Quantitative Chemical Proteomics for Identifying Candidate Drug Targets

Anal. Chem. 2003, 75, 2159-2165

Quantitative Chemical Proteomics for Identifying Candidate Drug Targets Yoshiya Oda,*,†,⊥ Takashi Owa,†,⊥ Toshitaka Sato,†,⊥ Brian Boucher,‡ Scott Daniels,‡ Hidenori Yamanaka,§ Yasuhiro Shinohara,§ Akira Yokoi,† Junro Kuromitsu,† and Takeshi Nagasu†

Laboratory of Seeds Finding Technology, Eisai Co., Ltd, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan, Applied Biosystems, 500 Old Connecticut Path, Framingham, Massachusetts 01701, and Amersham Biosciences K.K., Sanken Building, Hyakunincho 3-25-1, Shinjyuku, Tokyo 169-0073, Japan

We have developed a systematic strategy for drug target identification. This consists of the following sequential steps: (1) enrichment of total binding proteins using two differential affinity matrixes upon which are immobilized positive and negative chemical structures for drug activity, respectively; (2) covalent labeling of the proteins with a new cleavable isotope-coded affinity tag (ICAT) reagent, followed by proteolysis of the combined proteins; (3) isolation, identification, and relative quantification of the tagged peptides by liquid chromatography-mass spectrometry; (4) array-based transcription profiling to select candidate proteins; and (5) confirmation of direct interaction between the activity-associated structure and the selected proteins by using surface plasmon resonance. We present a typical application to identify the primary binding protein of a novel class of anticancer agents exemplified by E7070. Our results suggest that this approach provides a new aspect of quantitative proteomics to find specific binding proteins from protein mixture and should be applicable to a wide variety of biologically active small molecules with unidentified target proteins.

In biochemical studies to identify drug-binding proteins, compound-conjugated affinity matrix reagents have played an important role. For example, Schreiber et al. reported the identification of FK506-binding protein4 and mammalian histone deacetylase 15 using affinity matrixes. Compared with natural products, the affinity and specificity of synthetic small molecules to their protein targets are rather low in many cases. Thus, nonspecific interactions between a synthetic compound and its binding proteins often lead to difficulty in specifying the primary binding partners. To address this problem, we applied the fluorescent two-dimensional differential in-gel electrophoresis (2DDIGE) method6 and the newly developed cleavable isotope-coded affinity tag (ICAT) method to identify the primary binding protein(s) of a novel class of anticancer agents represented by E7070 as a model case. This compound, currently undergoing phase II clinical trials in Europe and the United States, was originally selected from sulfonamide-focused libraries by means of a phenotypic screen to assess cell cycle perturbation at the G1/S transition of P388 murine leukemia cells.7,8 Despite its unique cell cycle effect, the primary molecular target of E7070 has yet to be elucidated.

The results of the human genome project have made it possible to predict novel genes encoding putative drug targets and then to characterize a large number of small molecules from chemical libraries in a high-throughput, target-oriented manner.1 On the other hand, cell-based phenotypic screening of diverse compound collections represents a forward chemical-genetic approach to identify small molecules that modulate biological processes within living cells.2,3 Such unbiased drug screening is generally followed by critical research efforts to clarify the protein targets and molecular mechanisms involved in compound-induced phenotypes. To establish a more systematic methodology for these studies, new and efficient proteome technologies are required.

MATERIALS AND METHODS Preparation of Differential Chemical Affinity Matrixes. Figure 1A shows the chemical structures of E7070 (1), its analogues (2-4), and chemical probes used for target identification (5-8). Benzylamine variants of 1 and 2 were each dissolved in tetrahydrofuran and diluted with methanol and water to 4 mg/ mL final concentration. Affi-gel 10 (Bio-Rad) was added to each compound solution, and triethylamine was also used to adjust pH in the alkaline region. The amount of the immobilized compound was ∼8 mg/mL wet gel in each case. MTT Assay. HCT116-C9 and HCT116-C9-C1 cells9 were each seeded at 3.0 × 103 cells/well in 96-well microtiter plates and

