© The Author 2006. Published by Oxford University Press.
ARTICLE |
Antitumor Activity of ZSTK474, a New Phosphatidylinositol 3-Kinase Inhibitor
Affiliations of authors: Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Ariake, Koto-ku, Tokyo, Japan (SY, KY, TY); Research Laboratory, Zenyaku Kogyo Co., Ltd., Ohizumi-machi, Nerima-ku, Tokyo, Japan (SY, IK, HY, TM); Laboratory of Biological Chemistry, Department of Applied Biological Chemistry, Faculty of Agricultural and Life Science, Graduate School of Agricultural and Life Science, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, Japan (YF); The School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo, Japan (HG, SH)
Correspondence to: Takao Yamori, PhD, Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan (e-mail: yamori{at}jfcr.or.jp).
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ABSTRACT |
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Background: We previously synthesized a novel s-triazine derivative, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that strongly inhibited the growth of tumor cells. We identified its molecular target, investigated its effects on cellular signaling pathways, and examined its antitumor efficacy and toxicity in vivo. Methods: We used COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines and identified phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474. PI3K was immunoprecipitated from A549 cell lysates, and its activity was measured by assessing the incorporation of 32P into phosphatidylinositol. We used the crystal structure of the PI3K–LY294002 complex to model the binding of ZSTK474 to PI3K (where LY294002 is a known PI3K inhibitor). PI3K downstream activity was analyzed by immunoblotting. Antitumor activity of ZSTK474 was examined against A549, PC-3, and WiDr xenografts in nude mice. Phosphorylation of Akt, a serine/threonine protein kinase and a major signaling component downstream of PI3K, was assessed in vivo by immunohistochemistry. Results: PI3K was identified as a molecular target for ZSTK474 by COMPARE analysis. We confirmed that ZSTK474 directly inhibited PI3K activity more efficiently than the PI3K inhibitor LY294002. At concentrations of 1 µM, ZSTK474 and LY2194002 reduced PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control level. Molecular modeling of the PI3K–ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K. ZSTK474 inhibited phosphorylation of signaling components downstream from PI3K, such as Akt and glycogen synthase kinase 3
, and mediated a decrease in cyclin D1 levels. ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in critical organs. Akt phosphorylation was reduced in xenograft tumors after oral administration of ZSTK474. Conclusion: ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Phosphatidylinositol 3-kinase (PI3K), which is active in signal transduction, generates phosphatidylinositol-3,4,5-trisphosphate (PIP3) by phosphorylating phosphatidylinositol-4,5-bisphosphate (1,2). PI3K is involved in various cellular processes, including cell survival, vesicle transport, and cytoskeletal rearrangement (3,4). The importance of PI3K in tumorigenesis is supported by the following evidence. The chicken retrovirus ASV16 carries an oncogenic PI3K gene (5), and mutation and/or amplification of PI3K genes (6–10) has been reported in human and other mammalian cancer cells. Constitutive activation of PI3K may contribute to the malignant phenotype of signet ring carcinomas (11), and PTEN, a PIP3 phosphatase, has been identified as a tumor suppressor gene (12).
Many clinical studies also indicate that deregulation of PI3K pathway plays a role in various cancers (3,7) and that PI3K may thus be a good target for anticancer drug development (3). Validation of PI3K as a good target for drug development began with the use of dominant-negative mutants and RNA interference studies (13,14). Indeed, a number of compounds have been identified that inhibit PI3K, including wortmannin (15,16), which was originally isolated from soil bacteria and is toxic to fungi; the closely related viridin analogues (17,18); and LY294002, a morpholino derivative of the broad-spectrum kinase inhibitor quercetin (19). Because of inherent difficulties with the stability, solubility, and toxicity of these compounds, efforts are under way to develop new inhibitors of the PI3K pathway (20,21).
In previous work, we synthesized a chemical library of s-triazine derivatives and screened these compounds for their ability to inhibit tumor cell growth (22,23). Among more than 1500 s-triazine derivatives, we found a new compound, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that showed strong antiproliferative activity. However, its molecular target or its potential for a novel anticancer drug was unknown.
We previously established a panel of 39 human cancer cell lines (termed JFCR39) coupled to a drug activity database (24–26) that is comparable to the panel developed by the National Cancer Institute (27,28). We compared cell growth inhibition profiles against the JFCR39 (herein termed fingerprints) of more than 60 standard drugs including doxorubicin, fluorouracil, cytarabine, methotrexate, vincristine, cisplatin, paclitaxel, and irinotecan, by using COMPARE analysis (26), and we showed that COMPARE analysis is an information-intensive approach to identifying the molecular target of a new compound, as described by Paull et al. (28). This system can be used to predict the molecular target or the mode of action of test compounds by assessing the correlation coefficient between the fingerprints mediated by such test compounds and by various reference compounds with known modes of action, including PI3K inhibitors.
