High-throughput screening assay for inhibitors of heat ...

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 327 (2004) 176–183 www.elsevier.com/locate/yabio

High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity Martin G. Rowlands,a Yvette M. Newbatt,a Chrisostomos Prodromou,b Laurence H. Pearl,b Paul Workman,a and Wynne Ahernea,¤ a

Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK b Section of Structural Biology, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, UK Received 11 September 2003

Abstract The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510 § 70 M. The known HSP90 inhibitors geldanamycin and radicicol gave IC50 values of 4.8 and 0.9 M respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2–8% CV) and used to screen a compound collection of »56,000 compounds in 384-well format with Z0 factors between 0.6 and 0.8.  2003 Elsevier Inc. All rights reserved. Keywords: Heat-shock protein 90; ATPase; High-throughput screening; Geldanamycin; Malachite green; Phosphomolybdate

Heat-shock protein 90 (HSP90)1 is an abundant cellular protein (1–2% of total protein) and one of a number of molecular chaperones. HSP90 plays a key role in the response of cells to stress and is thought to be important in buVering the cell against the eVects of mutation [1]. In unchallenged cells, the protein is also responsible for maintaining the conformational stability, maturation, and hence activity of a set of speciWc key client proteins [1,2]. At least 40 clients of HSP90 have been described so far, and these fall into three main groups: steroid hormone receptors, protein (serine/threonine and tyrosine) kinases, and a collection of unrelated proteins that ¤

Corresponding author. Fax: +44-208-771-7899. E-mail address: [email protected] (W. Aherne). 1 Abbreviations used: HSP90, heat-shock protein 90; 17AAG, 17-allylamino, 17-demethoxygeldanamycin; DMAG, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; HTS, high-throughput screening; DMSO, dimethyl sulfoxide. 0003-2697/$ - see front matter  2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.10.038

include hTERT, the catalytic subunit of telomerase, and mutant p53 [1,2]. HSP90 client proteins all have important biochemical and regulatory roles in the cell and many are oncogenic including ErbB2, Raf-1, CDK 4, PKB/AKT, Polo1, and Met. The chaperone function of HSP90 is complex and involves binding to a series of cochaperones to form dynamic multimeric complexes [1,3]. It is now clear that HSP90 has intrinsic ATPase activity and that ATP binding and hydrolysis is essential for the activity of HSP90 [4,5]. Inhibition of HSP90 disrupts interactions with cochaperones and results in degradation of client proteins via the ubiquitin proteasome pathway. This leads to deregulation of processes fundamentally important in cancer, e.g., proliferation, cell cycle regulation, and apoptosis. Indeed, as several of the client proteins function in most if not all of the important molecular signaling pathways, it has been suggested [1] that HSP90