* Corresponding author. Phone: +81 298 47 7098. Fax: +81 298 47 7614. E-mail: [email protected]. † Eisai Co., Ltd. ‡ Applied Biosystems. § Amersham Biosciences K.K. ⊥ These authors contributed equally to this work. (1) Drews, J. Science 2000, 287, 1960-1964. (2) Haggarty, S. J.; Mayer, T. U.; Miyamoto, D. T.; Fathi, R.; King, R. W.; Mitchison, T. J.; Schreiber, S. L. Chem. Biol. 2000, 7, 275-286. (3) Stockwell, B. R. Trends Biotechnol. 2000, 18, 449-455. 10.1021/ac026196y CCC: $25.00 Published on Web 04/03/2003

© 2003 American Chemical Society

(4) Harding, M. W.; Galat, A.; Uehling, D. E.; Schreiber, S. L. Nature 1989, 341, 758-760. (5) Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996, 272, 408-411. (6) Unlu, M.; Morgan, M. E.; Minden, J. S. Electrophoresis 1997, 18, 20712077. (7) Owa, T.; Yoshino, H.; Okauchi, T.; Yoshimatsu, K.; Ozawa, Y.; Sugi, N. H.; Nagasu, T.; Koyanagi, N.; Kitoh, K. J. Med. Chem. 1999, 42, 3789-3799. (8) Owa, T.; Yoshino, H.; Yoshimatsu, K.; Nagasu, T. Curr. Med. Chem. 2001, 8, 1487-1503. (9) Yokoi, A.; Kuromitsu, J.; Kawai, T.; Nagasu, T.; Sugi, N. H.; Yoshimatsu, K.; Yoshino, H.; Owa, T. Mol. Cancer Ther. 2002, 1, 275-286.

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Figure 1. (A) Structures of E7070 and its analogues. (B) Anticancer activity assay. aIC50 value (drug concentration required for 50% inhibition of cell proliferation) was determined by the 3-day MTT assay performed twice in triplicate with each test compound. Errors were within (15% of the reported values. bRelative resistance value equals the IC50 for the E7070-resistant cell line HCT116-C9-C1 divided by the IC50 for the parental cell line HCT116-C9. (C) A typical SDSPAGE result after purification of binding proteins using chemical affinity matrixes. Whole protein extracts from HCT116-C9 cells were loaded onto the affinity columns to enrich binding proteins. Each binding protein pool was separated with a 10-20% T gel, and then the gel was stained with silver. 1, molecular markers; 2, eluents from the ethanolamine matrix; 3, eluents from the E7070-type matrix; 4, eluents from amide-type matrix. 2160

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precultured for 24 h. Test compounds were each dissolved at 40 mM in DMSO and further diluted with the culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 µg/mL)) to prepare 3-fold serial dilutions, with the maximum concentration being 200 µM after addition to each well. The obtained dilutions were added to the plates, and incubation was continued for another 3 days. The antiproliferative activity was determined by means of MTT colorimetric assay.10 Preparation of Cell Lysate. HCT116-C9 cells were grown to a density of 7 × 107 cells/15-cm diameter dish in the culture medium. Whole proteins were extracted with 5 mL of M-PER (Pierce) containing protease inhibitor cocktail (Roche Diagnostics) and 5 mM dithiothreitol (DTT). Enrichment of Binding Proteins. Cell lysate from 3.5 × 108 cells was loaded on 1 mL bed volume of each affinity matrix. After washing of the matrix with 25 mL of PBS including with 0.05% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 25 mL of PBS including 0.5 M sodium chloride, binding proteins were eluted with 6 M guanidine hydrochloride including 2% CHAPS and then dialyzed against 10 mM ammonium bicarbonate buffer. We repeated this procedure 10 times, and the final sample solutions were mixed and then dried by vacuum evaporation. The total amount of binding proteins was ∼2 mg from each affinity column. Identification of Proteins Enriched by Affinity Matrix. Proteins (100 µg) were dissolved in 8 M urea at pH 8.5 with Tris buffer, reduced by adding 5 mM DTT, and reacted with 25 mM acrylamide. After the reaction, the solution was diluted to 2 M urea with water, and 2 µg of trypsin was added. After digestion, tryptic peptides were separated on a cation exchange column, Mini-S (Amersham Pharmacia Biotech) with a linear gradient of sodium chloride. Fractions were collected every minute (total 40 fractions), desalted with an on-line trap column, and then analyzed with reversed phase LC-MS/MS. 2D-DIGE Analysis. Binding proteins (100 µg) eluted from matrixes 7 and 8 were labeled with Cy5 and Cy3 reagent, respectively, using a commercial kit supplied by Amersham Biotech. The labeled samples were mixed and separated on a 2D gel (the first dimension: 18 cm immobilized pH gradient gel, pH 3-10; the second dimension: 15% T SDS-PAGE). 2D-DIGE experiments were perfoemed four times to confirm reproducibility. The in-gel digestion procedure was based on the protocol that we previously reported.11 ICAT Analysis. Binding proteins (100 µg) eluted from matrixes 7 and 8 were labeled with isotopically light- and heavyICAT reagents, respectively, and treated according to the protocol recommended by Applied Biosystems. DNA Microarray Analysis. HCT116-C9 cells were plated at 5.0 × 106 cells/dish in 10-cm diameter dishes with 10 mL of the culture medium. After 24-h preincubation, the cells were treated for 12 h with 8 µM of each test compound or with 0.015% DMSO (control). The following microarray experiments were performed using Affymetrix HuGene FL arrays according to established protocols. All quantitative data were obtained using the Affymetrix GeneChip software.12 (10) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63. (11) Katayama, H.; Nagasu, T.; Oda, Y. Rapid Commun. Mass Spectrom. 2001, 15, 1416-1421.