The purpose of this study was to identify the molecular target of ZSTK474 with the aid of COMPARE analysis, to investigate the effects of ZSTK474 on cellular signaling pathways, and to examine the antitumor efficacy and toxicity of ZSTK474 in vivo.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chemicals
ZSTK116, ZSTK474, ZSTK781, and ZSTK1178 were synthesized in the Research Laboratory, Zenyaku Kogyo Co., Ltd. (Tokyo, Japan). For in vitro studies, these compounds were dissolved in dimethyl sulfoxide. ZSTK474 was suspended in 5% hydroxypropylcellulose in water as a solid dispersion form for animal experiments. Other anticancer drugs and chemicals were purchased as follows: doxorubicin and 5-fluorouracil from Kyowa Hakko Kogyo (Tokyo, Japan); cisplatin from Nippon Kayaku (Tokyo, Japan); irinotecan from Yakult Honsha (Tokyo, Japan); and wortmannin, LY294002, tetramethylrhodamine isothiocyanate-conjugated phalloidin, Igepal CA-630, and platelet-derived growth factor from Sigma (St. Louis, MO).
Cell Lines
A panel of 39 human cancer cell lines (termed JFCR39) (25,26) and B16F10 melanoma cells (29) were previously described. All cell lines were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C in humidified air containing 5% CO2. Cell lines OVCAR3, A549, PC-3, and WiDr, which were used for in vitro detailed analysis or for in vivo study, originated from an ovarian cancer, non–small-cell lung cancer, prostate cancer, and colon cancer, respectively. For in vivo study, A549, PC-3, and WiDr were grown as subcutaneous tumors in nude mice.
Analysis of Cell Proliferation Inhibition
The inhibition of cell proliferation was assessed by measuring changes in total cellular protein in a culture of each cell line in the JFCR panel of cell lines after 48 hours of drug treatment by use of a sulforhodamine B assay (30). The 50% growth inhibition (GI50) value of the drug was calculated as described previously (26,27). The graphic representation of a drug's mean differential growth inhibition for the cell line panel was based on a calculation that used a set of GI50 values, as described previously (28,31). To analyze the correlation between the mean graphs of drug A and drug B, the COMPARE computer algorithm was developed as previously described by Paull et al. (28). The Pearson correlation coefficient between the mean graphs of drug A and drug B was calculated (n = 39).
PI3K Assay in Tumor Cell Extracts
The PI3K assay has been previously described by Fukui et al. (32). In brief, A549 cells were lysed in a buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630 (Sigma), the lysates were centrifuged at 20 000g and 4 °C for 10 minutes, and the supernatants were used as cell lysate (protein = 2–4 mg/mL). To immunoprecipitate PI3K, we incubated 200 µL of cell lysate with anti-p85 polyclonal antibody (1 : 200 dilution; Upstate, Charlottesville, VA) and protein G–agarose (5 µL; Upstate). The anti-p85 polyclonal antibody recognizes the p85 regulatory subunit of PI3K. Class Ia catalytic subunits of PI3K (p110
, p110, and p110
) constitutively associate with the p85 subunit. Therefore, PI3K, PI3K, and PI3K can be immunoprecipitated by the anti-p85 polyclonal antibody. Agarose beads containing immunoprecipitates were washed twice with buffer A (20 mM Tris–HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Igepal CA-630), once with buffer B (500 mM LiCl and 100 mM Tris–HCl at pH 7.5), once with distilled water, and once with buffer C (100 mM NaCl and 20 mM Tris–HCl at pH 7.5). Immunoprecipitates were suspended in 20 µL of buffer C containing phosphatidylinositol at 200 µg/mL. The mixture was preincubated with or without ZSTK474, LY294002, or wortmannin, as indicated, at 25 °C for 5 minutes. [
-32P]ATP (2 µCi per assay mixture; final concentration, 20 µM) and MgCl2 (final concentration, 20 mM) were added to start the reaction. The reaction mixture was incubated at 25 °C for 20 minutes. During this incubation, formation of phosphatidylinositol-3-phosphate was linear (data not shown). Phosphorylated products of phosphatidylinositol were separated by thin-layer chromatography and visualized by autoradiography. The phosphatidylinositol-3-phosphate region was scraped from the plate, and radioactivity was also measured with liquid scintillation spectroscopy (LS6500 Scintillation System; Beckman Instruments, Fullerton, CA). The level of inhibition for each compound was determined as the percentage of 32P counts per minute obtained without the test compound. To asses the reversibility of PI3K inhibition, the PI3K immunoprecipitates were treated with 1 µM ZSTK474, 10 µM LY294002, or 0.1 µM wortmannin for 5 minutes, and the reaction mixture was divided into two aliquots. One aliquot was washed four times with a buffer containing 100 mM NaCl and 20 mM Tris–HCl (pH 7.5) to remove free ZSTK474, LY294002, or wortmannin from the PI3K, and the PI3K activity of the washed precipitate was measured. The PI3K activity of the other aliquot was measured without washing.
