 and MDA468/NQ16 cell sonicates were prepared by probe sonication in ice-cold 25 mM Tris-HCl (pH 7.4) containing 250 mM sucrose and 5 µM flavin adenine dinucleotide.
Inhibition of NQO1 by ES936. The inhibition of NQO1 by ES936, a potent mechanism-based inhibitor of NQO1, was achieved using a single dose of 100 nM ES936, which was nontoxic and resulted in >96% inhibition of NQO1 activity after 4 h in MDA468/NQ16 cells (Dehn et al., 2003).
High-Performance Liquid Chromatography and Liquid Chromatography/Mass Spectrometry Analysis. The metabolism of the benzoquinone ansamycins by NQO1 was analyzed by high-performance liquid chromatography (HPLC) on a Luna C18 5-µm, 4.6 x 250-mm reverse-phase column (Phenomenex, Torrance, CA) at room temperature. HPLC conditions were as follows: buffer A, 50 mM ammonium acetate (pH 4) containing 10 µM D(-)-penicillamine; buffer B, methanol (100%). Both buffers were continuously bubbled with argon, gradient, 30 to 90% B over 10 min, and then 90% B for 5 min (flow rate of 1 ml/min). The sample injection volume was 50 µl. Liquid chromatography/mass spectrometry (LC/MS) was performed using positive ion electrospray ionization, and the mass spectra were obtained with a PE Sciex API-3000 triple quadrupole MS (Foster City, CA) with a turbo ion spray source interfaced to a PE Sciex 200 HPLC system. Samples were chromatographed on a Luna C18 5-µm, 50 x 2-mm reverse-phase column (Phenomenex, Torrance, CA) using a gradient elution consisting of a 2-min initial hold at 20% B, followed by an increase to 80% B over 20 min at a flow rate of 200 µl/min and a sample injection volume of 20 µl. Solvent A was 10 mM ammonium acetate containing 0.1% (v/v) acetic acid (pH 4.4), and solvent B was 10 mM ammonium acetate in acetonitrile containing 0.1% (v/v) acetic acid. The mass spectrometer settings were turbo ion spray temperature of 250°C, spray needle voltage at 4500 V, declustering potential at 35 V, and focus plate at 125 V. Mass spectra were continuously recorded from 150 to 1000 atomic mass units every 3 s during the chromatographic analysis.
Hsp90 ATPase Activity Assay. Inhibition of yeast Hsp90 ATPase was measured as described previously (Rowlands et al., 2004). In brief, 2.5 µg of purified yeast Hsp90 was incubated in 100 mM Tris-HCl (pH 7.4) containing 20 mM KCl, 6 mM MgCl2, 200 µM NADH, the appropriate benzoquinone ansamycin (2 and 4 µM) with or without 0.33 µg of rhNQO1, and 2 µM ES936. Reactions (25 µl) were started by the addition of 1 mM ATP and allowed to proceed at 37°C for 3 h. Reactions were then diluted with 225 µl of 100 mM Tris-HCl (pH 7.4) containing 20 mM KCl and 6 mM MgCl2 mixed thoroughly, and 80 µl was transferred to each well (96-well plate) followed by 20 µl of malachite green reagent. After 10 min, trisodium citrate (83 mM) was added to stabilize the color, and plates were read at 650 nm.
Growth Inhibition Assays. Growth inhibition was measured using the MTT assay. Cells were seeded at 2 x 103 per well (96-well plate) in complete medium for 16 h. The cells were then pretreated with 100 nM ES936 or an equal amount of dimethyl sulfoxide (DMSO) for 30 min and then exposed to the appropriate benzoquinone ansamycin for 4 h, after which cells were washed free of drug and incubated in fresh medium for an additional 72 h. The increase in drug exposure time from 4 h to 72 h (continuous exposure) was performed for 17AAG and 17DMAG. Cell viability was measured by the MTT assay as described previously (Winski et al., 2001). Inhibition of rhNQO1 by ES936 was >98%.