Surface Plasmon Resonance (SPR) Analysis. Interactions of selected proteins with four different compound structures were examined using the BIACORE3000 system (Biacore, Uppsala, Sweden). Benzylamine variants of compounds 1-4 were each immobilized on the activated sensor chip CM5 according to the manufacturer’s instructions. LC-Mass Spectrometry. HPLC was performed with a 150 × 0.1 mm C18 capillary column, and the eluent was directed to an ESI ion trap mass spectrometer (ThermoFinnigan model LCQ) or an ESI QqTOF mass spectrometer (Applied Biosystems model QSTAR pulsar i) at a flow rate of 1 µL/min after flow splitting. MS/MS spectra were obtained in a data-dependent mode in which the highest-intensity peaks in each MS scan were chosen for collision-induced dissociation. MS/MS data were analyzed by MASCOT (Matrix Sciences) and Sonar MS/MS (ProteoMetrics). RESULTS AND DISCUSSION To design differential chemical affinity matrixes, we first examined the structure-activity relationship for the E7070 class of anticancer agents. Thus, cell growth inhibition assays according to the MTT method10 were performed with an E7070-sensitive human colon cancer cell line HCT116-C9 and its E7070-resistant subclone HCT116-C9-C1.9 The IC50 and relative resistance values for compounds 1-6 are listed in Figure 1B. As compared with E7070, the amide analogue 2 and the 2-Cl analogue 4 were less potent by 2 orders of magnitude as growth inhibitors of HCT116C9. HCT116-C9-C1 did not display significant cross-resistance to these two compounds. Although the 3-H analogue 3 was 48-fold less active than E7070 toward HCT116-C9, the 9-fold crossresistance of HCT116-C9-C1 to this compound should be noted. The BODIPY FL-tagged derivatives 5 and 6 were originally designed as fluorescent chemical probes to examine the cellular localization of this series of compounds. Interestingly, despite the dramatic structural modification to the 4 (para) position of the benzene ring of 5, significant activity and specificity toward HCT116-C9 were retained, as compared with HCT116-C9-C1. In addition, the amide analogue 6, even at 100 µM, did not exhibit any potent activity against both cell lines. These results suggest that the location of the BODIPY FL moiety may be independent of a critical interaction with the putative protein target(s). On the basis of the above results, we designed two chemical affinity matrixes, 7 (positive) and 8 (negative), and used them differentially to find specific binding proteins to the E7070 class of anticancer agents. However, as shown in Figure 1C, many proteins were found on both matrixes, whereas an ethanolamineconjugated matrix as another negative control matrix did not enrich any proteins under our experimental conditions. This means that most of the proteins retained by 7 and 8 bind to the compound structures themselves, but not to the solid support. Handa et al. reported that the latex beads developed in their laboratories could considerably reduce nonspecific interactions between numerous sticky proteins and the solid support.13 In our case, however, the complicated result was attributable to the high (12) Lockhart, D. J.; Dong, H.; Byrne, M. C.; Follettie, M. T.; Gallo, M. V.; Chee, M. S.; Mittmann, M.; Wang, C.; Kobayashi, M.; Horton, H.; Brown, E. L. Nat. Biotechnol. 1996, 14, 1675-1680. (13) Shimizu, N.; Sugimoto, K.; Tang, J.; Nishi, T.; Sato, I.; Hiramoto, M.; Aizawa, S.; Hatakeyama, M.; Ohba, R.; Hatori, H.; Yoshikawa, T.; Suzuki, F.; Oomori, A.; Tanaka, H.; Kawaguchi, H.; Watanabe, H.; Handa, H. Nat. Biotechnol. 2000, 18, 877-881.