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inhibition would target all of the six traits of cancer [6], leading to a combinatorial approach to the treatment of this disease. Inhibition of the ATPase activity of HSP90 leads to antitumor activity in vitro and in vivo [7,8]. The Wrst class of HSP90 inhibitors to be discovered was the benzoquinone ansamycins, which include the natural products, herbimycin A and geldanamycin [9,10]. Geldanamycin binds competitively to the ATP binding site and blocks HSP90 function, thereby altering the assembly of the HSP90, client protein, and cochaperone complex and leading to degradation of the client proteins. Geldanamycin is a potent antiproliferative agent but is unsuitable for clinical use due to its marked hepatotoxicity and narrow therapeutic window [8]. However, the geldanamycin analogue 17-allylamino, 17-demethoxygeldanamycin (17AAG) retains the potent antitumor activity of the parent compound [11,12] with reduced liver toxicity [13] and is currently in clinical study in our Centre and in U.S. Centers [14–17]. Second generation HSP90 inhibitors are required to overcome the known limitations of 17AAG, including metabolism by polymorphic enzymes [12,18], resistance caused by P-glycoprotein expression [12], limited oral bioavailability [19], and poor solubility necessitating the use of a cumbersome formulation. The pharmacological properties of a series of geldanamycin and 17AAG analogues have been investigated [20–22] and the analogue 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) shows promise [23]. Another natural product, radicicol, is also a potent inhibitor of HSP90 ATPase activity [24] but the molecule has a number of unfavorable pharmacological properties and in vivo instability. However, radicicol oximes have shown activity in animal models [25]. Finally a fully synthetic small-molecule, purinebased inhibitor, PU3, rationally designed with the help of molecular modeling, has also been described [26]. PU3 and its analogues [27] are less potent than 17AAG but are more soluble and may have more favorable pharmaceutical properties. High-throughput screening (HTS) now plays a pivotal role in molecular mechanism-based cancer drug discovery [28,29]. The aim of the present study was to develop a high-throughput screen for inhibitors of HSP90 ATPase activity and to identify small-molecule inhibitors that could have the potential to be developed as therapeutic agents with an improved pharmacological proWle compared to those of existing compounds. Assays for HTS need to be sensitive, cost-eVective, miniaturized, and amenable to automation. The conventional assay for HSP90 ATPase activity, a pyruvate/lactate dehydrogenase coupled-enzyme assay [5], based on that described previously [30] is not suitable for HTS and a more appropriate assay was sought. Preliminary experiments explored various methods to measure the inhibition of HSP90 ATPase activity including the use of

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the luciferin/luciferase assay of ATP concentration [31] and the Escherichia coli phosphate binding protein to measure inorganic phosphate [32,33]. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green [34–36], was, however, utilized successfully. The development and optimization of the assay together with its application to screen a diverse small-molecule compound collection are reported here.

Materials and methods Materials Immulon 96-well and Cliniplate 384-well clear, polystyrene, Xat-bottomed plates were purchased from Thermo Labsystems, Basingstoke, UK. Malachite green, polyvinyl alcohol (USP), and ammonium molybdate (ACS grade) were obtained from Sigma Chemical Co., Poole, UK. ATP, sodium salt of special quality, was from Boehringer–Mannheim, UK. All aqueous solutions were prepared with AR water from BDH and to minimize contamination with inorganic phosphate, glassware and pH meters were rinsed with double-distilled water before use. Plasticware was used wherever possible. Absorbance was read at 620 nm on a Wallac Victor 2 plate reader (Perkin–Elmer Life Sciences). Compounds for screening were from commercial suppliers and included compounds archived from within the Centre and through Cancer Research UK. Preparation of yeast HSP90 protein Histidine-tagged yeast HSP90 was transformed into E. coli and puriWed (190%) by metal aYnity, gel Wltration, and ion-exchange chromatography [5]. The protein was divided into aliquots (1 mg) and stored at ¡80 °C for up to 6 months with no loss of activity. ATPase activity was monitored by a coupled-enzyme assay in which the phosphorylation of ADP by pyruvate kinase is coupled to the reduction of the resulting pyruvate by lactate dehydrogenase at the expense of NADH [5]. Colorimetric determination of ATPase activity The assay procedure was based on that previously described [35,36,41]. On the day of use, the malachite green reagent was prepared and contained malachite green (0.0812%, w/v), polyvinyl alcohol (2.32%, w/v; dissolves with diYculty and requires heating), ammonium molybdate (5.72%, w/v, in 6 M HCl), and AR water, mixed in the ratio 2:1:1:2. The reagent is initially dark brown, but on standing for »2 h at room temperature changes to a golden yellow and is ready for use. The assay buVer was 100 mM Tris–HCl, 20 mM KCl, 6 mM