Immunoblot Analysis. MDA468 and MDA468/NQ16 cells were grown in 100-mm plates in complete medium to 
70% confluency. For Hsp70 and Raf-1 analysis, cells were treated with DMSO or 17DMAG (50-100 nM) in 10 ml of complete medium for 24 h. After drug treatment, cells were washed in phosphate-buffered saline and then lysed by the addition of radioimmuno-precipitation assay buffer [50 mM Tris-HCl, pH 7.4, 0.5% (v/v) NP40] containing 1 Mini protease tablet (protease inhibitor mixture; Roche, Indianapolis, IN) and phosphatase inhibitors (30 mM NaF, 40 mM β-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM orthovanadate, and 1 mM EGTA). Lysates were probe-sonicated (2 s) on ice and then centrifuged to remove cellular debris. Protein concentrations were determined on supernatant by the method of Lowry et al. (1951). Samples were heated to 70°C in 2x Laemmli SDS sample buffer, and proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (precast minigel; Bio-Rad, Hercules CA) and then transferred to 0.4-µm polyvinylidene difluoride membranes. Membranes were blocked in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Tween 20, and 5% nonfat milk for a minimum of 1 h at room temperature. Anti-Hsp70 and anti-Raf antibodies were added for 1 h at room temperature. All the primary antibodies were diluted 1:1000, except actin (1:5000). Horseradish peroxidase-labeled secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA) were diluted 1:5000 and added for 30 min. Proteins were visualized using enhanced chemiluminescence detection.
Molecular Modeling of the Benzoquinone Ansamycin and Corresponding Hydroquinone Ansamycin in the Amino-Terminal Domain of the Yeast Hsp90 Crystal Structure. Molecular modeling studies were performed on a Silicon Graphics Octane 2 workstation using the InsightII software package version 2000 (Accelrys, Inc., San Diego, CA). The crystallographic coordinates for the 2.5-Å structure of the amino-terminal domain of yeast Hsp90, Protein Data Bank no. 1A4H (Prodromou et al., 1997), were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The Builder Module was used to add hydrogen to the protein structure, and the ionizable residues were corrected for physiologic pH. The benzoquinone ansamycin and the corresponding hydroquinone structures were constructed and assigned the correct atom type and bond order from the cocrystallized GM structure. Once constructed, the ligands were in turn positioned, using the coordinated system of the protein, in the nucleotide-binding domain of Hsp90; the ligand assembly was then associated with the Hsp90 protein structure. For the molecular mechanics and molecular dynamics calculations, the Discover Module was used, and the potentials and charges of the Hsp90-ligand complex were corrected using consistent valence forcefield (Dauber-Osguthorpe et al., 1988). The Hsp90-ligand complex was then minimized using the conjugate gradient method (1000 iterations). The Docking Module was used to perform the intermolecular energy calculation to determine the nonbonded interaction energy between the Hsp90 protein and the appropriate ligand. An interface 6-Å radius subset encompassing the ligand-binding domain was selected, and both van der Waals and electrostatic energies were calculated with a specified cutoff of 8 Å.
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; Guo et al., 2005). The identification of the hydroquinone ansamycin product, from the chemical or NQO1-mediated reduction of the benzoquinone ansamycin, was confirmed by LC/MS, giving ions for GMH2 at m/z 580.5 [M+NH4+]+, 17AGH2 at m/z 548.2 [M+H+]+, 17DMAGH2 at m/z 619.3 [M+H+]+, and 17AEP-GAH2 at m/z 645.3 [M+H+]+ (Fig. 1D, data shown for 17DMAG; for other compounds, see supplemental data). All the hydroquinone ansamycins formed by the reduction of the benzoquinone ansamycins in this series were susceptible to autoxidation. The rate of autoxidation of the hydroquinone ansamycin was dependent on the substituent at the 17-position; from our studies, the GM hydroquinone from this series was the most stable (data not shown). The autoxidation of the hydroquinone ansamycin could be impeded by continuously gassing the HPLC buffers during analysis with nitrogen or argon and by the addition of a copper chelator to the HPLC buffers (see under Materials and Methods).
