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. 2011 May;79(5):823–832. doi: 10.1124/mol.110.070086

A Mechanistic and Structural Analysis of the Inhibition of the 90-kDa Heat Shock Protein by the Benzoquinone and Hydroquinone AnsamycinsS⃞

Philip Reigan 1, David Siegel 1, Wenchang Guo 1, David Ross 1,✉
PMCID: PMC3082934  PMID: 21285336

Abstract

The benzoquinone ansamycins inhibit the ATPase activity of the 90-kDa heat shock protein (Hsp90), disrupting the function of numerous client proteins involved in oncogenesis. In this study, we examine the role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the metabolism of trans- and cis-amide isomers of the benzoquinone ansamycins and their mechanism of Hsp90 inhibition. Inhibition of purified human Hsp90 by a series of benzoquinone ansamycins was examined in the presence and absence of NQO1, and their relative rate of NQO1-mediated reduction was determined. Computational-based molecular docking simulations indicated that the trans- but not the cis-amide isomers of the benzoquinone ansamycins could be accommodated by the NQO1 active site, and the ranking order of binding energies correlated with the relative reduction rate using purified human NQO1. The trans-cis isomerization of the benzoquinone ansamycins in Hsp90 inhibition has been disputed in recent reports. Previous computational studies have used the closed or cocrystallized Hsp90 structures in an attempt to explore this isomerization step; however, we have successfully docked both the trans- and cis-amide isomers of the benzoquinone ansamycins into the open Hsp90 structure. The results of these studies indicate that both trans- and cis-amide isomers of the hydroquinone ansamycins exhibited increased binding affinity for Hsp90 relative to their parent quinones. Our data support a mechanism in which trans- rather than cis-amide forms of benzoquinone ansamycins are metabolized by NQO1 to hydroquinone ansamycins and that Hsp90-mediated trans-cis isomerization via tautomerization plays an important role in subsequent Hsp90 inhibition.

Introduction

The 90-kDa heat shock protein (Hsp90) is overexpressed in cancer cells and is implicated in their survival by maintaining the active conformation of key oncogenic proteins, including ErbB2, Raf-1, Akt, hypoxia-inducible factor-1α, and mutant p53 (Maloney and Workman, 2002). The function of Hsp90 is complex and involves homodimerization, recruitment of cochaperones, accessory proteins, and the client protein, operating in a dynamic “chaperone cycle” dependent on the ATPase activity of Hsp90 (Pearl and Prodromou, 2006). Hsp90 is an important anticancer target, and the conserved ATP-binding domain of Hsp90 is also the binding site of the natural products geldanamycin (GM) and radicicol and a range of semisynthetic and synthetic compounds (Roe et al., 1999; Maloney and Workman, 2002). These compounds prevent Hsp90 from cycling between ADP- and ATP-bound conformations, resulting in the degradation of multiple oncogenic client proteins via the ubiquitin-proteasome pathway and ultimately growth arrest or apoptosis (Maloney and Workman, 2002).

The benzoquinone ansamycin class of Hsp90 inhibitors include GM and its semisynthetic derivative 17-allylamino-17-demethoxy-geldanamycin (17AAG), which have been shown to bind to Hsp90 with micromolar affinity in vitro and with nanomolar activity in vivo (Roe et al., 1999; Chiosis et al., 2003) and have demonstrated selectivity toward tumor cells (Chiosis and Neckers, 2006). The benzoquinone ansamycins exist in two isomeric forms: in solution they adopt an almost planar trans-amide conformation (Schnur and Corman, 1994), whereas cocrystallization studies indicate the formation of the C-shaped cis-amide isomer in the nucleotide-binding site of Hsp90 (Stebbins et al., 1997; Jez et al., 2003). Isomerization of the benzoquinone ansamycins has been proposed to be a key requirement for Hsp90 inhibition (Lee et al., 2004). An early cocrystallization study suggested that Hsp90 may bind to the benzoquinone ansamycins because they resemble misfolded peptides (Stebbins et al., 1997). Although the nucleotide-binding site of Hsp90 is not a recognized peptide-binding domain, the Hsp90-mediated isomerization of the benzoquinone ansamycins has been supported by quantum and molecular mechanic studies (Jez et al., 2003; Lee et al., 2004) and the cis-trans isomerization of nonproline peptide bonds (Schiene-Fischer et al., 2002). Other studies have proposed that a cochaperone may have isomerase activity (Pearl and Prodromou, 2000; Kamal et al., 2003), and a recent study has disputed the isomerization of the benzoquinone ansamycins as a requirement for Hsp90 inhibition (Onuoha et al., 2007).

In addition to trans-cis isomerization, the C17 substituents of the benzoquinone ansamycins are extensively metabolized in vivo (Egorin et al., 1998), and the 19-position is prone to glutathionylation (Cysyk et al., 2006; Guo et al., 2008). The redox active quinone moiety is susceptible to one- and two-electron reduction by flavin-containing reductases (Guo et al., 2005; Lang et al., 2007). The direct two-electron reduction of benzoquinone ansamycins catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase, EC: 1.6.99.2) generating their hydroquinone derivatives circumvents the formation of semiquinone radicals and reactive oxygen species (Ross, 2004). Furthermore, NQO1 is expressed at high levels in many solid tumors (Siegel and Ross, 2000), and the expression of NQO1 has been found to correlate with 17AAG sensitivity (Kelland et al., 1999), offering the potential for drug activation with tumor selectivity (Rooseboom et al., 2004).

In our previous studies, we reported the metabolism of a series of benzoquinone ansamycins by recombinant human (rh)NQO1 generating the corresponding hydroquinone ansamycins (Scheme 1), and these were more potent inhibitors of yeast Hsp90 ATPase activity than their parent quinones (Guo et al., 2005; Guo et al., 2006). This potentiated inhibition was rationalized by molecular modeling simulations that displayed increased direct hydrogen bond interactions between the hydroquinone ansamycins and the amino acid residues in the nucleotide-binding site of Hsp90 (Guo et al., 2005, 2006). In this study, we extend our previous investigations using yeast Hsp90 to human Hsp90 (yeast Hsp90 has ∼60% homology with human Hsp90). We have examined the relative rate of rhNQO1-mediated reduction of a series of benzoquinone ansamycins and the inhibition of purified human Hsp90 by both benzoquinone and hydroquinone ansamycins. Computational-based molecular docking was used to investigate the conformation of the benzoquinone ansamycins in the NQO1 active site and the structural properties that influence the rate of NQO1-mediated reduction. The interaction of the trans- and cis-amide isomers of the benzoquinone and hydroquinone ansamycins in the nucleotide-binding site of human Hsp90 was also investigated using molecular docking to explore the potential mechanism of Hsp90-mediated trans-cis isomerization.