PI3K Assay for Recombinant Catalytic Subunits of PI3K
PI3K assay for recombinant p110, the catalytic subunit of PI3K, was described previously by Gray et al. (33). In a reaction volume of 20 µL, human p110 isoforms , -, or - (Upstate) were incubated with 10 µM phosphatidylinositol-4,5-bisphosphate and 10 µM ATP in assay buffer for 30 minutes at room temperature. Stop buffer (5 µL) containing EDTA and biotinylated PIP3 was added, followed by 5 µL of detection buffer containing europium-labeled anti–glutathione S-transferase (GST) monoclonal antibody, GST-tagged general receptor for phosphoinositides (GRP1) PH domain, and streptavidin allophycocyanin. Plus and minus kinase control wells were also included. The plate was read in the time-resolved fluorescence mode, and the homogenous time-resolved fluorescence (HTRF) signal was determined according to the following formula: HTRF = 10 000 x (emission at 665 nm/emission at 620 nm).
Molecular Modeling of the PI3K–ZSTK474 Complex
Conformational analysis of ZSTK474 was performed first with the FlexX docking program (34), which was used to construct a model of the PI3K–ZSTK474 complex structure. This program treats ring systems, such as the morpholino groups in ZSTK474, as rigid structures. The conformational analysis was done with the CAMDAS, version 2.1, program (conformational analyzer with molecular dynamics and sampling) (35). Conditions used for CAMDAS calculation were similar to those described previously (36). Twelve dihedral angles with respect to two morpholino rings were used to cluster similar conformations. As a result of the CAMDAS calculation, 64 different conformers were obtained.
The crystal structure of the PI3K–LY294002 complex (Protein Data Bank Identification Code 1E7V [PDB] ) was used to obtain the PI3K template structure for docking. The docking calculation of each ZSTK474 conformation with PI3K was performed with the FlexX program. A total of 10 482 different docking models were generated. Each docking model was then evaluated with the following five different score functions: the F-score (34), the D-score (37), the G-score (38), the ChemScore (39), and the PMF-score (40). These functions were implemented in the SYBYL C-Score module. Finally, a new index, termed the average of auto-scaled score, was calculated to rank all docking models. This index has been recently defined in our laboratory to more adequately rank docking models (41). In the top 20 docking models that were ranked according to the average of auto-scaled scores, all ZSTK474 molecules were found to similarly interact with PI3K. The only difference among the 20 models was with respect to the conformation of the two morpholino rings of ZSTK474. Therefore, we present one of these 20 models, in which two morpholino rings of ZSTK474 take a chair conformation, because this model has the lowest internal energy.
Assay for Chromatin Condensation
OVCAR3 cells were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum for 24 hours and then cultured for 48 hours with or without 10 µM ZSTK474. Cells were collected, fixed in 1% glutaraldehyde in phosphate-buffered saline (PBS) at 4 °C for 24 hours, stained with 167 µM Hoechst 33342 (Sigma), and examined by fluorescence microscopy to identify apoptotic cells.
Flow Cytometry Analysis
Cells were harvested, washed with ice-cold PBS, and fixed in 70% ethanol. Cells were then washed twice with ice-cold PBS again, treated with RNase A (500 µg/mL; Sigma) at 37 °C for 1 hour, and stained with propidium iodide (25 µg/mL; Sigma). The DNA content of the cells was analyzed with a flow cytometer (FACScalibur, Becton Dickinson, Franklin Lakes, NJ).
Membrane Ruffling
Murine embryonic fibroblasts were treated with or without 1 µM ZSTK474 for 15 minutes and then stimulated with platelet-derived growth factor (10 ng/mL) for 5 minutes. Cells were fixed with 3% formaldehyde at room temperature for 30 minutes and permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. Filamentous actin in cells was stained with tetramethylrhodamine isothiocyanate-conjugated phalloidin at 0.1 µg/mL, and cells were examined by fluorescence microscopy.
Analysis of PIP3 Production
PIP3 production in intact cells was measured as described previously by Kimura et al. (42). In brief, to label murine embryonic fibroblasts, medium was replaced with prewarmed phosphate-free Dulbecco's modified Eagle medium with 25 mM HEPES (pH 7.4) containing [32P]orthophosphate (0.1 mCi/mL), and cultures were incubated at 37 °C for 4 hours. Lipids were extracted with chloroform and separated by thin-layer chromatography. PIP3 spots were visualized by autoradiography and identified by standards synthesized by PI3K in vitro (data not shown). Radioactivity in the spots was quantitated with a PhosphorImager SI system (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis
A549 cells were lysed in a buffer containing 10 mM Tris–HCl at pH 7.4, 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 50 mM Na3VO4, 5 mM EDTA, aprotinin at 100 Kal U/mL, 1 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 0.1% sodium dodecyl sulfate. Proteins in cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by electroblotting onto a polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ). Rabbit polyclonal antibodies for Akt, phosphorylated Akt (phosphorylated residue Ser-473), phosphorylated glycogen synthase kinase 3 (GSK-3; phosphorylated residue Ser-9), phosphorylated FKHR (phosphorylated residue Thr-24)/phosphorylated FKHRL1 (phosphorylated residue Thr-32), phosphorylated TSC-2 (phosphorylated residue Ser-1462), phosphorylated mTOR (phosphorylated residue Ser-2448), phosphorylated p70S6K (phosphorylated residue Thr-389), phosphorylated MEK1/2 (phosphorylated residues Ser-217/221), and phosphorylated ERK1/2 (phosphorylated residues Thr-202/Tyr-204) were purchased from Cell Signaling Technology (Beverly, MA) and were diluted 1 : 1000. Rabbit anti–cyclin D1 polyclonal antibody (1 : 200 dilution) was purchased from BioSource International (Camarillo, CA). Horseradish peroxidase–conjugated goat anti–rabbit immunoglobulin G (Cell Signaling Technology) was used as a secondary antibody. Immunoreactive bands were identified with the ECL-plus Western Blotting Detection System (Amersham Biosciences).