protein binding nature of the compounds attached to the beads. Although we tested various conditions for affinity purification, a substantial improvement was not obtained. In consequence, we identified a total of 285 proteins from the protein pool enriched by 7 using two-dimensional HPLC-MS/MS analysis.14,15 E7070 actually shows very high protein binding (more than 99%) to human blood serum, suggesting that it may bind to various proteins in a nonspecific manner. Of the 285 proteins identified, many were highly abundant proteins, such as tubulins and molecular chaperons. It was difficult to remove these abundant proteins, which were always observed on both 7 and 8. This situation made it difficult to differentiate matrix 7-specific binding proteins from proteins binding nonspecifically to both matrixes. Therefore, quantitative proteome analysis was particularly important for identifying the true target among the numerous nonspecific binders. To get quantitative information on proteins binding to 7 and 8, we first applied 2D-DIGE. As shown in Figure 2, whole protein extracts from HCT116-C9 cells were aliquoted into two pools. One pool was loaded onto the positive matrix 7, and the other pool, onto the negative matrix 8. After washing, the binding proteins were eluted and were labeled with fluorescence dyes Cy5 and Cy3, respectively. These two pools were mixed in equivalent amounts, and analyzed with 2D-DIGE. Consequently, matrix 7-specific and also matrix 8-specific spots were illuminated on the gel at the same time. Mass spectrometry measurement identified cytosolic malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) B as matrix 7-selective binding proteins, as shown in Figure 3. The 2D-DIGE is a very attractive procedure, because it is easy to find specific protein spots in a differential manner. However, several basic, very large or poorly soluble proteins may not be detected on 2D gels,16 and when two proteins overlap in one spot, the quantitative information is ambiguous. On the basis of the above considerations, we next employed the ICAT method,17,18 which should overcome the limitations of 2D-DIGE. Since all proteins containing Cys residues are selectively modified with the ICAT reagent and the digested peptides are enriched on an avidin column, the complexity of the mixture is significantly reduced. In our protocol, proteins binding to 7 and 8 were labeled with ICAT-d0 and ICAT-d8, respectively, in the same way as for 2D-DIGE (see Figure 2). In consequence, cytosolic MDH was identified as a matrix 7-selective binding protein, and RNA-binding protein regulatory subunit was identified as a matrix 8-selective one. The results were consistent with the 2D-DIGE data with a few exceptions, such as LDH, which was identified as a matrix 7-selective binding protein by the 2D-DIGE method, but not by this ICAT method. Although 230 proteins (345 peptides) were identified in the pool labeled with ICAT-d0, this sample contained at least 285 proteins, as described above. Therefore, there is still a possibility that the true protein target(s) might have been overlooked in this conventional ICAT method. (14) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., 3rd. Nat. Biotechnol. 1999, 17, 676-682. (15) Washburn, M. P.; Wolters, D.; Yates, J. R., 3rd. Nat. Biotechnol. 2001, 19, 242-247. (16) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395. (17) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (18) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946951.

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Figure 2. Schematic representation of the procedure for chemical quantitative proteomics. Binding proteins to chemical affinity matrix 1 (i.e., E7070-type) or chemical affinity matrix 2 (i.e., amide-type) were labeled with different tags (Cy3 and Cy5 or ICAT reagents), combined, digested, separated, and analyzed as desribed in the text.

Figure 3. Two-dimensional electrophoresis profile of binding proteins labeled with Cy3 and Cy5. Whole protein extracts from HCT116-C9 cells were loaded onto the chemical affinity matrixes to enrich binding proteins. Binding proteins to the amide-type matrix were labeled with Cy3 (dark blue), and those to the E7070-type matrix, with Cy5 (green). The specific spots for Cy3 and Cy5 were each excised and identified by means of mass spectrometry. The spots and their names are indicated in this figure.

Aebersold et al. have recently developed a solid-phase isotope tagging reagent to improve the conventional ICAT method.19 According to their report, each of the protein pools was labeled with a photocleavable tag on the solid support after tryptic digestion. However, protein digestion can show poor reproducibility, and thus, the two protein pools should be (19) Zhou, H.; Ranish, J. A.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2002, 20, 512-515.