Scheme 1.

Scheme 1.

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The NQO1-mediated reduction of the benzoquinone ansamycins.

Materials and Methods

Materials.

GM, 17-demthoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin (17DMAG), and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin (17AEP-GA) were obtained from Invitrogen (Carlsbad, CA), and 17AAG and 17-(amino)-17-demethoxygeldanamycin (17AG) were obtained from the National Cancer Institute and Kosan Biosciences (Hayward, CA). 2,6-Dichlorophenol-indophenol, NADH, NADPH, bovine serum albumin, and D(−)penicillamine were obtained from the Sigma-Aldrich (St. Louis, MO). Malachite green phosphate assay kit was obtained from BioAssay Systems (Hayward, CA). 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936) (Winski et al., 2001) was supplied by Professor Christopher J. Moody (School of Chemistry, University of Nottingham, Nottingham, United Kingdom). Human Hsp90 was a kind gift from Professor David Toft (Mayo Clinic College of Medicine, Rochester, MN). Recombinant human NQO1 was purified from Escherichia coli as described previously (Beall et al., 1994). The activity of rhNQO1 was 4.5 μmol 2,6-dichlorophenol-indophenol per minute per milligram.

HPLC and LC/MS Analysis.

The NQO1-mediated reduction of the benzoquinone ansamycins was monitored by HPLC on a Luna C18 5 μm, 4.6 × 250 mm reversed-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, acetonitrile (100%). Both buffers were continuously bubbled with argon, gradient, 30% buffer B to 90% buffer B over 10 min and then 90% buffer B for 5 min (flow rate of 1 ml/min). The sample injection volume was 50 μl. LC/MS was performed using positive-ion electrospray ionization, and mass spectra were obtained using a PE Sciex API-3000 triple quadrupole MS (Applied Biosystems, Foster City, CA) with a turbo ion spray source interfaced to a PE Sciex 200 HPLC system. Samples were separated on a Luna C18 5 μm, 50 × 2 mm reverse-phase column (Phenomenex) using a gradient elution consisting of a 2 min initial hold at 20% buffer B followed by an increase to 80% buffer B over 20 min at a flow rate of 200 μl/min and a sample injection volume of 20 μl. Solvent A was made up of 10 mM ammonium acetate containing 0.1% (v/v) acetic acid, pH 4.4, and solvent B was composed of 10 mM ammonium acetate in acetonitrile containing 0.1% (v/v) acetic acid. Mass spectra were continuously recorded from 150 to 1000 atomic mass units every 3 s during the chromatographic analysis, with a turbo ion spray temperature of 250°C, spray needle voltage at 4500 V, declustering potential at 35 V, and focus plate at 125 V.

Reduction of Benzoquinone Ansamycins by rhNQO1.

Benzoquinone ansamycin 50 μM, NADH 200 μM, and rhNQO1 (1.65 μg of for GM, 17AAG, and 17AG; 3.3 μg for 17DMAG; 6.6 μg for 17AEP-GA) in 50 mM potassium phosphate buffer, pH 7.4 (1 ml), containing 1 mg/ml bovine serum albumin were incubated at 37°C for 10 min. Reactions were monitored by HPLC, and the reduction rate was determined by measuring NADH oxidation at 340 nm during the reaction. The reaction was stopped with an equal volume of methanol. The apparent Km values for 17AAG and 17DMAG (0.004–0.8 mM) were determined using NADH 0.5 mM and rhNQO1 10 μg, incubated in 50 mM potassium phosphate buffer, pH 7.4, containing FAD (1 μM), 0.4% (v/v) Tween 80, and 0.4% (v/v) Triton X-100 at 30°C and by monitoring quinone-dependant oxidation of NADH. Reactions were terminated by diluting the reaction 10 times with ice-cold methanol, and the resulting NADH concentrations were determined by fluorescence spectroscopy (excitation, 340 nm; emission, 460 nm). Km values were determined using Prism software (GraphPad Software Inc., San Diego, CA).

Hsp90 ATPase Activity Assay.

Inhibition of human Hsp90 ATPase activity was measured as described previously (Rowlands et al., 2004; Guo et al., 2005). Briefly, 2.5 μg of purified human Hsp90 was incubated in 100 mM Tris-HCl, pH 7.4, containing 20 mM KCl, 6 mM MgCl2, 200 μM NADH, benzoqinone ansamycin (2 and 4 μM) with or without rhNQO1 0.33 μg, and with or without 2 μM ES936. Reactions (25 μl) were started by the addition of 1 mM ATP and allowed to proceed at 37°C for 12 h. Reactions were then diluted with 225 μl of 100 mM Tris-HCl, pH 7.4, containing KCl 20 mM and 6 mM MgCl2 mixed thoroughly, and 80 μl was transferred to a 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.

Molecular Modeling.

All simulations were performed using Discovery Studio software (version 2.5; Accelrys, San Diego, CA). The crystallographic coordinates of the 1.8-Å human NQO1 structure cocrystallized with ES936 (PDB 1KBQ), the 2.2Å open human Hsp90 structure (PDB 1YES), and the 1.9-Å human Hsp90 structure cocrystallized with GM (PDB 1YET) (Stebbins et al., 1997; Winski et al., 2001) were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org). The cis-amide isomers of the benzoquinone ansamycins were constructed from GM isolated from the human Hsp90-GM cocrystallized complex, the trans-amide isomers were constructed from trans-17-azetidinyl-17-demethoxygeldanamycin (Schnur and Corman, 1994) obtained from the Cambridge Structural Database (http://www.ccdc.cam.ac.uk), and the corresponding hydroquinone ansamycins were generated by modifying the quinone ring structure. The ionizable residues were corrected for physiological pH, and the potentials and charges of the complexes were corrected using CHARMm in all simulations (Brooks et al., 2009).

NQO1 Docking.