Animal Experiments
Animal care and treatment was performed in accordance with the guidelines of the animal use and care committee of the Research Laboratory, Zenyaku Kogyo, and conformed to the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the above committee. Male BDF1 mice and female nude mice with BALB/c genetic backgrounds were purchased from Charles River Japan, Inc. (Yokohama, Japan). Mice were maintained under specific pathogen–free conditions and provided with sterile food and water ad libitum. We used 56 BDF1 mice. Each BDF1 mouse was subcutaneously injected with 5 x 105 B16F10 melanoma cells. Seven days after inoculation, animals were divided randomly into test groups (each with seven mice), and the administration of the drugs, as indicated, began (day 0). ZSTK474 at 100, 200, or 400 mg/kg of body weight was orally administered daily from days 0 to 13. Reference drugs were irinotecan, cisplatin, doxorubicin, and 5-fluorouracil. Each reference drug was administered intravenously from day 0 at the maximum tolerable dose by use of the following optimal schedules: irinotecan at 100 mg/kg administered on days 0, 3, and 7; cisplatin at 10 mg/kg on day 0; doxorubicin at 12 mg/kg on day 0; and 5-fluorouracil at 50 mg/kg on days 0, 3, and 7. Human tumor xenografts were generated with A549 lung cancer cells, PC-3 prostate cancer cells, and WiDr colon cancer cells and were grown as subcutaneous tumors in nude mice. Forty-five nude mice were used in these experiments. Each nude mouse was subcutaneously inoculated with a tumor fragment of 3 mm x 3 mm x 3 mm. When tumors reached a volume of 100–300 mm3, animals were divided randomly into test groups (each with five mice) (day 0). ZSTK474 (400 mg/kg) was administered orally from day 0 until day 13 except for days 3 and 10. For longer administration periods, ZSTK474 at 400 mg/kg was orally administered daily from days 0 to 26, except for days 6, 13, and 20. Then, the mice were photographed on day 28. In another experiment, ZSTK474 at a higher dosage of 800 mg/kg was orally administered in the same schedule, and the body weight changes of the mice were monitored to observe toxicity.
Measurement of Tumor Volume
The length (L) and width (W) of the subcutaneous tumor mass were measured by calipers in live mice, and the tumor volume (TV) was calculated as: TV = (L x W2)/2. The tumor volume at day n was expressed as relative tumor volume (RTV), according to the formula RTV = TVn/TV0, where TVn is the tumor volume at day n, and TV0 is the tumor volume at day 0. Tumor regression (T/C%) on day 14 was determined by use of the RTV values as follows: T/C% = 100 x (mean RTV of treated group)/(mean RTV of control group). To assess toxicity, we measured the body weight of the tumor-bearing mice. After the chronic administration of ZSTK474, mice were subjected to necropsy examination, including the histopathologic evaluation of bone marrow.
Bone Marrow in Mice Femur
Right-side femurs were fixed with 10% neutral formalin and decalcified with Plank–Rychlo fluid (43). After embedding in paraffin, 3-µm sections of bone marrow were stained with hematoxylin and eosin.
Immunohistochemistry
When the volume of tumors reached 100–300 mm3, ZSTK474 at 400 mg/kg was orally administered once. Mice were killed 4 hours after ZSTK474 administration, and the tumors were excised. Tumor tissue was fixed in 10% neutral formalin and embedded in paraffin. The 6-µm sections were deparaffinized in xylene and then in a 100% to 50% ethanol series. Immunohistochemistry-specific phosphorylated Akt (phosphorylated on Ser-473) antibody and phosphorylated Akt (phosphorylated on Ser-473) blocking peptide were purchased from Cell Signaling Technology. The tissue sections were analyzed by immunohistochemistry for the expression of phosphorylated Akt (Ser-473). For control experiment, phosphorylated Akt (Ser-473) blocking peptide was incubated with phosphorylated Akt (Ser-473) antibody at 4 °C for 2 hours before incubating with the tissue section. The ABC method (Vectastain ABC system kit; Vector Laboratories, Burlingame, CA) was used for immunohistochemical staining, with the sections also being counterstained with hematoxylin.
Statistical Analysis
Pearson correlation coefficients were calculated for the COMPARE analysis and statistical correlation. The two-sided Mann–Whitney U test was used to test the statistical significance of the antitumor efficacy of ZSTK474 in relative tumor growth ratio on days 4, 7, 11, and 14. The number of samples is indicated in the description of each experiment. All statistical tests were two-sided.
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RESULTS |
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Measurement of the Growth-Inhibitory Activity of ZSTK474 and Identification of its Molecular Target
We synthesized and screened a chemical library of s-triazine derivatives based on their ability to mediate growth inhibition of tumor cells. We found that ZSTK474 showed potent antiproliferative activity. The chemical structure of ZSTK474 is shown in Fig. 1.