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MgCl2, pH 7.4. The kinetic analysis of the HSP90 ATPase activity, shown in Fig. 2, was carried out using a nonlinear regression Wt of the experimental points to the Michaelis–Menten equation. To obtain Km and V values, the Eadie–Hofstee linear transformation (V against V/ [s]) was used, with the slope D ¡Km and the intercept on the x axis D V/Km. High-throughput screening The test compounds were diluted from mother plates (10 mM in 100% (v/v) DMSO) into daughter plates (200 M in 2.0% % (v/v) DMSO); 5 l of each compound solution was added to each well (equivalent to a Wnal concentration of 40 M) of the Immulon 96-well assay plate. The Wrst and last rows of the 96-well plate contained the appropriate concentration of DMSO only and represent the control and background values, respectively. ATP was dissolved in the assay buVer to give a stock concentration of 2.5 mM and stored at room temperature. A 10-l aliquot of ATP solution was added to each well to give a Wnal assay concentration of 1 mM. Just before use, HSP90 protein was thawed on ice and suspended in chilled assay buVer to a stock concentration of 0.30 mg/ml, and the solution was kept on ice. The incubation was started by adding 10 l of the stock HSP90 to each well (except for the background wells which received 10 l of assay buVer), giving a Wnal assay volume of 25 l. This was equivalent to 2.5–3.75 g (1.45– 1.60 M) of HSP90 depending on the diVerent batches of protein used. The plates were shaken (approximately 2 min) using a plate shaker (e.g., Wellmixx (Thermo Labsystems) or MTS4 (IKA-Schuttler)) sealed with plastic Wlm and incubated for 3 h at 37 °C. To stop the incubation, 80 l of the malachite green reagent was added to each well and the plate shaken again. Following the addition of 10 l of 34% sodium citrate to each well, the plate was shaken once more and left to stand at room temperature for about 15 min, and the absorbance at 620 nm was measured. An identical procedure was used to run the assay in the 384-well plates, except that all the volumes were halved. For screening, this assay was readily automated using a RapidPlate 96/384 (Zymark Ltd., Runcorn, UK) for adding the compounds and a 96/384 Multidrop (Labsystems, Cambridge, UK) for the addition of the other reagents. Signal to noise (S/N) was calculated using the equation (mean signal ¡ mean background)/ standard deviation of the background, and the suitability of the assay for screening was assessed using the ZI factor [37]. For IC50 determinations, a range of stock concentrations of the compound in DMSO was prepared. Five appropriate concentrations were used depending on the relative potency of each compound. A 1-l aliquot of each concentration was transferred to the wells of the assay plate and 4 l of assay buVer added.

Results Optimization of HSP90 ATPase assay Initial experiments conWrmed that there was a linear relationship between the absorbance at 620 nm of the phosphomolybdate–malachite green complex and the concentration of inorganic phosphate (from 0.5 to 2.8 nmol of inorganic phosphate in assay volume of 25 l). A disadvantage of this method for assaying ATPase activities is that the acidic conditions and the catalytic eVect of molybdate lead to nonenzymic hydrolysis of ATP, producing inorganic phosphate and causing an increase in absorbance. It has previously been demonstrated [38] that this eVect could be overcome by the addition of sodium citrate after the molybdate reagent. Our preliminary experiments showed that sodium citrate has no eVect on the linearity of the standard curve. Addition of sodium citrate prevents the nonenzymic hydrolysis of ATP, which causes the increase in absorbance, and the color of the complex formed in the prescence of sodium citrate is stable for up to 2 h. All subsequent assays were carried out with the sodium citrate being added to the assay after the malachite green reagent. Colored compounds (red, green, blue, brown, and yellow) from the National Cancer Institute Diversity Set, at a nominal concentration of 40 M, were tested on the malachite green reagent for interference to the absorbance. Brown and yellow compounds gave a small increase (»15%) in the absorbance at 620 nm (data not shown). Using the assay format as described under Materials and methods, the results in Fig. 1 show that the release of inorganic phosphate from ATP by the yeast HSP90 protein was linear with time of incubation, up to 4 h at 37 °C. Linearity with the amount of protein in the assay was also seen, up to 3.75 g per 25-l assay volume, which corresponds to 1.6 M HSP90. It was found that DMSO at concentrations up to 10% (v/v) did not aVect the stability of the color signal or the enzyme reaction (data not shown). Fig. 2 shows a typical plot of the initial rate, calculated using the standard curve to convert the absorbance units to enzyme velocity, against the ATP concentration. From the Eadie–Hofstee plot, a mean Km of 510 § 70 m (mean § SD) and V of 0.76 § 0.12 mol of inorganic phosphate produced/h/mg of protein were obtained from three independent experiments. The catalytic activity of the pure enzyme was 0.47 min¡1. Based on these observations, the substrate concentration for subsequent assays was Wxed at 1 mM ATP and a 3 h incubation at 37 °C was used. Radicicol, geldanamycin, and 17AAG exhibit competitive inhibition of HSP90 ATPase activity [5,24]. The IC50 values for the three natural products under the optimized conditions of the malachite green assay (Table 1) were determined from a range of inhibitor concentrations (0.1–100 M). The results were then compared to