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mixed with each other before tryptic digestion to normalize the variation in digestion efficiency. Hence, we finally tried a new cleavable ICAT method for comprehensive analysis. The experimental flowchart is shown in Figure 4A. In consequence, 377 proteins (656 peptides) were identified as matrix 7-binding proteins, as summarized in Figure 4B. This number (377) is larger than those of the conventional ICAT method (230) and multidimensional LC-MS/MS (285). The reason for

Figure 4. (A) Strategy for quantitative protein analysis using cleavable ICAT reagents. Two protein samples to be compared were labeled at cysteine residues with C0-ICAT and C9-ICAT reagents, respectively, and then combined. The mixture was subjected to proteolysis, then biotin tag-containing peptides were enriched on an avidin column, the tag was cleaved by adding trifluoroacetic acid, and the peptides were analyzed by LC-MS/MS. (B) The top 20 proteins specifically bound to the E7070-type matrix. A smaller value of binding ratio means more specific binding to the E7070-type matrix.

Figure 5. (A) Typical sensorgrams obtained from the real-time interactions between the E7070-type sensor chip and various recombinant proteins. MDH, malate dehydrogenase; GPI, glucose-6-phosphate isomerase; LDH, lactate dehydrogenase. (B) The sensorgrams indicate the interactions between cytosolic MDH and immobilized chemical structures. RU, resonance unit. Immobilized amounts were 170 RU for the E7070type chip, 165 RU for the 3-H-type one, 328 RU for the 2-Cl-type one, and 184 RU for the amide-type one. Cytosolic MDH (0.6 µM) and 1 µM LDH, GPI, and mitochondrial MDH were flowed over the surface on each chip at 40 µL/min.

this improvement is considered to be as follows. The mass tag after cleavage by trifluoroacetic acid weighs 227 Da for the C0-light form. Because of the small size and the chemical nature of the tag, the observed peptide fragmentation patterns in MS/MS were not complicated. On the other hand, undesirable fragmentation of peptides was frequently observed in the conventional ICAT spectra (data not shown). The reason new

cleavable ICAT identified more proteins, as compared to 2D-LC-MS/MS, is that the ICAT could reduce the complexity of the peptide mixture.17,19 Thus, we identified several matrix 7-selective binding proteins, as listed in Figure 4B, with high confidence. It should be mentioned that the improved ICAT method also identified cytosolic MDH as one of the most selective binders to matrix 7. Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

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Table 1. Array-Based Transcription Profiling (A) Functional Classification of E7070-Induced Alterations in Gene Expression category of E7070 (8 µM, 12 h)-affected genes no. of probe sets Up-Regulation (at Least 2-fold)

18

mRNA processing/translation cell signaling miscellaneous

5 2 11

Down-Regulation (at Least 2-fold)

163

metabolism cell cycle control cell signaling/immune response mRNA processing/translation transcription/chromatin modulation Golgi function/membrane trafficking proteolysis cytoskeleton apoptosis miscellaneous

30 23 23 16 13 8 6 4 2 38

(B) Summary of Transcriptional Changes Induced by Test Compounds 1-4a compound/fold change value accession no.

gene name

1

M55905

mitochondrial NAD(P)+ dependent malic enzyme mitochondrial aspartate aminotransferase cytosolic aspartate aminotransferase medium-chain acyl-CoA dehydrogenase (MCAD) carnitine palmitoy transferase I type I muscle glycogen synthase neuron-specific (γ) enolase mitochondrial intermediate peptidase precursor NADH dehydrogenase (ubiquinone) Fe-S protein 1 glutathione synthetase

-3.0

M22632 M37400 M91432 Y08682 J04501 X51956 U80034 X61100 U34683 M95809 U11791 X06745 L47276 D38553 U18291 U49844

M59911 X53587 U40282 M29550 U81554 Z17227 M97935 a

basic transcription factor 62-kDa subunit (BTF2) cyclin H DNA polymerase alpha subunit R- topoisomerase II truncated form hCAP-H CDC16Hs FRAP-related protein (FRP1/ATR) integrin R-3 integrin β-4 integrin-linked kinase calcineurin A1 CaM kinase II γ IL-10 receptor β (CRF2-4) transcription factor ISGF-3 (STAT 1)