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To identify potential molecular targets for ZSTK474, we used COMPARE analysis, an approach that is based on chemosensitivity measurements from the JFCR39 panel of cell lines. We treated cells in the JFCR39 panel with ZSTK474 and examined its effect on cell proliferation. In this way, we obtained the fingerprint for ZSTK474 (Fig. 2). The COMPARE analysis revealed that the fingerprint of ZSTK474 correlated with the fingerprints of noanticancer drug currently in use. However, the fingerprint of ZSTK474 did correlate statistically significantly with that of LY294002, a PI3K inhibitor (r = .766 and P<.001), as shown in Fig. 2, and with that of wortmannin, another well-characterized PI3K inhibitor (r = .519 and P = .001). Thus ZSTK474 appears to act as a PI3K inhibitor, although its structure is different from that of LY294002 or of wortmannin (Fig. 1).
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We also compared the potency of ZSTK474 in cell growth inhibition with those of LY294002 and wortmannin. The mean logarithm of GI50 for ZSTK474 was –6.49 (at 0.32 µM ZSTK474), which was substantially lower than that observed with LY294002 (–5.13 at 7.4 µM) or wortmannin (–5.00 at 10 µM), indicating that ZSTK474 had stronger cell growth inhibitory activity than either of these two known PI3K inhibitors.
Inhibition of PI3K Activity by ZSTK474
We next examined the ability of ZSTK474 to inhibit PI3K activity. We selected A549 cells as the source of PI3K because these cells had an average level of sensitivity to ZSTK474 among cells in the JFCR39 panel. For these experiments, we used PI3K that had been immunoprecipitated from A549 cell lysates with an anti-p85 polyclonal antibody. ZSTK474 inhibited PI3K activity in a dose-dependent manner (Fig. 3, A). At concentrations of 1 µM, ZSTK474 and LY2194002 reduced the PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control activity. The respective concentrations of ZSTK474, LY294002, and wortmannin that inhibited 50% of the PI3K activity (IC50) were 37, 790, and 11 nM. Therefore, as a PI3K inhibitor, ZSTK474 had a 20-fold greater activity than that of LY294002, but its activity was similar to that of wortmannin. However, ZSTK474 reversibly inhibited PI3K activity, as does LY294002, whereas wortmannin irreversibly inhibited PI3K activity (Fig. 3, B). ZSTK474 did not substantially inhibit the activity of 139 other protein kinases (Supplementary Table 1, available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue8), indicating that ZSTK474 is highly specific to PI3K.
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There are four subtypes of p110, the catalytic subunit of PI3K—
, , , and . The specificity of ZSTK474 to these subtypes was of interest because a high frequency of mutations in the PIK3CA gene, encoding p110, in human cancers has been recently reported (7,8). With the PI3K assay described above, it was not possible to determine subtype specificity because the precipitates obtained with the anti-p85 polyclonal antibody could contain a mixture of p110, -, and -. To determine the subtype specificity of ZSTK474, we examined ZSTK474 for its activity against recombinant p110, -, and -. We found that ZSTK474 inhibited the activities of p110, -, and -, with respective IC50 values of 17, 53, and 6 nM (Supplementary Fig. 1 available at http://jncicancerspectrum.oxfordjournals.org/jnci/content/vol98/issue8), indicating that ZSTK474 was apparently not subtype specific. Correlation of PI3K Inhibition with Growth-Inhibitory Activity of s-Triazine Derivatives
ZSTK474 is an s-triazine derivative, as are ZSTK1178, ZSTK781, and ZSTK116, and all have different PI3K inhibitory activities. We investigated whether the PI3K inhibitory activities (IC50) of these four s-triazine derivatives were related to their growth inhibitory activities (assessed as the mean logarithm of GI50). A statistically significantly high correlation (r = .98 and P = .023) was observed between these two inhibitory activities (Fig. 3, C), suggesting that ZSTK474 inhibited cell proliferation by inhibiting PI3K activity.
Molecular Modeling of the PI3K–ZSTK474 Complex
Because all previously described PI3K inhibitors apparently bind to the ATP-binding site of p110, the catalytic subunit of PI3K (44), we investigated whether ZSTK474 also binds to this site by using a model structure, the crystal structure of the PI3K–LY294002 complex, to generate a PI3K template structure for docking analysis (44). Analysis of this model structure indicated that there are three hydrogen-bonding interactions between PI3K and ZSTK474 (i.e., K802 NZ, one of the morpholino oxygens; S806 OG, the other morpholino oxygen; and V882 NH, the benzimidazole nitrogen [Fig. 4, A]).
