Proteomics accelerating the identification of the target molecule of bioactive small molecules

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Proteomics accelerating the identification of the target molecule of bioactive small molecules Konstanty Wierzba, Makoto Muroi and Hiroyuki Osada Failures in many drug development programs in the past decades were related to unspecified mechanism of action and poor pharmacokinetic (PK) properties. Recent developments are focused on well defined targets, improved PK profiles, however, not much is known about off-target effects, especially those responsible for diminishing drug activity. Steadily increasing application of proteomics in drug development should expose clinically relevant proteins for the analysis of drug effects, to show what group of patients will respond and who should not be treated with an agent. Address Chemical Library Validation Team, Chemical Biology Core Facility, Chemical Biology Department, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Corresponding author: Osada, Hiroyuki ([email protected])

Current Opinion in Chemical Biology 2011, 15:57–65 This review comes from a themed issue on Omics Edited by Kate Carroll and Pieter Dorrestein

The classical drug development was based on the well established target molecules, for instance thymidylate synthase for 5-fluorouracil analogs [3], topoisomerase II for podophyllotoxins [4], DNA for anthracyclines, to name a few. The further wide explorations of those compounds led to the discovery of numerous other mechanisms presumably responsible for anticancer activity. The question arose as to whether one must identify the only one and specific mechanism of action or many others as well to ideally match them to the exact type of neoplastic disease. The latter case without any doubt is the most reasonable, but requires a great deal of data that recently can be manageable due to a rapid technological development in the field of proteomics. Clinical experience gathered so far resulted in establishing of the treatment protocols, for instance estrogen receptor negative breast cancer is not treated with tamoxifen, the antagonist of the ER [5], also the patient with metastatic colorectal cancer expressing mutated KRAS gene cannot be treated with the anti-EGFR mAb cetuximab due to insensitivity to EGFR inhibitors [6].

Available online 4th November 2010 1367-5931/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2010.10.009

Introduction The WHO predicted in 2000 more than 10 million new cases of cancers to be followed by 7 million deaths worldwide [1]. The most recent survey indicates the declining incidence of certain cancers in western countries while still ascending in several less developed countries [2]. So far, the most effective choice against cancer is surgical intervention, however, in many instances, for example advanced tumors, hematologic neoplasia, the chemotherapy is an ultimate choice but temporal one. There are many active anticancer drugs; however, their mode of action remains not fully clarified yet. On the contrary, we lack information if a tumor possesses the Achilles heel molecular target and what protective mechanisms the treated cancer will employ against the drug. Briefly, to be prepared for continuing the effective pharmacologic interventions, we must predict the sequence of molecular events taking place during chemotherapy, both in cancer and in host’s body. www.sciencedirect.com

The very first information on well molecularly characterized drug and cancer type relationships can be obtained from publically available databases. Given that the widely used anticancer drugs represent well defined mechanisms of action, we made an attempt to construct a global heat map connecting the cancers of different origin with those drugs, to know how often a drug is explored in a particular cancer, reflecting the interest of researchers or clinicians (Figure 1). Visual inspection provides us an image of drug–cancer connections; one may identify several islands grouping the drugs of similar mechanism of action. In the case of antimetabolites, the 5-FU type drugs correlated with solid cancers, while purine type and antifolates are connected to blood neoplasia. Hormonal drugs are clearly localized in the island of hormonally regulated tissues, indicating the initialization of target oriented drug design with that group of drugs. Recently developed molecular targeted drugs, like sorafenib and glivec, were well explored in renal cancer and hepatocellular carcinoma and stomach cancer and leukemias, respectively. Certain caution is necessary in the case of brain cancers, their clinical responses to any agent primary depend upon an ability to pass through the blood–brain barrier, hardly permeable for many drugs, rather than to specific interaction with a target. This simple data analysis gives us the opportunity of preliminary prediction of molecular pathways involved in cancerous processes in cancers of Current Opinion in Chemical Biology 2011, 15:57–65

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Figure 1

Heat map showing correlation between main types of anticancer drugs and cancers of different organ origin. Data are expressed as the relative exploration rate (ER%), a coefficient derived based on the PubMed reports. Because of the lower number of reports, the different scale was applied for kinase inhibitors and hormonal drugs in order to show difference between individual compounds. Given that scientific output is strongly connected with disease burden in a population, the number of scientific reports may correlate with either total number of new patients or with the incidence of disease. Furthermore, the drugs with certain degree of selectivity toward a disease are preferentially used for the treatment or for experimental purposes, and this fact is reflected by the number of reports. Therefore, based on this assumption, the number or reports was extracted from the PubMed, using the coupled key words [‘name’ cancer and ‘name’ drug], without employing any filtration no matter whether a report was related to either an experimental or clinical study, positive or negative results. Since the total number of reports does not reflect a specific drug–cancer connection, the number of reports were adjusted to the cancer incidence rate to estimate the ‘exploration rate (ER)’ and ‘relative exploration rate (rER)’, the latter representing the ER adjusted to the total number of reports representing a particular drug and general expression of cancer.