2

3

Metabolism NC

4

function

-1.5

NC

malate, TCA cycle

-2.0

NC

NC

NC

malate-aspartate shuttle

-2.1

NC

NC

NC

malate-aspartate shuttle

-2.5

NC

-1.9

NC

lipid

-3.4

NC

-2.3

-1.6

lipid

-3.5 -3.2 -5.7

NC NC NC

NC -2.0 NC

NC NC NC

carbohydrate carbohydrate mitochodrial processing

-2.9

NC

-1.6

NC

respiratory electron transfer

-3.1

NC

-1.8

NC

redox

Cell Cycle Control -4.2 NC -1.6

NC

TFIIH, transcription, NER

-5.1 -4.0 -2.0

NC NC NC

-2.3 -2.0 NC

NC NC NC

TFIIH, transcription, NER DNA replication DNA replication, DNA repair

-3.9 -3.5 -2.6

NC NC NC

NC NC NC

NC NC NC

mitotic chromosome condensation mitosis, protein turnover (APC/C) cell cycle checkpoint, DNA repair

NC NC NC NC NC NC NC

adhesion adhesion adhesion Ca2+-linked Ca2+-linked IL-10 signaling IFN signaling, transcription factor

Cell Signaling/Immune Response -2.6 NC NC -4.2 NC -1.6 -2.5 NC NC -2.5 NC NC -5.3 NC -1.8 -3.9 NC NC -3.1 NC -1.7

8 µM, 12 h. NC means no change, based on the Affymetrix software algorithm.

In parallel with these proteome experiments, we also conducted DNA microarray analysis20 to examine transcriptional effects of E7070 on HCT116-C9 cells.9,20 After 12 h of treatment at 8 µM, E7070 caused characteristic down-regulation of sets of genes involved in cellular metabolism, cell cycle control, cell signaling/ (20) Owa, T.; Yokoi, A.; Yamazaki, K.; Yoshimatsu, K.; Yamori, T.; Nagasu, T. J. Med. Chem. 2002, 45, 4913-4922.

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immune response, and so on (Table 1A). Further gene expression monitoring (at 8 µM for 12 h) was carried out with each of the analogues 2-4. We highlighted 24 genes, listed in Table 1B, because all of them were down-regulated in a time-dependent manner from the 6-h to 24-h time points, at least in HCT116-C9 cells treated with 0.8 µM E7070, a pharmacologically relevant and clinically achievable drug concentration (unpublished results by

T.O., A.Y., and J.K.). Analogues 2 and 4 exerted almost no effect on the expression of the 24 genes. Analogue 3 caused clear downregulation of one-half of these genes, although the fold change value for 3 was less than that for E7070 in each of the genes. These results were in accordance with the MTT assay data. Although mRNA expression of multiple genes critical to cell cycle control was actually repressed by E7070 treatment, components of the cell cycle machinery were not found as matrix 7-selective binding proteins in our 2D-DIGE and ICAT experiments. The quantitative proteome analyses indicated that several metabolic enzymes, such as cytosolic MDH, could be candidates as targets for the E7070 class of anticancer agents. In this respect, it is noteworthy that subsets of metabolic genes involved in energy production and the cellular redox environment were profoundly down-regulated by treatment with E7070 (and also the 3-H analogue 3). In addition, it has been repeatedly shown that energy and redox states in cells are closely connected with cell cycle control and immune response signals. Judging from the results of the quantitative proteome analyses and the implications of the gene expression monitoring study, we focused on some metabolic enzyme proteins, including cytosolic MDH, as potential protein targets of the E7070 class of anticancer agents. Therefore, we prepared the recombinant proteins and examined the direct interaction by using SPR analysis,13 as shown in Figure 5A. In the experiment, benzylamine variants of E7070 were immobilized on the SPR sensor chip surface. Cytosolic MDH displayed a strong direct interaction with the E7070-type sensor chip, whereas the other metabolic enzyme proteins examined did not interact significantly with the identical chip. LDH was a specific binder to the E7070-type matrix 7 in the 2D-DIGE analysis. In addition, the new cleavable ICAT analysis identified LDH as a binding protein somewhat selective to the same matrix 7. However, SPR measurement did not suggest specific binding of this protein to the E7070-type structure. From these results, it appears that recombinant LDH does not interact with this compound structure, though the possibility remains that the endogenous molecule may have some interaction, either directly or indirectly. In relation to this, there were actually two or more spots of LDH on the 2D-DIGE gel, suggesting that LDH bound to matrix 7 might have undergone some modification. A further examination was performed with three additional sensor chips: the amide-type, the 3-H-type, and the 2-Cl-type. In the cytosolic MDH flow, the 3-H-type chip gave a sensorgram that was similar to the E7070-type one, but was attenuated. On the other hand, the amide-type and 2-Cl-type chips did not show any strong interaction with cytosolic MDH. These findings mean that the structure and activity correlations in the series of chemical structures evaluated here match exactly with the order of the affinity to cytosolic MDH. Hence, we suggest that cytosolic MDH is likely to be at least one of the true cellular targets of the E7070type anticancer agents. However, in our experiments, according to a common assay protocol with recombinant cytosolic MDH, the enzyme inhibition of E7070 was undetectable as to the enzymatic activity that catalyzes the reductive process of oxalo(21) Burgos, C.; Gerez de Burgos, N. M.; Rovai, L. E.; Blanco, A. Biochem. Pharmacol. 1986, 35, 801-804. (22) Knockaert, M.; Meijer, L. Biochem. Pharmacol. 2002, 64, 819-825. (23) Knockaert, M.; Wieking, K.; Schmitt, S.; Leost, M.; Grant, K. M.; Mottram, J. C.; Kunick, C.; Meijer, L. J. Biol. Chem. 2002, 277, 25493-25501.