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We next compared the binding modes of ZSTK474 with those of ATP in the active site of PI3K. Two of the three hydrogen bonds between ZSTK474 and PI3K, V882 NH and S806 OG, mimicked those in the ATP–PI3K complex; therefore, the overall arrangements of ZSTK474 and ATP in the PI3K active site are very close (Fig. 4, B). The benzimidazole nitrogen of ZSTK474 forms a hydrogen bond with V882 NH. This hydrogen bond mimics the hydrogen bond between ATP N1 and V882 NH. As discussed previously (45), all kinase inhibitors appear to have a hydrogen bond acceptor in a position equivalent to ATP N1. These features of the ZSTK474–PI3K complex support the hypothesis that ZSTK474 binds to the ATP-binding site of PI3K. In contrast, ZSTK474 and LY294002 appear to bind to different sites in the ATP-binding pocket (Fig. 4, C), with LY294002 forming two hydrogen bonds between PI3K at V882 NH and K833 NZ.
As shown in Fig. 4, D and E, the amino acid residues Met-804 (M804), Trp-812 (W812), and Met-953 (M953) of PI3K create a unique space in the ATP-binding pocket that is larger than the corresponding portion of the ATP-binding pocket of other protein kinases (44). In our models, this space was occupied by the bulky 8-phenyl group of LY294002 (Fig. 4, D), as previously indicated by Walker et al. (44), or by the triazine and morpholino groups of ZSTK474 (Fig. 4, E).
Inhibition of Membrane Ruffling by ZSTK474
It is widely accepted that membrane ruffling is regulated by PI3K by means of a pathway that is independent of Akt, a serine/threonine protein kinase. We examined ZSTK474 for its effect on membrane ruffling induced by platelet-derived growth factor in murine embryonic fibroblast cells. We found that membrane ruffling was induced 5 minutes after platelet-derived growth factor was added to the cultures (Fig. 5, A). When cells were incubated for 15 minutes with 1 µM ZSTK474 before the addition of platelet-derived growth factor, both membrane ruffling and the formation of PIP3, a measure of PI3K activity, were blocked (Fig. 5, B). Thus, ZSTK474 appears to inhibit PI3K activity and also to block membrane ruffling.
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Effects of ZSTK474 on Apoptosis and Cell Cycle
Because PI3K and its downstream components appear to mediate antiapoptotic signals, we examined the effect of ZSTK474 on the induction of apoptosis. At 10 µM, a higher concentration than its GI50, ZSTK474 induced apoptosis in OVCAR3 cells (Fig. 6, A and B). However, 10 µM ZSTK474 did not induce apoptosis in A549 cells but instead mediated complete G1-phase arrest (Fig. 6, C). Therefore, it appears that induction of apoptosis by ZSTK474 is a weak and cell type-dependent event. Here the induction of G1-phase arrest by ZSTK474 might lead to the inhibition of tumor growth in vivo, which was indeed observed in the animal experiments with A549 below.
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Inhibition of Akt Phosphorylation and Downstream Signaling by ZSTK474
The time course of ZSTK474-mediated inhibition of the phosphorylation of Akt, one of the major targets of PI3K, and inhibition of other downstream signaling components was examined in A549 cells by immunoblotting. Treatment with 0.5 µM ZSTK474 decreased the level of phosphorylated Akt (Ser-473) within 5 minutes without altering overall Akt protein levels and then decreased the level of phosphorylated GSK-3 (Ser-9) and of cyclin D1 protein expression within 30 minutes (Fig. 7, A). We next investigated the effect of various concentrations of ZSTK474 from 0 to 2.0 µM on the levels of downstream signaling components in the Akt pathway. ZSTK474 inhibited the phosphorylation of forkhead in rhabdomyosarcoma-like 1 (FKHRL1) on Thr-32, forkhead in rhabdomyosarcoma (FKHR) on Thr-24, tuberous sclerosis complex 2 (TSC-2) on Ser-1462, mammalian target of rapamycin (mTOR) on Ser-2448, and p70 ribosomal protein S6 kinase (p70S6K) on Thr-389 and decreased the expression of cyclin D1 protein in a dose–dependent manner (Fig. 7, B). In contrast, phosphorylation of extracellular signal-regulated kinases (ERK1/2) and mitogen-activated protein kinase/extracellular signal–regulated kinase kinases (MEK1/2) were not inhibited by ZSTK474, suggesting that ZSTK474 did not inhibit the RAS–ERK pathway (Fig. 7, A and B). Thus ZSTK474 also appears to act through the Akt pathway.
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Antitumor Efficacy of ZSTK474 In Vivo
We evaluated the antitumor activity and toxicity of ZSTK474 by use of a mouse cancer model and three human cancer xenograft models. When administered orally for 2 weeks, ZSTK474 inhibited the growth of subcutaneously implanted mouse B16F10 melanoma tumors in a dose-dependent manner. Almost complete tumor growth inhibition (i.e., tumor regression) was observed at higher dosages. Administration of ZSTK474 at 100, 200, or 400 mg/kg produced tumor regression of 28.5% (95% CI = 21.4% to 35.7%), 7.1% (95% CI = 2.7% to 11.5%), or 4.9% (95% CI = 3.2% to 6.5%) on day 14, respectively. This growth inhibition was superior to that of the four major anticancer drugs examined—irinotecan, cisplatin, doxorubicin, and 5-fluorouracil (each administered at their respective maximum tolerable doses). Tumor regression in mice of treated with irinotecan, cisplatin, doxorubicin, or 5-fluorouracil was 96.0% (95% CI = 63.5% to 128.4%), 35.7% (95% CI = 28.6% to 42.9%), 24.0% (95% CI = 16.4% to 31.5%), or 68.3% (95% CI = 21.4% to 115.3%) on day 14, respectively (Fig. 8, A). Moreover, the myelosuppression induced by ZSTK474 at 400 mg/kg was marginal (Fig. 8, B).