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The malachite green Hsp90 ATPase activity assay has been previously used as a high-throughput screen to evaluate Hsp90 inhibition (Rowlands et al., 2004). It was used here to examine the differences in inhibitory activity of this series of benzoquinone ansamycins and their respective hydroquinones on the yeast Hsp90 ATPase reaction (Fig. 2). In these assays, purified yeast Hsp90 and ATP were incubated with the appropriate benzoquinone ansamycin (2 and 4 µM) and NADH in the presence and absence of rhNQO1 over a 3-h period, after which reactions were terminated, and the concentration of inorganic phosphate was measured using the malachite green assay (Rowlands et al., 2004). A significant decrease in ATPase activity was observed with each of the benzoquinone ansamycins examined in the presence of NQO1 compared with that obtained for the benzoquinone ansamycins with NADH, and this could be prevented by pretreatment with ES936. At these concentrations, minimal inhibition of the Hsp90-mediated ATPase reaction was observed for 17DMAG and 17AEP-GA, which further highlighted the effect of the hydroquinone ansamycin on Hsp90 inhibition. The results from this ATPase assay showed that for this series of benzoquinone ansamycins, the hydroquinone ansamycin, generated by NQO1, was the more potent Hsp90 inhibitor.
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These studies were extended to a cell-based system, and the formation of the hydroquinone ansamycins was investigated in cell sonicates prepared from the isogenic MDA468 and MDA468/NQ16 human breast cancer cell lines. These cell lines have been used previously to examine the role of NQO1 in antitumor quinone metabolism (Dehn et al., 2004), including 17AAG (Guo et al., 2005). The parental MDA468 cell line is deficient in NQO1 (<10 nmol DCPIP/min/mg) because of homozygous expression of the NQO1*2 polymorphism (Traver et al., 1997). The MDA468/NQ16 cell line was generated by the stable transfection of the parental MDA468 cell line with human NQO1, resulting in high NQO1 activity (>1800 nmol DCPIP/min/mg). The HPLC analysis of MDA468 and MDA468/NQ16 cell sonicates treated with these benzoquinone ansamycins yielded similar results to those obtained previously with 17AAG (Guo et al., 2005). The hydroquinone ansamycins were not detected in the MDA468 cell sonicates (Fig. 3A, data shown for 17DMAG; for other compounds, see supplemental data). Whereas the MDA468/NQ16 cell sonicates readily generated the hydroquinone ansamycin (Fig. 3B, data shown for 17DMAG; for other compounds, see supplemental data), which was NADH- or NADPH-dependent, and the formation of the hydroquinone could be prevented by ES936 (Fig. 3C, data shown for 17DMAG; for other compounds, see supplemental data).
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Molecular modeling studies of this series of benzoquinone ansamycins and their corresponding hydroquinones into the ATP-binding site of the yeast Hsp90 crystal structure revealed significant differences in the binding energies post-minimization. The nonbonded interaction energy is the sum of the van der Waals and electrostatic energies, the measure of the affinity between the Hsp90 protein and the ligand investigated. In all the benzoquinone ansamycins examined, the hydroquinone had a greater nonbonded interaction energy than the parent quinone. These data support the hypothesis that the hydroquinone ansamycin is a more potent inhibitor of Hsp90. After minimization, the Hsp90-ligand complex was visualized to identify important amino acid residues in the ATP-binding domain that interact via hydrogen bonding with the ligand investigated; there was no significant change in the global conformation of Hsp90. A number of protein-ligand and through-solvent interactions lock the macrocycle of benzoquinone and the hydroquinone ansamycins in an overall conformation similar to that reported for the original yeast Hsp90-GM cocrystallized structure (Prodromou et al., 1997). The Hsp90-hydroquinone ansamycin complex revealed additional direct hydrogen-bonding interactions between the hydroquinone ansamycin and the Hsp90 protein, accounting for the greater interaction energy (Table 2, data shown for 17DMAG; for other compounds, see supplemental data), compared with the parent quinone. In the ATP-binding domain of yeast Hsp90, the C21 ketone of the benzoquinone ansamycins hydrogen bonds with the amine of Lys98, and the C18 ketone of the quinone ring system interacts with a water molecule that in turn contacts Asp40 (Fig. 5, A and C, data shown for 17DMAG; for other compounds, see supplemental data). The C21 hydroxyl of the hydroquinone ansamycins GMH2, 17AGH2, and 17AEP-GAH2 also hydrogen bonds with the amine of Lys98, and the C18 hydroxyl of the hydroquinone ring system directly hydrogen bonds to Asp40, whereas the C21 hydroxyl of 17AAGH2 and 17DMAG2 does not interact directly with Lys98. The hydrogen atom of the C18 hydroxyl, in all the hydroquinone ansamycins examined, interacts with an oxygen atom of the carboxylate side chain of Asp40, which results in a more compact C-clamp conformation around helix-2, and in the case of the 17-amino-substituted ansamycins, the hydroquinone allows the amide of the ansa ring to interact with the backbone nitrogen of Phe124 (Fig. 5, B and D, data shown for 17DMAG; for other compounds, see supplemental data). Although no direct interactions were observed with yeast Hsp90 and the 17-substituent of the benzoquinone ansamycins or their respective hydroquinones, the side chain is orientated into solvent and does not seem to directly interfere with the binding of the ligands to the Hsp90 binding pocket.