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Fig. 1. (A) HSP90 ATPase activity as determined by the malachite green reagent as a function of time and protein using the procedure described under Materials and methods. Each point is the mean of four determinations, with error bars omitted for clarity. (B) Data from (A) plotted as HSP90 ATPase activity against protein for the 3-h time point with the error bars included. Table 1 Potency of radicicol, geldanamycin, and 17AAG against yeast HSP90 ATPase activity using the coupled-enzyme assay and the malachite green assay Compound

Radicicol Geldanamycin 17AAG

Coupled-enzyme assay % Inhibition at 2 M

IC50 (M)

88 51 20

0.5 5.0 ND

Malachite green assay IC50 (M, mean § SD) 0.9 § 0.4 4.8 § 0.8 8.7 § 2.3

ND, not done. Table 2 Typical values for the screening performance of the malachite green assay for HSP90 ATPase activity in polystyrene multiwell plates

Fig. 2. Michaelis–Menten and Eadie–Hofstee plots to show the variation of HSP90 ATPase activity with substrate concentration. At each concentration of ATP, the activity was linear with time using 2.5 g protein and 3 h of incubation (data not shown). In the Michaelis–Menten plot, the points are the mean § SD of n D 4.

those obtained using the coupled-enzyme assay system. Initially, the compounds were assayed at 2 M and percentage inhibition was determined. Radicicol and geldanamycin were then assayed across a range of concentrations to produce IC50 values (Table 1). The three compounds gave a similar order of potency in the two assay systems, namely radicicol1geldanamycin117AAG and thus the results validate the malachite green assay as a means of identifying inhibitors of HSP90 ATPase.

Mean signal Signal CV Mean background Background CV Signal/Noise Z0 factor

96-well

384-well

0.775 3.4% 0.154 3.9% 23.0 0.84

1.66 5.6% 0.263 5.6% 14.6 0.75

The malachite green protocol provided a simple assay that was readily automated for screening, and acceptable performance characteristics were obtained in both 96and 384-well formats (Table 2). S/N ratios were above 10 and variability was less than 6% CV for both total signal and no-enzyme blanks, resulting in Z0 factors of 10.7. Thus this represents an assay that is highly suitable for high-throughput screening. Screening assays All compounds were screened at a Wnal nominal concentration of 40 M in 0.4% (v/v) DMSO. Hits were

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scored if the absorbance was reduced by more than 50% compared to the signal produced by the enzyme controls and conWrmation assays were carried out in duplicate at 40 M using compound from the mother plate. Each assay plate was run with a positive control that consisted of four wells containing geldanamycin at a Wnal concentration of 40 M. To test the performance of the assay under automated conditions, 40 plates (3280 compounds) were screened in 96-well format. Although no conWrmed hits were obtained, the values for CV (2–8%) and Z0 factors (0.6–0.8) were similar to those observed during assay development. At this point the assay was converted to 384-well plate format. The remaining 53,440 compounds in our collection were screened in 167 384-well plates. Fig. 3 shows an example of the assay characteristics of a typical screening batch of 15 384-well plates with the absorbance values for the control wells (totals and blanks). The positive control of geldanamycin at 40 M produced »90% inhibition of the HSP90 ATPase activity as demonstrated by the absorbance values close to those of the blank values. The hit rate from the 53,440 compounds was 0.3% (150 compounds), but the activity of only 2 (CCT018159 and CCT018158) chemically related compounds was reconWrmed at 40 M. A search of the compound collection identiWed three other structurally related analogues, two of these were less potent than the original hits and the third (CCT016391) was inactive. The dose response curves for the compound series identiWed in the highthroughput screen are shown in Fig. 4, together with geldanamycin for comparison. The values for inhibition of the HSP90 ATPase under the screening conditions and the IC50 data derived from the concentration response