The ES936 molecules were removed from the NQO1 structure, and the anionic form of reduced FAD was constructed from each FAD cofactor (Reigan et al., 2007). The binding site was defined as whole residues within a 5-Å radius subset encompassing the FAD cofactor. The flexible docking program was used to dock the trans-amide isomers of the benzoquinone ansamycins into the NQO1 active site, followed by simulated annealing step to optimize interactions (Koska et al., 2008). In addition to the benzoquinone ansamycin, flexibility was allowed in the following active site R-groups: Leu103, Gln104, Trp105, Phe106, Thr148, Gly149, Gly150, Ser151, Met154, Tyr155, His161, Pro68, Gly122, Tyr126, Thr127, Tyr128, Met131, Phe178, Phe232, and the FAD cofactor. An in situ minimization using the conjugate gradient method (1000 iterations with a root mean square cutoff of 0.001 kcal/mol) coupled with the Poisson-Boltzmann surface area implicit solvent model, was used to calculate binding energy for each docked pose (Feig et al., 2004).

Hsp90 Docking.

The cis- and trans-isomers of the benzoquinone and hydroquinone ansamycins were docked separately into the nucleotide-binding site of the open human Hsp90 structure, defined as whole residues within a 4-Å radius subset. The flexible docking program was used to dock the benzoquinone and hydroquinone ansamycins into the nucleotide-binding site of Hsp90, which was followed by a minimization using the conjugate gradient method (1000 iterations with a root mean square cutoff of 0.001 kcal/mol). Flexibility was allowed in the following active site R-groups: Ile26, Glu47, Leu48, Ser50, Asn51, Ser52, Asp54, Ala55, Lys58, Ile96, Gly97, Met98, Asp102, Leu103, Asn105, Asn106, Leu107, Gly108, Ile110, Lys112, Gly132, Gly135, Val136, Gly137, Phe138, Tyr139, Val150, and Thr184.The interaction of the cis-amide isomers of the benzoquinone and hydroquinone ansamycins with Hsp90 were further examined using the human Hsp90 structure cocrystallized with GM. The cocrystallized GM structure was removed, and the cis-amide isomers of the benzoquinone and hydroquinone ansamycins were in turn docked into the nucleotide-binding domain of Hsp90 using the same docking method for the open Hsp90 structure. The Poisson-Boltzmann implicit solvent model used in NQO1 docking was used to calculate the binding energy for each docked pose.

Results

NQO1-Mediated Reduction of Benzoquinone Ansamycins.

The benzoquinone ansamycins GM, 17AAG, 17AG, 17DMAG, and 17AEP-GA were all reduced by purified rhNQO1, using either NADH or NADPH as cofactors, to the corresponding hydroquinone ansamycins without further metabolism. The reduction of the benzoquinone ansamycins by rhNQO1 was monitored spectrophotometrically, and the generation of the polar hydroquinone ansamycin was proportional to the loss of benzoquinone ansamycin (see Supplemental Data). The hydroquinone ansamycins were identified by LC/MS, and mass ions were observed for 17-(allylamino)-17-demethoxygeldanamycin hydroquinone (17AAGH2) at m/z 603.7 [M+NH4]+, geldanamycin hydroquinone (GMH2) at m/z 580.5 [M+NH4]+, 17-(amino)-17-demethoxygeldanamycin hydroquinone (17AGH2) at m/z 548.2 [M+H]+, 17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin hydroquinone at m/z 619.3 [M+H]+, and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin hydroquinone (17AEP-GAH2) at m/z 645.3 [M+H]+ (see Supplemental Data). The relative rhNQO1-mediated reduction rate for this series of benzoquinone ansamycins was dependent on the C17 substituent and fastest for 17AAG followed by 17AG, GM, 17DMAG, and then 17AEP-GA (Fig. 1). The NQO1-dependent formation of the hydroquinone ansamycin from the parent quinone was prevented by ES936, a mechanism-based inhibitor of NQO1 (see Supplemental Data). Apparent Km values were determined for the NQO1-mediated reduction of 17AAG (189 μM) and 17DMAG (262 μM) (see Supplemental Data).

Fig. 1.

Fig. 1.

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Relative NQO1-mediated reduction rate of the benzoquinone ansamycins. Columns, mean (n = 3); bars, S.D. GM, 17AG, 17DMAG, and 17AEP-GA were significantly different from 17AAG: *, p < 0.01, **, p < 0.001. Statistical analysis was performed using one-way analysis of variance with Tukey multiple comparison test.

Molecular Docking of the trans-Amide Isomers of the Benzoquinone Ansamycins into Human NQO1.

Molecular docking simulations demonstrated that the trans-amide isomers of this benzoquinone ansamycin series could be accommodated within the active site of NQO1. The reduced anionic form of the FAD cofactor was used in these docking studies because this represents the NQO1 enzyme at the beginning of the catalytic cycle, when the enzyme can accept the quinone substrate before reduction to the hydroquinone (Li et al., 1995; Cavelier and Amzel, 2001). The flexible docking program generated 50 structural complexes for 17AAG, 41 for 17AG, 21 for GM, 15 for 17DMAG, and 11 for 17AEP-GA. The resultant complexes were evaluated based on the calculated binding energy, the number of hydrogen bond interactions between NQO1 and the benzoquinone ansamycin, and the position of the quinone ring in the active site of NQO1. A recurring high-ranking binding conformation was identified for each benzoquinone ansamycin in this series, in which the quinone moiety was parallel to the isoalloxazine ring of the flavin cofactor, optimizing π–π interactions and increasing the van der Waals contribution to the binding energy. In this conformation, the C21 carbonyl of the benzoquinone ansamycin displayed a hydrogen bond with Tyr128, the C17 substituent occupied the cleft formed by Tyr155, His161, His194, Phe232, and Phe236, and the majority of the ansa ring was outside of the active site (Fig. 2). In this series of benzoquinone ansamycins, 17AAG had the most favorable binding energy followed by 17AG, GM, 17DMAG, and then 17AEP-GA, for this conformation (Table 1). In addition, the binding energies obtained for the highest ranked pose for each benzoquinone ansamycin correlates with the distance between the C21 carbonyl of the quinione and Tyr128, which would suggest that the benzoquinone ansamycins with small C17 substituents are accommodated by NQO1 more readily that benzoquinone ansamycins with large C17 substituents. The docking of the cis-amide isomers of the benzoquinone ansamycins was attempted, but this was unsuccessful because of steric conflicts with active site residues.

Fig. 2.

Fig. 2.