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). They also described increased inhibitory activity of GM and 17AAG in NQO1-transfected BE cells relative to NQO1-null BE parental cells and suggested that NQO1 might play a role in the cellular metabolism of these benzoquinone ansamycins (Kelland et al., 1999). We extended these studies for 17AAG by examining the purified rhNQO1-mediated reduction of 17AAG; identifying the reduction product, 17AAGH2, by HPLC and LC/MS; assessing the inhibition of Hsp90 ATPase activity of 17AAG and 17AAGH2 generated by NQO1 and abrogated by ES936; examining toxicity in isogenic MDA468 and MDA468/NQ16 cells; and investigating the interactions of 17AAG and 17AAGH2 in the ATP binding domain of Hsp90 by computational modeling (Guo et al., 2005).
In this article, we have examined a series of benzoquinone ansamycins, differing in structure only at the 17-position, to determine whether the "active" hydroquinone ansamycin hypothesis can be applied to a range of the common benzoquinone ansamycin compounds, including 17DMAG, which is currently in phase I trials in patients with metastatic, unresectable solid tumors and lymphomas. In each case, we achieved reduction of the benzoquinone ansamycin to the corresponding hydroquinone ansamycin using purified rhNQO1; the resulting hydroquinone was identified by HPLC and LC/MS. To confirm that the hydroquinone ansamycins, generated by NQO1, were more potent Hsp90 inhibitors than their respective parent quinones, we used purified yeast Hsp90 to assess the ATPase activity. Indeed, as expected, the hydroquinone ansamycins were more potent inhibitors of the yeast Hsp90 ATPase reaction. The inhibition of ATPase activity by the hydroquinone ansamycins could be abrogated by use of ES936, a mechanism-based inhibitor of NQO1, confirming the role of the hydroquinone moiety in potentiating Hsp90 inhibition.
The NQO1-dependent formation of the hydroquinone ansamycins was detected by HPLC analysis in MDA468/NQ16 sonicates but not in MDA468 sonicates and could be blocked by use of ES936. Furthermore, the effect of this series of benzoquinone ansamycins on growth inhibition was increased in MDA468/NQ16 cells relative to the NQO1-deficient parental MDA468 cell line, which was consistent with previous data (Kelland et al., 1999; Guo et al., 2005). The inhibition of NQO1 with ES936 in MDA468/NQ16 cells resulted in growth inhibitory activity of the benzoquinone ansamycins to approximately those observed in the parental MDA468 cells. In addition, the nonquinone Hsp90 inhibitor, radicicol, displayed essentially the same growth inhibitory profile in both MDA468 and MDA468/NQ16 cells. These data clearly show that the increased sensitivity in cells containing elevated NQO1 levels is caused by the reduction of the benzoquinone ansamycin to the more active hydroquinone form, in concurrence with previous data (Guo et al., 2005). The increased-fold potentiation of 17AAG and 17DMAG in MDA468/NQ16 relative to MDA468 cells with increased exposure time may be a result of increased accumulation of the hydroquinone ansamycin, generated by NQO1, in cells over time, caused by the decreased lipid solubility of the hydroquinone form relative to the parent quinone. The differences in -fold potentiation between 17AAG and 17DMAG could result from the rate of NQO1-mediated reduction because 17AAG is reduced at a faster rate relative to 17DMAG (data not shown) or from the stability of the hydroquinone ansamycin formed. Therefore, the marked increase in -fold potentiation, particularly with 17AAG, could be significant with respect to antitumor effect in tumors containing high levels of NQO1. The amount of NQO1 needed to generate adequate levels of hydroquinone ansamycin sufficient for optimal growth inhibitory activity will vary from cell to cell and will depend on a variety of factors, including the reduction rate, the stability of the hydroquinone generated, and the rate of repair of any cellular damage resulting from Hsp90 inhibition.