acetate to malate using NADH as a cofactor. The reason for this would appear to be due to a very poor solubility (low micromolar or less) of E7070 in an assay buffer. Conventional cytosolic MDH assay requires at least 10-100 µM NADH for monitoring changes in the absorbance at 340 nm.21 Given that E7070 binds to cytosolic MDH in an NADH-competitive fashion, its poor solubility would be a serious drawback to the enzyme inhibition in the assay using such high concentrations of NADH. To address this possibility, a BIAcore-based competition assay was further conducted. We immobilized the core structure of E7070 on a sensor chip of BIAcore according to the same procedure as described in Figure 5 and flowed cytosolic MDH with NADH/NAD/NADP/NADPH. As a result, NADH inhibited the binding of the E7070 pharmacophore and cytosolic MDH at low micromolar concentrations. In contrast, NAD and NADPH needed a several hundred micromolar range to compete with the pharmacophore on the enzyme, and NADP was incapable of the competition even at 2 mM. This observation suggests that E7070 shares the binding site on cytosolic MDH with NADH, a cofactor of this enzyme. Moreover, the difficulty in detecting the enzyme inhibition of E7070 may be attributable to its very poor solubility in water as mentioned above. With reference to the present study, Knockaert and Meijer et al. have recently reported that mitochondrial MDH was identified as an unexpected intracellular target of paullones that constitute a family of potent and apparently selective cyclin-dependent kinase and GSK-3 inhibitors.22,23 They also have used affinity chromatography very effectively to identify in vivo targets of this novel class of cell cycle inhibitors. Mitochondrial and cytosolic MDHs are known to play a key role in a variety of metabolic events, including malate-aspartate shuttle, TCA cycle, gluconeogenesis, etc. Thus, one of the most interesting implications of their finding combined with ours is a potential involvement of the MDH-related metabolic processes in cell cycle regulatory machinery and mechanisms of antitumor action. Biochemical and molecular biological studies are now in progress to examine the precise connection between the function(s) of cytosolic MDH and the anticancer activity of E7070. CONCLUSIONS Current proteomics analysis for drug discovery has concentrated on comparing cells and tissues in two different states, for example, normal versus disease. In the present study, however, we have focused on differential quantitative analysis to discover a drug-binding target(s) using compound-immobilized affinity matrixes. Our integrated strategy should be of wide utility for identification of the targets of biologically active small molecules. The methodology presented here provides a new tool for chemical proteomics in post-genomic medicinal science. ACKNOWLEDGMENT We thank Mr. Tsuyoshi Tabata and Dr. Takatoshi Kawai for their development of the bioinformatics software, Dr. Hiroyuki Katayama for his assistance in immobilization of compounds on affinity resins, and Dr. Tony Hunt for coordinating the ICAT projects. Received for review October 2, 2002. Accepted March 6, 2003. AC026196Y Analytical Chemistry, Vol. 75, No. 9, May 1, 2003

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