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Flat-ribbon representation of human NQO1 in complex with the trans-amide isomer of 17AAG (A), GM (B), 17AG (C), 17DMAG (D), 17AEP-GA (E) (stick display style; colored by atom type, carbon atoms colored yellow) in the reduced anionic FAD (stick display style; colored by atom type, carbon atoms colored orange) site, key amino acid residues (stick display style; colored by atom type) and hydrogen bond interactions (black dashed lines) are displayed.

TABLE 1.

Interaction and associated energies of the trans-amide isomers of the benzoquinone ansamycins in complex with NQO1

Compound ΔVvdw ΔVelect ΔGbind H-Bond Interaction
 H-Bond Distance
Amino Acid Ligand
kcal/mol Å
17AAG −11.5 −56.2 −18.3 Tyr128 C21 = O 2.48
GM −8.1 −68.0 −15.5 Tyr128 C21 = O 2.50
17AG −13.6 −56.1 −16.1 Tyr128 C21 = O 2.52
17DMAG −12.0 −52.7 −12.7 Tyr128 C21 = O 2.54
17AEP-GA −5.7 −39.4 −8.7 Tyr128 C21 = O 2.57
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ΔVvdw, van der Waals energy; ΔVelect, electrostatic energy; ΔGbind, binding energy.

Inhibition of Human Hsp90 ATPase Activity.

The inhibition of human Hsp90 ATPase activity by this series of benzoquinone and hydroquinone ansamycins was evaluated using the malachite green phosphate assay (Rowlands et al., 2004). The inhibition of human Hsp90 ATPase activity was assessed with only 17AAG and 17AAGH2 (Guo et al., 2005). The benzoquinone ansamycins did inhibit the ATPase activity of human Hsp90 at the concentrations investigated; however, inhibition significantly increased when the benzoquinone ansamycins were incubated with rhNQO1, and this increased inhibition was abrogated by pretreatment with ES936 (Fig. 3). These results clearly demonstrate that the hydroquinone ansamycins generated by NQO1 were more potent inhibitors of Hsp90 ATPase activity than the corresponding benzoquinone ansamycins, in agreement with our previous data using yeast Hsp90 (Guo et al., 2005, 2006).

Fig. 3.

Fig. 3.

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Inhibition of human Hsp90 ATPase activity by 17AAG and 17AAGH2. Human Hsp90 ATPase activity was measured in reactions with either vehicle [dimethyl sulfoxide (DMSO)] or 17AAG (A), GM (B), 17AG (C), 17DMAG (D), or 17AEP-GA (E) (2 and 4 μM) in the presence and absence of rhNQO1. The reactions were analyzed after 12 h, and phosphate concentrations were measured using the malachite green assay. □, benzoquinone ansamycin and NADH; ■, benzoquinone ansamycin, NADH, and rhNQO1; Inline graphic, benzoquinone ansamycin, NADH, NQO1, and ES936. Columns, mean (n = 3); bars, S.D. Hsp90 ATPase activity in incubates containing benzoquinone ansamycin, NADH, and NQO1 were statistically different from incubates containing benzoquinone ansamycin and NADH or benzoquinone ansamycin, NADH, NQO1, and ES936: *, p < 0.05, one-way analysis of variance with Tukey for pairwise comparison. Data for 17AAG has been presented previously (Guo et al., 2005); the experiment was repeated, and the data are shown here to complete the comparison of all compounds in this structural series.

Molecular Docking of the trans- and cis-Amide Isomers of the Benzoquinone and Hydroquinone Ansamycins into Human Hsp90.

Previous studies had reported that it was not possible to dock the trans-amide isomers of the benzoquinone ansamycins into the Hsp90 structure; however, these studies used the human Hsp90 structure cocrystallized with GM or 17DMAG (Jez et al., 2003; Lee et al., 2004). In this study, the trans- and cis-amide isomers of the benzoquinone and hydroquinone ansamycins were successfully docked into the nucleotide-binding domain of the open Hsp90 structure (Stebbins et al., 1997). The calculated binding energies values indicate greater stability between the hydroquinone ansamycin and Hsp90 than the parent quinone. Similar high-scoring conformations were observed across the benzoquinone and hydroquinone ansamycin series for the trans- and cis-amide isomers, all with favorable dock scores and binding energies for binding to the Hsp90 protein (Table 2 data shown for 17AAG; for other compounds, see Supplemental Data). The overall conformation of the quinone or hydroquinone and the ansa ring were similar for the trans-amide isomers of the benzoquinone and hydroquinone ansamycins in the nucleotide-binding domain of the open human Hsp90 structure (Fig. 4, A and B, representations shown for 17AAG; for other compounds, see Supplemental Data). However, differences were observed in the orientation of the C17 substituent outside of the binding pocket, with the exception of 17AG and 17AGH2, the C17 substituents of the benzoquinone ansamycins were almost perpendicular to the plane of the quinone, and in the case of the hydroquinone ansamycins, they extended in the plane of the hydroquinone. There were few hydrogen bond interactions between the trans-amide isomers and the Hsp90 protein, the most notable being the interactions between the C18 carbonyl of 17AAG and 17AG with Ser113 and the C18 hydroxyl of 17AGH2 and 17AEP-GAH2 with Gly135. Interestingly, although no hydrogen bond interactions were observed between Lys112 and the hydroquinone ansamycins, with the exception of 17AEP-GAH2, the NH3+ group of Lys112 was directed toward the hydroquinone ring. This was in contrast to the benzoquinone ansamycins, in which the NH3+ group of Lys112 was directed away from the quinone ring.

TABLE 2.

Interactions and associated energies for the trans-/cis-amide isomers of 17AAG and 17AAGH2 in complex with the open human Hsp90 structure

Compound Dock Score ΔVvdw ΔVelect ΔGbind H-Bond Interaction
 H-Bond Distance
Amino Acid Ligand
kcal/mol Å
trans-17AAG 60.4 −10.5 −58.1 −16.2 Ser113 C18=O 1.76
trans-17AAGH2 86.2 −13.8 −75.6 −24.6
cis-17AAG 56.7 −15.3 −57.5 −26.9 Phe138 C1=O 2.26
Asp54 C17-NHR1 2.23
Lys112 C21=O 1.80
Asp93 C24-NH2 1.91
cis-17AAGH2 89.7 −12.9 −65.8 −34.9 Phe138 C1=O 2.14
Asp54 C17-NHR1 2.08
Asp54 C18-OH 1.60
Lys112 C21-OH 1.96
Asp93 C24-NH2 2.06
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ΔVvdw, van der Waals energy; ΔVelect, electrostatic energy; ΔGbind, binding energy.