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We then examined the antitumor activity of ZSTK474 against A549, PC-3, and WiDr human xenografts, which originated from a non–small-cell lung cancer, a prostate cancer, and a colon cancer, respectively. Chronic oral administration of ZSTK474 completely inhibited growth of A549, PC-3, and WiDr xenografts at a dose of 400 mg/kg (Fig. 8, C [upper panels] and D), and it induced the regression of A549 xenograft tumors. To assess toxicity, we measured the body weight of the tumor-bearing mice. Their weight was slightly reduced by administration of ZSTK474, but the reduction was tolerable (no reduction, 17% reduction, and 3% reduction of the day 0 weight on day 14 in A549-bearing mice, in PC-3-bearing mice, in WiDr-bearing mice, respectively) (Fig. 8, C [lower panels]). Dermal toxicity has been reported when LY294002 administered to nude mice (46). However, we observed no dermal toxicity with ZSTK474 even after more than 28 days, as shown in Fig. 8, D. Furthermore, chronic oral administration of ZSTK474 at a higher dose (800 mg/kg) over 4 weeks again reduced body weight within a tolerable range (14% reduction of the day 0 weight on day 28), without toxic effects in critical organs. Thus ZSTK474 appeared to have good therapeutic efficacy in vivo.
Inhibition of the Phosphorylation of Akt by ZSTK474 In Vivo
To confirm that ZSTK474 inhibits the PI3K pathway in vivo, the phosphorylation status of Akt in the subcutaneous A549 tumor tissue was immunohistochemically determined by an anti-Akt antibody specific for phosphorylation at Akt amino acid residue Ser-473. We found that administration of ZSTK474 reduced the level of Akt phosphorylation in tumor tissue (Fig. 8, E).
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, we demonstrated that a new compound, ZSTK474, is a potent inhibitor of PI3K that differs structurally from the well-known PI3K inhibitors LY294002 and wortmannin. We also demonstrated that orally administered ZSTK474 has therapeutic efficacy against human cancer xenografts in mice. To the best of our knowledge, this is the first report of an orally administered PI3K inhibitor that has strong antitumor activity without severe toxicity in vivo. The PI3K pathway is activated in various cancer types, and therefore PI3K is a promising therapeutic target of cancer (3,4,21). Thus ZSTK474 appears to be a new anticancer drug candidate targeting PI3K.
From a methodology viewpoint, another novelty of this study is our use of an information-intensive approach, COMPARE analysis, to identify the target of ZSTK474. The COMPARE analysis predicted that the target of ZSTK474 was PI3K, which was later confirmed by direct inhibition of PI3K activity with ZSTK474. We have previously identified the targets of new compounds MS-247 (26) and FJ5002 (47) as topoisomerase I/II and telomerase, respectively, by using COMPARE analysis. This study further confirms that the COMPARE analysis is a powerful tool that can predict the molecular targets of new compounds.
Molecular modeling of the PI3K–ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K through three hydrogen bonds, which is a finding consistent with the ZSTK474-mediated inhibition of PI3K being reversible. Because ZSTK474 formed three hydrogen bonds with PI3K, instead of the two hydrogen bonds formed between LY294002 and PI3K, ZSTK474 appears to be a stronger competitor for the ATP-binding pocket of PI3K than LY294002. Indeed, the PI3K inhibitory activity of ZSTK474 was approximately 20-fold stronger than that of LY294002. Another important feature of ZSTK474 is its high specificity for PI3K. When tested at 30 µM, ZSTK474 did not inhibit the activity of 139 other protein kinases examined. The three-dimensional space in the ATP-binding pocket of PI3K is larger than the corresponding spaces found in the ATP-binding pockets of other protein kinases (44). The triazine and morpholino groups of ZSTK474 fit well into this space. Therefore, these moieties may be important in the specificity of ZSTK474 for PI3K. To further confirm the binding of ZSTK474 to the ATP-binding pocket of PI3K, a biochemical experiment that examines whether ZSTK474 competitively inhibits the binding of ATP to its binding site is required.
PI3K plays an important role in the proliferation of tumor cells. We compared ZSTK474 with other PI3K inhibitors, LY294002 and wortmannin, for its ability to inhibit cell proliferation. At concentrations of less than 1 µM, ZSTK474 potently inhibited the growth of various cancer cell lines. The growth inhibitory activity of ZSTK474 was more than 10-fold stronger than that of wortmannin, which may be due to wortmannin's being somewhat unstable in cell culture medium (48), or of LY294002, which may be due to the PI3K inhibitory activity of LY294002 being more than 10-fold weaker than that of ZSTK474. Furthermore, the PI3K inhibitory activities of other s-triazine derivatives statistically significantly correlated with their cell growth inhibitory activities. These results suggested that ZSTK474 inhibited cell growth by inhibiting PI3K, with subsequent inhibitory effects on downstream signaling molecules.