Fig. 4. Dose response curves for the compound series that inhibits HSP90 ATPase activity. CCT018156, 䉲; CCT018157, 䊊; CCT018158, 䊉; CCT018159, 䊐; CCT016391, 䉭; geldanamycin, 䊏. Each point is the mean of four determinations § SD. Table 3 Potency of inhibition in screening assay and IC50 values for the compound series Compound

% Inhibition in screen at 40 M

IC50(M)

CCT018156 CCT018157 CCT018158 CCT018159 CCT016391 Geldamycin 17AAG

25 26 64 79 0 90 80

46.1 § 2.8 60.8 § 6.7 22.3 § 2.5 8.9 § 0.72 1100 4.8 § 0.8 8.7 § 2.3

curves in Fig. 4 are shown in Table 3. More detailed pharmacology of these compounds with respect to inhibition of HSP90 ATPase activity and cell proliferation will be published in full elsewhere. Preliminary experiments using a cell-based enzyme-linked immunosorbent assay have shown that the molecular signature [11] expected following HSP90 inhibition was obtained with the active compound CCT018159 but not with the inactive analogue CCT016391 [39]. Discussion

Fig. 3. Assay performance in one batch of screening (15 384-well plates), 䉲, Totals (enzyme controls); 䉭, positive control of 20 M geldanamycin (each value is the mean § SD of n D 4); 䊏, blanks. For the totals and blanks, each value is the mean § SD of n D 16.

HSP90 is an exciting new target in cancer drug discovery that oVers the potential for combinatorial treatment of this disease by simultaneous disruption of several key oncogenic signaling pathways. This could lead to broad-spectrum antitumor activity and reduce the likelihood of drug resistance arising [1]. The ATPase activity of HSP90 is essential for its chaperone function and several inhibitors of this activity have been described [1]. Although these inhibitors are not ideal clinical agents and their properties limit therapeutic opportunities, some evidence of clinical eYcacy with 17AAG has been obtained [16]. New HSP90 inhibitors


are required to fully realize the potential of this therapeutic approach. We therefore developed a highthroughput assay for inhibition of HSP90 ATPase activity and used this method to screen our compound collection to Wnd novel inhibitors that would provide the basis of a drug development program. The established coupled-enzyme assay for measuring the ATPase activity of HSP90 is unsuitable for screening as it is too cumbersome for high-throughput, and the presence of two other enzymes could confound the results obtained. We investigated the use of the luciferase measurement of ATP as the basis of a screen but because of the high concentration of ATP required for HSP90 ATPase activity and its slow turnover, very low S/N ratios were obtained. The PBP binding assay for inorganic phosphate also proved to be diYcult to use because of high background signals, presumably because of inorganic phosphate contamination in water and plasticware. Enzymes can be used to remove contaminating phosphate [32,33] but this was again thought to be undesirable in a HTS setting. However, the PBP assay has recently been applied successfully to study human HSP90 ATPase activity in a nonscreening setting [40]. The malachite green assay was therefore our assay of choice. This method is based on the reaction of phosphomolybdate with the dye malachite green, and has been used previously to assay protein tyrosine phosphatase [35], phosphoinositide phosphatases [36] and, in a 96well-plate format, myosin ATPase [41]. High-throughput screening for inorganic pyrophosphatases in a 96-wellplate format [42] and ultra-high-throughput screening for ATPase activity in 384- and 1536-well-plate formats [43], using the malachite green reagents, have been reported. However, the present study is the Wrst report of the use of the malachite green assay to screen a compound collection to detect inhibitors of HSP90 ATPase activity. The assay was reproducible, cost eVective, suitable for automation, and easily converted from 96- to 384-well plates. Instability of the signal was overcome by the addition of sodium citrate after the malachite green reagent. Using the protocol described, the signal to background and Z0 factors were highly acceptable for screening with Z0 and Z factors over »0.5. The assay was used to screen a total of »60,000 compounds. A substrate concentration of 1 mM was required to achieve a good assay performance (high OD value) even though this was equivalent to »2£ Km for ATP. The kinetic values obtained, namely a Km for ATP hydrolysis of 513 M and a Kcat of 0.47 min¡1, are consistent with other values reported in the literature for yeast HSP90 ATPase, eg., 300 M and 0.35 min¡1 [44], 830 M and 0.53 min¡1 [45], and 172 M and 0.44 min¡1 [46]. The overall unconWrmed hit rate was 0.3%. The activity of only two chemically related hits was reconWrmed in later assays. The reasons for this are unclear but random dispensing errors are the most likely reason. To maximize