Fig. 4.

Fig. 4.

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The human Hsp90–17AAG/17AAGH2 complex. The nucleotide binding domain of open human Hsp90 in complex with the trans-amide isomer of 17AAG (A) and 17AAGH2 (B) and the cis-amide isomer of 17AAG (C) and 17AAGH2 (D). The nucleotide binding domain of bound human Hsp90 in complex with the cis-amide isomer of 17AAG (E) and 17AAGH2 (F). 17AAG and 17AAGH2 (stick display style; carbon atoms are colored yellow) in the nucleotide-binding domain of Hsp90, key amino acid residues (stick display style; colored by atom type), and hydrogen bond interactions (black dashed lines) are displayed.

The conformations of the cis-amide isomers of the benzoquinone and hydroquinone ansamycins were similar in the nucleotide-binding site of the open Hsp90 structure, and with the exception of GM and GMH2, the C17 substituents of the benzoquinone and hydroquinone ansamycins adopted a similar arrangement outside of the binding pocket. A greater number of hydrogen bond interactions was observed between the hydroquinone ansamycins with a C17 amino substituent and the open Hsp90 structure compared with the corresponding benzoquinone ansamycins (Fig. 4, C and D, representations shown for 17AAG; for other compounds, see Supplemental Data). The hydroquinone ansamycins displayed additional hydrogen bond interactions between the C18 hydroxyl and Asp54, and this hydrogen bond was observed with GMH2; however, no hydrogen bond interactions were observed between the C17 substituent of GM or GMH2 and the open Hsp90 structure. GM was the only benzoquinone ansamycin to display an additional hydrogen bond between the C1 amino and Gly135, resulting in GM and GMH2 having an equal number of hydrogen bond interactions with the open Hsp90 structure. The docking of the trans- and cis-amide hydroquinone ansamycins in this series into the open Hsp90 structure resulted in more favorable dock scores and calculated binding energy values than the corresponding benzoquinone ansamycins, and this was largely due to the increased contribution of electrostatic energy (Table 2 data shown for 17AAG; for other compounds, see Supplemental Data).

The docking of the benzoquinone and hydroquinone ansamycins into the GM bound form of human Hsp90 (Stebbins et al., 1997) resulted in conformations and interactions similar to those reported in the cocrystallized structures (Stebbins et al., 1997; Jez et al., 2003). In these studies, only the cis-amide isomers of the benzoquinone and hydroquinone ansamycins were used, because the trans-amide isomers could not be positioned into the nucleotide-binding domain without steric conflicts with active site residues. Consistent with previous studies, using the yeast Hsp90 crystal structure (Guo et al., 2005, 2006), the hydroquinone ansamycins displayed additional hydrogen bond interactions with the human Hsp90 protein compared with the parent quinone (Fig. 4, E and F, representations shown for 17AAG; for other compounds, see Supplemental Data). The C18 hydroxyl interacts with the carboxylate side chain of Asp40 and allows for further interaction between the C17 substituent and the Asp54 and Lys58. These additional hydrogen bond interactions between the hydroquinone ansamycin and the Hsp90 protein result in a more compact C-clamp conformation around helix 2, and these additional interactions translate into more favorable calculated binding energies than those of the parent quinones (Table 3 data shown for 17AAG; for other compounds, see Supplemental Data), providing a structural explanation as to why the hydroquinone ansamycins are more potent Hsp90 inhibitors.

TABLE 3.

Interactions and associated energies for the cis-amide isomers of 17AAG and 17AAGH2 in complex with the bound human Hsp90 structure

Compound Dock Score ΔVvdw ΔVelect ΔGbind H-Bond Interaction
 H-Bond Distance
Amino Acid Ligand
kcal/mol Å
cis-17AAG 61.4 −12.2 −50.5 −13.4 Phe138 C1=O 2.04
Lys58 C11-OH 1.88
Asp54 C17-NHR1 2.37
Lys112 C21=O 1.71
Asp93 C24-NH2 1.87
cis-17AAGH2 88.3 −13.0 −64.9 −16.9 Phe138 C1=O 1.95
Lys58 C11-OH 1.92
Asp54 C17-NHR1 2.18
Asp54 C18-OH 1.65
Lys112 C21-OH 2.01
Asp93 C24-NH2 1.85
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ΔVvdw, van der Waals energy; ΔVelect, electrostatic energy; ΔGbind, binding energy.

Discussion

The amide bond in the ansa ring of the benzoquinone and hydroquinone ansamycins restricts rotation and, consequently, only trans- and cis-amide conformations are possible. The benzoquinone ansamycins adopt the trans-amide conformation in solution, because this is energetically favored (Schnur and Corman, 1994; Jez et al., 2003), and cocrystallization studies have clearly shown the cis-amide isomer is bound to Hsp90 (Stebbins et al., 1997; Jez et al., 2003), indicating that trans-cis isomerization of the benzoquinone ansamycins must occur during the binding process. However, a recent study concluded that no isomerization step occurs in the binding of the benzoquinone ansamycins to Hsp90 (Onuoha et al., 2007) and proposed that small quantities of the cis-amide isomer in solution, identified in an NMR study (Thepchatri et al., 2007), may bind directly to Hsp90, and in vivo potency could be due to the direct binding of the hydroquinone of the cis-amide isomer. This counters the results of a study examining the effects of conformationally constrained cis-amide chimeric inhibitors of Hsp90 (Duerfeldt et al., 2009) and our molecular docking studies in which the cis-amide isomer would not dock into NQO1. However, the results of a binding affinity study using BODIPY-geldanamycin in the absence and presence of reducing agents reported by Onuoha et al. may, in fact, support Hsp90-mediated isomerization. The binding affinity was time-dependent in the absence of reducing agents, indicating that the benzoquinone ansamycins weakly bind Hsp90 initially and then undergo a structural change, in agreement with a prior study (Gooljarsingh et al., 2006). The non–time-dependent increased binding affinity in the presence of reducing agents may be due to the formation of the hydroquinone and the cis-amide isomer, because reducing agents were used to catalyze tautomerization (Clarke et al., 1984; Schmid, 1993). The Hsp90-mediated isomerization of the benzoquinone ansamycins has also been supported by quantum and molecular mechanic studies (Jez et al., 2003; Lee et al., 2004) and the identification of nonprolyl peptide cis-trans isomerases (Bouckaert et al., 2000; Schiene-Fischer et al., 2002). Lee et al. (2004) proposed that Lys112 and Ser113 of Hsp90 interact with the C1 amide of the benzoquinone ansamycin promoting isomerization via tautomerization, and based on the loss of GM binding to a Ser113 Hsp90 point mutant, they determined that Ser113 was essential for Hsp90-mediated trans-cis isomerization. However, the mutation of Ser113 may in turn affect the conformation of Lys112 in the nucleotide-binding site. Although the initial benzoquinone ansamycin binding pose described by Lee et al. was not identified in our docking studies, and no interactions were observed between the C1 amide of the benzoquinone or hydroquinone ansamycins and Lys112 or Ser113 residues of Hsp90, the results of our docking studies provide support for the model of Hsp90-mediated trans-cis isomerization through promoting tautomerization and suggest that Lys112 is an important mediator in this process.