PI3K participates in the induction of the membrane ruffling. ZSTK474 inhibited platelet-derived growth factor–induced membrane ruffling in murine embryonic fibroblast cells, which is an Akt-independent process, and inhibited the generation of PIP3. These results demonstrate that ZSTK474 inhibited PI3K activity in intact cells.
Because the PI3K pathway is considered to mediate antiapoptotic signals in tumor cells, ZSTK474 was expected to induce apoptosis efficiently by inhibiting PI3K. However, we observed weak or no induction of apoptosis after ZSTK474 treatment in OVCAR3 cells or A549 cells, respectively. Therefore, induction of apoptosis by ZSTK474 appears to be a cell type–dependent event, probably because of the differential status of the PI3K signaling pathway in various cancer cell lines.
One of the major downstream effectors of PI3K is Akt, a serine/threonine protein kinase. Therefore, we investigated the effect of PI3K on the activation of Akt and its key downstream components. ZSTK474 reduced, in a time-dependent manner, phosphorylation of the protein kinases Akt and GSK-3 and the expression of cyclin D1 protein. Akt inhibits cyclin D1 degradation by regulating the activity of GSK-3 (49). After its phosphorylation by GSK-3, cyclin D1 is targeted for degradation by the proteasome. Akt phosphorylates GSK-3 directly and blocks its kinase activity, thereby allowing cyclin D1 to accumulate in cells (49). ZSTK474 inhibited the phosphorylation of other downstream signaling components that are involved in regulating cell proliferation (i.e., FKHRL1, FKHR, TSC-2, mTOR, and p70S6K) in a dose-dependent manner. These results indicated that the inhibition of PI3K by ZSTK474 was followed by a decrease in relevant downstream signaling, which may in turn facilitate cell growth inhibition. In fact, we observed complete G1-phase arrest in A549 cells after ZSTK474 treatment. In contrast, ZSTK474 did not inhibit phosphorylation of either ERK1/2 or MEK1/2, suggesting that ZSTK474 does not inhibit components in the RAS–ERK pathway.
Orally administered ZSTK474 displayed potent antitumor activity against human cancer xenografts in mice, without evidence of critical toxicity. Known PI3K inhibitors—such as LY294002 (46,50–52), wortmannin (53,54), and viridin analogues (54)—have previously been examined for their antitumor activities. In most reports, LY294002, which was administered intraperitoneally because it is insoluble in water, had antitumor activity when administered at an early stage (i.e., within 7 days after inoculation of tumor cells) (50), but it also induced severe dermal toxicity (46). Wortmannin also had antitumor activity, but again it achieved sufficient efficacy only when administered at an early stage (53), and it caused liver toxicity (54). An analogue of wortmannin, PX-866, has recently been developed (54) that has better efficacy and lower toxicity than wortmannin. However, this agent still produces liver toxicity (54). In this study, we found that ZSTK474 had strong antitumor efficacy against advanced-stage tumors (i.e., >15 days after inoculation of tumor cells) and that ZSTK474 did not produce noticeable toxicity in critical organs after daily administration for more than 14 days. We also confirmed reduction of Akt phosphorylation after ZSTK474 administration. Thus, the reduction of Akt phosphorylation may be a useful biomarker marker to monitor the efficacy of ZSTK474 in future investigations of ZSTK474.
Although ZSTK474 indicated favorable features for a novel anticancer drug candidate as described above, various preclinical studies remain before ZSTK474 can be evaluated in a clinical trial, which include more precise investigation of the molecular pharmacology, analyses of the pharmacokinetics and the pharmacodynamics, and careful investigation of the toxicity. Investigation of the toxicity derived from PI3K inhibition is especially important because PI3K is expressed in various normal tissues. These studies are currently under way.
In conclusion, we demonstrated that the compound ZSTK474 is a new PI3K inhibitor and that it has strong antitumor activity against human cancer xenografts without critical toxicity. It appears that ZSTK474 can be orally administered over long periods, which may be important for the treatment of cancer. Thus, ZSTK474 merits further investigation as an anticancer drug.
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NOTES |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We thank Dr. R. H. Shoemaker and Dr. K. D. Paull for discussion on the establishment of JFCR39 and COMPARE analysis; Ms. Y. Nishimura, Ms. M. Seki, Ms. Y. Mukai, Ms. M. Okamura, and Dr. Y. Yoshida for technical assistance; Mr. T. Watanabe, Dr. Y. Tsuchida and Mr. K. Saito for synthesis of compounds; and Mr. M. Takehara and Dr. M. Takahashi for advice on the study.
Supported by grants-in-aid of the Priority Area "Cancer" from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, to T. Yamori (11177101); grants-in-aid for Scientific Research (B) from Japan Society for the Promotion of Science to T. Yamori (17390032); and a grant from National Institute of Biomedical Innovation, Japan, to T. Yamori (05-13).
Funding to pay the Open Access publication charges for this article was provided by the Japanese Foundation for Cancer Research.
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Manuscript received September 16, 2005; revised January 23, 2006; accepted March 3, 2006.
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