Computational-based modeling studies of the benzoquinone ansamycins and the hydroquinone ansamycins in the nucleotide-binding domain, of the N-terminal domain, and of the yeast Hsp90 crystal structure displayed a greater number of hydrogen bond interactions between the Hsp90 protein and the hydroquinone ansamycin, resulting in increased total interaction energies compared with the parent quinone. The benzoquinone ansamycin and hydroquinone ansamycin series investigated in this study differed only in substitution at the 17-position, and in all instances, the 17-substituent pointed into solvent and did not appear to interact directly with the Hsp90 protein. The greater interaction energies of the hydroquinone ansamycins can be explained by the additional direct hydrogen bond contacts with the Hsp90 protein, mainly because of the hydrogen bond donor contribution of the hydroquinone moiety, which results in a more compact C-shaped conformation that is evident from the hydrogen bonding interaction between the C18 hydroxyl and the Asp40 residue of helix-2; the strength of this interaction, as indicated by the hydrogen bond length, seems to correlate with the electrostatic contribution to the nonbonded interaction energy across this hydroquinone ansamycin series. The more compact conformation of the hydroquinone ansamycin around helix-2, the greater protein-hydroquinone ansamycin hydrogen bond contacts, and the resultant greater interaction energies for the hydroquinone ansamycins relative to the parent quinone for this series provide explanation as to why the hydroquinone ansamycins are more potent Hsp90 inhibitors.
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A rational extension to this research would be to develop a prodrug approach (Scheme 2) to deliver the more potent hydroquinone ansamycin intracellularly. The generation of a series of hydroquinone ansamycin prodrugs would initially circumvent the NQO1-mediated reduction of the benzoquinone ansamycin and may additionally improve the solubility and bioavailability characteristics of the Hsp90 inhibitor. An ideal prodrug candidate would generate, via intracellular enzymatic hydrolysis, a relatively stable hydroquinone ansamycin, which is also a potent inhibitor of Hsp90 and in its quinone form is a good substrate for NQO1 to facilitate reduction back to the more potent Hsp90 inhibitor, the hydroquinone ansamycin. In addition, the hydroquinone ansamycin has increased water solubility and a decreased tendency to cross cell membranes, leading to increased accumulation of the more potent Hsp90 inhibitor, the hydroquinone ansamycin, in tumor cells containing NQO1 (Workman, 2003; Guo et al., 2005). This prodrug approach may further improve the selectivity of the Hsp90 inhibitor, in that the hydrolyzing enzyme may be specific to or more abundant in a certain tissue type.
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Footnotes |
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W.G. and P.R. contributed equally to this work.
ABBREVIATIONS: Hsp90, heat shock protein 90; GM, geldanamycin; 17AAG, 17-(allylamino)-17-demethoxygeldanamycin; 17DMAG, 17-demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin; 17AG, 17-(amino)-17-demethoxygeldanamycin; NQO1, NAD(P)H:quinone oxidoreductase; 17AAGH2, 17-(allylamino)-17-demethoxygeldanamycin hydroquinone; ES936, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl-]indole-4,7-dione; 17AEP-GA, 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin; DCPIP, 2,6-dichlorophenol-indophenol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BSA, bovine serum albumin; rhNQO1, recombinant human NQO1; HPLC, high-performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; DMSO, dimethyl sulfoxide.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: David Ross, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, C-238, 4200 East 9th Avenue, Denver, CO 80262. E-mail: david.ross{at}uchsc.edu
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