The benzoquinone ansamycin series examined in our study were metabolized by rhNQO1 to generate the corresponding hydroquinone ansamycins, and these were more potent inhibitors of purified human Hsp90 than their parent quinones. The potentiated inhibition of Hsp90 by the hydroquinone ansamycins was rationalized by the increased hydrogen bond interactions between the hydroquinone ansamycins and the Hsp90 protein using computational-based modeling. The interaction of the trans- and cis-amide isomers of the benzoquinone and hydroquinone ansamycins in the open human Hsp90 structure provide support for Hsp90-mediated isomerization. In all cases, the hydroquinone ansamycins had more favorable binding energies than the parent quinones, indicating that both the trans- and cis-amide isomers of the hydroquinone ansamycins would exhibit increased binding affinity for Hsp90. Modeling studies support that the trans-amide isomer of the benzoquinone and hydroquinone ansamycins bind to Hsp90, and this would be a critical initial step for Hsp90-mediated isomerization. This could also rationalize the results of mutational studies (Lee et al., 2004), because mutating the Lys112 and/or the Ser113 residues could impede Hsp90-mediated isomerization, but it would not obstruct the binding of the trans-amide isomer. The high-scoring complexes of the trans-amide isomers in the open human Hsp90 structure, identified in our study, may represent the initial binding conformation of the benzoquinone and hydroquinone ansamycins. The C18 carbonyl of the trans-amide isomer of 17AAG and 17AG displayed a hydrogen bond interaction with Ser113. The deprotonation of the amide nitrogen of the benzoquinone ansamycins may allow the C1 amide or the C18 carbonyl to abstract a proton from Ser113, promoting tautomerization of the amide via a proton shuttle between the quinone and the amide (Scheme 2A). In response to the change in the electronic properties of the quinone ring, Lys112 may then move to protonate the nitrogen of the imine. The destabilization of the hydroquinone ring is not required for the isomerization of the hydroquinone ansamycins (Scheme 2B), because Lys112 is in close proximity to the nitrogen of the C1 amide to donate a proton if required (Fig. 5). The interaction between the Lys112 residue and the lone pair of electrons of the amide nitrogen acts to deconjugate the resonance of the amide bond and promote trans-cis isomerization.

Scheme 2.

Scheme 2.

Open in a new tab

The proposed mechanism of trans-cis isomerization of the benzoquinone ansamycins (A) and the hydroquinone ansamycins (B). Arrows with broken lines indicate bond rotation.

Fig. 5.

Fig. 5.

Open in a new tab

The interaction of trans-17AAG (A) and trans-17AAGH2 (B) with Lys112 in the nucleotide-binding domain of human Hsp90.

This proposed acid catalyzed trans-cis isomerization mechanism is similar to that described for the peptidyl-prolyl isomerases (PPIases). Although the precise mechanism of peptidyl-prolyl cis-trans isomerization catalyzed by PPIases remains to be fully elucidated, it is widely accepted that the protonation of the imine nitrogen is the fundamental step in enzyme-catalyzed isomerization (Schmid, 1993; Hur and Bruice, 2002). Cis-trans isomerization of prolyl peptides by the cyclophilin PPIases involves the stabilization of the amide oxygen by an asparagine residue, the proline ring is held in position by the phenyl group of a phenylalanine residue, and the guanidino group of a conserved arginine residue weakens the double bond character of the C-N bond (Hur and Bruice, 2002). Similar interactions were observed between the trans-amide isomers of the benzoquinone and hydroquinone ansamycins and the open Hsp90 structure, with Ser113 and Phe138 stabilizing benzoquinone and hydroquinone ansamycin binding, and the NH3+ group of Lys112 is available to protonate the imine nitrogen promoting the keto tautomer. The intrinsic structural properties of the ansamycin ring, the anchoring of the carbamate through interaction with Asp90 at the base of the nucleotide-binding domain, and the interaction of the ansa ring with amino acid residues via through-bond interactions with the specific solvent network in the nucleotide-binding domain may also aid to drive trans-cis isomerization (Pappenberger et al., 2001). The confirmation of Hsp90-mediated trans-cis isomerization of the benzoquinone and hydroquinone ansamycins is important because it could be exploited in the development of new Hsp90 inhibitors, and the effect of conformationally restrained cis-amide isomers of benzoquinone ansamycin analogs on Hsp90 inhibition has been reported (Duerfeldt et al., 2009).

The rate of NQO1-mediated reduction of this benzoquinone ansamycin series was dependent on the C17 substituent and was fastest for 17AAG because of the small size of the C17 substituent and the additional interactions between the allyl group and active site residues. The allyl substituent of 17AAG is almost perpendicular to the plane of the phenoxy of Tyr155, further increasing the van der Waals contribution to the calculated binding energy through π–π interactions. The C17 amino and methoxy substituents of 17AG and GM are small enough to be accommodated by the NQO1 active site; however, the large 2-(dimethylamino)ethyl and 2-(pyrrolidinyl-1-yl)ethyl substituents of 17DMAG and 17AEP-GA are directed away from His161 and Tyr155, and their steric bulk would restrict the quinone from entering the active site, and this is reflected in the relative reduction rate. The difference between the relative rates of NQO1-mediated reduction of 17AAG and 17DMAG was approximately 3-fold. Interestingly, the ranking order of the calculated binding energy associated with the benzoquinone ansamycin binding conformation was also in agreement with the order of the relative reduction rate using purified rhNQO1.