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the number of hits identiWed, the screen was run at a nominal compound concentration of 40 M. Although the eVect of color on the assay was minimal, other nonspeciWc compound eVects may have contributed to this high false-positive rate. A structure search of our library showed that there were three other compounds with activity in the same chemical series as CCT018159, the most potent hit identiWed. The lower activity of two of these analogues, CCT018156 and CCT018157, caused them to just miss being identiWed as hits (Table 3) in the primary screen. A third analogue, CCT016391, was inactive at concentrations up to 100 M. In summary, the malachite green assay for inorganic phosphate has been developed, validated, and used to screen a compound collection for inhibitors of yeast HSP90. The screen identiWed one compound series of HSP90 ATPase inhibitors, and a drug discovery project in which the potency and other pharmacological properties of the compound series will be optimized has been initiated. Acknowledgments We are grateful to Dr. Fabrice Turlais for help with evaluating the screening data. This work was funded by Cancer Research UK. References [1] A. Maloney, P. Workman, Hsp90 as a new therapeutic target for cancer therapy: the story unfolds, Expert Opin. Biol. Ther. 2 (2002) 3–24. [2] L. Neckers, Hsp90 inhibitors as novel cancer therapeutic agents, Trends Mol. Med. 8 (2002) 555–561. [3] J.S. Issacs, Xu Wanping, L. Neckers, Heat shock protein 90 as a molecular target for cancer therapeutics, Cancer Cell 3 (2003) 213–217. [4] C. Prodromou, S.M. Roe, R. O’Brien, J.E. Ladbury, P.W. Piper, L.H. Pearl, IdentiWcation and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone, Cell 90 (1997) 65–75. [5] B. Panaretou, C. Prodromou, S.M. Rose, R. O’Brien, J.E. Ladbury, P.W. Piper, L.H. Pearl, ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo, EMBO J. 17 (1998) 4829–4836. [6] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [7] T.W. Schulte, L.M. Neckers, The benzoquinone ansamycin 17allylamino, 17-demethoxygeldanamycin binds to HSP90 and shares important biologic activities with geldanamycin, Cancer Chemother. Pharmacol. 42 (1998) 273–279. [8] J.G. Supko, R.L. Hickman, M.R. Grever, Malspeis, Preclinical pharmacologic evaluation of geldanamycin as an antitumour agent, Cancer Chemother. Pharmacol. 36 (1995) 305. [9] C. Deboer, P.A. Meulman, R.J. Wnuk, D.H. Peterson, Geldanamycin, a new antibiotic, J. Antibiot. (Tokyo) 23 (1970) 442. [10] L. Neckers, T.W. Schulte, E. Mimnaugh, Geldanamycin as a potential anti-cancer agent: its molecular target and biochemical activity, Invest. New Drugs 17 (1999) 361–373.

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