The selective generation of the hydroquinone ansamycins within the cancer cell is desirable, because this will result in potentiated Hsp90 inhibition, and increased cell retention and accumulation compared with the parent quinone. The properties of the C17 substituent and the metabolism of the benzoquinone ansamycin, by NQO1 and other oxidoreductases, should be considered in the development of further benzoquinone ansamycins. The rational progression of this approach would be to develop a stable, water-soluble prodrug that protects the hydroquinone group and is activated via intracellular enzymatic hydrolysis, generating a relatively stable hydroquinone ansamycin and potent Hsp90 inhibitor, which in its quinone form would be an efficient substrate for NQO1.

Supplementary Material

Data Supplement
supp_79_5_823__index.html (3.2KB, html)

Acknowledgments

We acknowledge Dr. Daniel L. Gustafson and Joeseph Zirolli for their assistance with mass spectral determinations.

S⃞

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

This work was supported by the National Institutes of Health National Cancer Institute [Grant R01-CA51210].

The authors disclose a pending patent interest in hydroquinone ansamycins and their prodrugs (international application number PCT/US2005/031524).

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.110.070086.

ABBREVIATIONS:

Hsp90
90-kDa heat shock protein
17AAG
17-(allylamino)-17-demethoxygeldanamycin
17AAGH2
17-(allylamino)-17-demethoxygeldanamycin hydroquinone
17AG
17-(amino)-17-demethoxygeldanamycin
17AGH2
17-(amino)-17-demethoxygeldanamycin hydroquinone
17DMAG
17-demethoxy-17-[[2-(dimethylamino)ethyl]amino]-geldanamycin
17AEP-GA
17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin
17AEP-GAH2
17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin hydroquinone
ES936
5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione
GM
geldanamycin
GMH2
geldanamycin hydroquinone
NQO1
NAD(P)H:quinone oxidoreductase 1
PDB
Protein Data Bank
PPIases
peptidyl-prolyl isomerases
LC/MS
liquid chromatography/mass spectrometry
HPLC
high-performance liquid chromatography
rh
recombinant human.

Authorship Contributions

Participated in research design: Reigan and Ross.

Conducted experiments: Reigan, Siegel, and Guo.

Performed data analysis: Reigan and Siegel.

Wrote or contributed to the writing of the manuscript: Reigan and Ross.

References

  1. Beall HD, Mulcahy RT, Siegel D, Traver RD, Gibson NW, Ross D. (1994) Metabolism of bioreductive antitumor compounds by purified rat and human DT-diaphorases. Cancer Res 54:3196–3201 [PubMed] [Google Scholar]
  2. Bouckaert J, Dewallef Y, Poortmans F, Wyns L, Loris R. (2000) The structural features of concanavalin A governing non-proline peptide isomerization. J Biol Chem 275:19778–19787 [DOI] [PubMed] [Google Scholar]
  3. Brooks BR, Brooks CL, 3rd, Mackerell AD, Jr., Nilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, Caflisch A, Caves L, Cui Q, Dinner AR, Feig M, Fischer S, Gao J, Hodoscek M, Im W, Kuczera K, Lazaridis T, Ma J, Ovchinnikov V, Paci E, Pastor RW, Post CB, Pu JZ, Schaefer M, Tidor B, Venable RM, Woodcock HL, Wu X, Yang W, York DM, Karplus M. (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30:1545–1614 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cavelier G, Amzel LM. (2001) Mechanism of NAD(P)H:quinone reductase: Ab initio studies of reduced flavin. Proteins 43:420–432 [PubMed] [Google Scholar]
  5. Chiosis G, Huezo H, Rosen N, Mimnaugh E, Whitesell L, Neckers L. (2003) 17AAG: low target binding affinity and potent cell activity–finding an explanation. Mol Cancer Ther 2:123–129 [PubMed] [Google Scholar]
  6. Chiosis G, Neckers L. (2006) Tumor selectivity of Hsp90 inhibitors: the explanation remains elusive. ACS Chem Biol 1:279–284 [DOI] [PubMed] [Google Scholar]
  7. Clarke ED, Wardman P, Wilson I. (1984) Cis-trans isomerization of the (5-nitro-2-furyl)acrylamide, AF-2, initiated by ascorbate, glutathione, Fe(II) and OH-. Biochem Pharmacol 33:83–87 [DOI] [PubMed] [Google Scholar]
  8. Cysyk RL, Parker RJ, Barchi JJ, Jr., Steeg PS, Hartman NR, Strong JM. (2006) Reaction of geldanamycin and C17-substituted analogues with glutathione: product identifications and pharmacological implications. Chem Res Toxicol 19:376–381 [DOI] [PubMed] [Google Scholar]
  9. Duerfeldt AS, Brandt GE, Blagg BS. (2009) Design, synthesis, and biological evaluation of conformationally constrained cis-amide Hsp90 inhibitors. Org Lett 11:2353–2356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Egorin MJ, Rosen DM, Wolff JH, Callery PS, Musser SM, Eiseman JL. (1998) Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations. Cancer Res 58:2385–2396 [PubMed] [Google Scholar]
  11. Feig M, Onufriev A, Lee MS, Im W, Case DA, Brooks CL., 3rd (2004) Performance comparison of generalized born and Poisson methods in the calculation of electrostatic solvation energies for protein structures. J Comput Chem 25:265–284 [DOI] [PubMed] [Google Scholar]
  12. Gooljarsingh LT, Fernandes C, Yan K, Zhang H, Grooms M, Johanson K, Sinnamon RH, Kirkpatrick RB, Kerrigan J, Lewis T, et al. (2006) A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow, tight binding inhibition by geldanamycin and its analogues. Proc Natl Acad Sci USA 103:7625–7630 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guo W, Reigan P, Siegel D, Ross D. (2008) Enzymatic reduction and glutathione conjugation of benzoquinone ansamycin heat shock protein 90 inhibitors: relevance for toxicity and mechanism of action. Drug Metab Dispos 36:2050–2057 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guo W, Reigan P, Siegel D, Zirrolli J, Gustafson D, Ross D. (2005) Formation of 17-allylamino-demethoxygeldanamycin (17-AAG) hydroquinone by NAD(P)H:quinone oxidoreductase 1: role of 17-AAG hydroquinone in heat shock protein 90 inhibition. Cancer Res 65:10006–10015 [DOI] [PubMed] [Google Scholar]
  15. Guo W, Reigan P, Siegel D, Zirrolli J, Gustafson D, Ross D. (2006) The bioreduction of a series of benzoquinone ansamycins by NAD(P)H:quinone oxidoreductase 1 to more potent heat shock protein 90 inhibitors, the hydroquinone ansamycins. Mol Pharmacol 70:1194–1203 [DOI] [PubMed] [Google Scholar]
  16. Hur S, Bruice TC. (2002) The mechanism of cis-trans isomerization of prolyl peptides by cyclophilin. J Am Chem Soc 124:7303–7313 [DOI] [PubMed] [Google Scholar]
  17. Jez JM, Chen JC, Rastelli G, Stroud RM, Santi DV. (2003) Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90. Chem Biol 10:361–368 [DOI] [PubMed] [Google Scholar]
  18. Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, Burrows FJ. (2003) A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 425:407–410 [DOI] [PubMed] [Google Scholar]
  19. Kelland LR, Sharp SY, Rogers PM, Myers TG, Workman P. (1999) DT-Diaphorase expression and tumor cell sensitivity to 17-allylamino, 17-demethoxygeldanamycin, an inhibitor of heat shock protein 90. J Natl Cancer Inst 91:1940–1949 [DOI] [PubMed] [Google Scholar]
  20. Koska J, Spassov VZ, Maynard AJ, Yan L, Austin N, Flook PK, Venkatachalam CM. (2008) Fully automated molecular mechanics based induced fit protein-ligand docking method. J Chem Inf Model 48:1965–1973 [DOI] [PubMed] [Google Scholar]
  21. Lang W, Caldwell GW, Li J, Leo GC, Jones WJ, Masucci JA. (2007) Biotransformation of geldanamycin and 17-allylamino-17-demethoxygeldanamycin by human liver microsomes: reductive versus oxidative metabolism and implications. Drug Metab Dispos 35:21–29 [DOI] [PubMed] [Google Scholar]
  22. Lee YS, Marcu MG, Neckers L. (2004) Quantum chemical calculations and mutational analysis suggest heat shock protein 90 catalyzes trans-cis isomerization of geldanamycin. Chem Biol 11:991–998 [DOI] [PubMed] [Google Scholar]
  23. Li R, Bianchet MA, Talalay P, Amzel LM. (1995) The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. Proc Natl Acad Sci USA 92:8846–8850 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maloney A, Workman P. (2002) HSP90 as a new therapeutic target for cancer therapy: the story unfolds. Expert Opin Biol Ther 2:3–24 [DOI] [PubMed] [Google Scholar]
  25. Onuoha SC, Mukund SR, Coulstock ET, Sengerovà B, Shaw J, McLaughlin SH, Jackson SE. (2007) Mechanistic studies on Hsp90 inhibition by ansamycin derivatives. J Mol Biol 372:287–297 [DOI] [PubMed] [Google Scholar]
  26. Pappenberger G, Aygün H, Engels JW, Reimer U, Fischer G, Kiefhaber T. (2001) Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics. Nat Struct Biol 8:452–458 [DOI] [PubMed] [Google Scholar]
  27. Pearl LH, Prodromou C. (2000) Structure and in vivo function of Hsp90. Curr Opin Struct Biol 10:46–51 [DOI] [PubMed] [Google Scholar]
  28. Pearl LH, Prodromou C. (2006) Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu Rev Biochem 75:271–294 [DOI] [PubMed] [Google Scholar]
  29. Reigan P, Colucci MA, Siegel D, Chilloux A, Moody CJ, Ross D. (2007) Development of indolequinone mechanism-based inhibitors of NAD(P)H:quinone oxidoreductase 1 (NQO1): NQO1 inhibition and growth inhibitory activity in human pancreatic MIA PaCa-2 cancer cells. Biochemistry 46:5941–5950 [DOI] [PubMed] [Google Scholar]
  30. Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH. (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem 42:260–266 [DOI] [PubMed] [Google Scholar]
  31. Rooseboom M, Commandeur JN, Vermeulen NP. (2004) Enzyme-catalyzed activation of anticancer prodrugs. Pharmacol Rev 56:53–102 [DOI] [PubMed] [Google Scholar]
  32. Ross D. (2004) Quinone reductases multitasking in the metabolic world. Drug Metab Rev 36:639–654 [DOI] [PubMed] [Google Scholar]
  33. Rowlands MG, Newbatt YM, Prodromou C, Pearl LH, Workman P, Aherne W. (2004) High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal Biochem 327:176–183 [DOI] [PubMed] [Google Scholar]
  34. Schiene-Fischer C, Habazettl J, Schmid FX, Fischer G. (2002) The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase. Nat Struct Biol 9:419–424 [DOI] [PubMed] [Google Scholar]
  35. Schmid FX. (1993) Prolyl isomerase: enzymatic catalysis of slow protein-folding reactions. Annu Rev Biophys Biomol Struct 22:123–142 [DOI] [PubMed] [Google Scholar]
  36. Schnur R, Corman ML. (1994) Tandem [3,3]-sigmatropic rearrangements in an ansamycin-stereospecific conversion of an (S)-allylic alcohol to an (S)-allylic amine derivative. J Org Chem 59:2581–2584 [Google Scholar]
  37. Siegel D, Ross D. (2000) Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues. Free Radic Biol Med 29:246–253 [DOI] [PubMed] [Google Scholar]
  38. Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP. (1997) Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell 89:239–250 [DOI] [PubMed] [Google Scholar]
  39. Thepchatri P, Eliseo T, Cicero DO, Myles D, Snyder JP. (2007) Relationship among ligand conformations in solution, in the solid state, and at the Hsp90 binding site: geldanamycin and radicicol. J Am Chem Soc 129:3127–3134 [DOI] [PubMed] [Google Scholar]
  40. Winski SL, Faig M, Bianchet MA, Siegel D, Swann E, Fung K, Duncan MW, Moody CJ, Amzel LM, Ross D. (2001) Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches. Biochemistry 40:15135–15142 [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Data Supplement
supp_79_5_823__index.html (3.2KB, html)
supp_mol.110.070086_Suopplemental_Data.pdf (8.1MB, pdf)

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