different histology, assuming the frequency of exploration of a drug with defined mechanism of action is due to existence of specific pathways. The development of targeted agents brought many promising compounds to the light; however, their clinical usage is limited to hematologic cancers that have well established target, for instance tyrosine kinase domain of the abl kinase in chronic myelogenous leukemia is targeted by glivec [7]. The other kinase inhibitors appeared to be less effective against solid tumors and not free of severe Current Opinion in Chemical Biology 2011, 15:57–65

side effects. A multikinase target drug can hit several nodes in the pathways relevant to a particular disease resulting in the increase of efficacy or adverse effects, commonly observed in recently performed clinical trials. Therefore, the cytotoxic agents paradoxically are still of great importance in anticancer drug development. Moreover, the diversity of chemical libraries of targeted agents is limited, while the source of cytotoxic substances, the nature, is still far away from reaching its limits. The implementation of new active natural compounds to cancer therapy may face many obstacles mainly due to www.sciencedirect.com

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the unknown mechanism of action, however, a high density screening systems capable of the identification of a wide range of target molecules, including those responsible for the toxic effects, may solve that obstacle. In this review, we try to present recent proteomic methodologies enabling the identification of mechanism of action of a test compound, often through the comparison of the induced proteomic profile with those contained in the databases. Furthermore, the ability of cancer to overcome harmful effects of drugs will push researchers to early implement the detection of molecular signatures related to drug resistance and, when possible, the host factors determining the retention of drug in the body. These issues were not extensively explored so far in proteomic profiling, however, there is necessity to implement for the benefit of a patient. Proteomic analysis provides a valuable set of data on a direct binding and expression changes suggesting the mode of action of small molecules.

Chemical proteomics detecting a direct binding of small molecules to target proteins Chemical proteomics is one of the most powerful methods for detecting interactions between a compound and target proteins. Traditionally, the affinity beads conjugated via suitable functional group with small molecules have been used for obtaining binding proteins. A protein bound to affinity beads is washed and then eluted by the solution containing a free compound. Protein identification is performed by N-terminal amino acid sequencing using Edman degradation method or by a peptide mass finger printing using mass spectrometry (MS). Within this decade, tremendous improvement in protein identification by MS allows rapid identification of large number of proteins even if present at low levels. Chemical proteomics may determine the activity profile of a compound that may lead to discovery of a new target. Additionally, the peripheral technologies, such as the immobilization [8] and linker design [9], have been improved too. The CB30865 compound was discovered during screen for thymidylate synthase inhibitors, however its high cytotoxicity indicated the presence of additional target [10]. Chemical proteomic analysis performed by Fleischer et al. identified the target as Nampt, an enzyme performing the first step of nicotinamide conversion to NAD [11]. In this case, the direct interaction with protein made possible the identification of the target, despite the fact that the other techniques failed. The lack of a suitable functional group is an obstacle in small molecules immobilization to the solid surface; its blocking may decrease the binding affinity. Therefore, Kanoh et al. developed a nonselective universal coupling method, which enables the attachment of a variety of small molecules to solid surface using a photo affinity www.sciencedirect.com

reaction [12,13]. They assumed that a compound may contain several coupling sites and depending on the protein target certain functional groups remain intact, thus enabling the assay. However, some tests may provide false-negative results when the functional group is masked in this procedure. Using this method, Kawatani et al. identified glyoxalase I to be a binding protein for methyl-gerfelin, a compound of fungal origin, and demonstrated that osteoclastogenesis was suppressed by its inhibition [14]. This technique is commonly used in our laboratory; it allows systematic exploration of the interaction of test compounds with almost any disease-related protein. For that purpose, thousands of small molecules extracted from chemical library at the RIKEN Natural Products Depository (NPDepo) were printed on a glass plate, to prepare chemical arrays, which are exposed to the desired proteins being tagged with DsRed, a fluorescent marker [15]. Recently, using this method, Miyazaki et al., found a triphenyl compound (TPh A) as the inhibitor of pirin, a protein associated with cancer malignancy [16]. They demonstrated that TPh A inhibited migration of melanoma cells through suppressing SNAI2 expression, regulator of various cancer cells mobility. Reversing the case, a complex biological pathway related to cancer cell migration, more importantly a metastasis, can be traced using a capability of a new bioprobe, such as TPh A. The above presented techniques are highly suitable for compound possessing good binding affinity, but not free of certain obstacles, such as disturbances caused by washing procedures, functional groups shadowed by solid phase, labeling procedures. To overcome that problem, a new platform has been developed; the entire detection system is based upon the surface plasmon resonance (SPR) imaging technique [17]. A combination of SPR and the photo-cross-linked chemical array led to the discovery of several compounds targeting the influenza A nucleoprotein [18]. Further improvement of the assay system was achieved by introducing the cleavable linker for photo-cross-linked small-molecule affinity matrix that permits the efficient detection of proteins covalently bound to the immobilized small molecule [19].

Cell-based phenotype profiling method for target analysis The exposure of the cells to chemical substances induces specific changes allowing cell-based phenotypic profiling. Contrary to chemical proteomics, it may also provide valuable information on the physiological state of a cell being under therapeutic stress. The targeting is not limited to proteins but may include many other macromolecules. The analysis of a target is facilitated by well done validation procedure performed for various well-known Current Opinion in Chemical Biology 2011, 15:57–65

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inhibitors, such as anticancer drug; this allows the construction of the phenotypes database to be used as a reference. The search of the database with the mean of an algorithm reveals the closest match of newly found phenotype to a reference compound. From pharmacological view point it is very valuable, however, not widely used due to the requirement of sophisticated instruments and high costs as well. Therefore, less complicated cellbased assays are widely used for analysis of drug effects. The differential sensitivity of the panel of cancer cell lines such as the NCI60 [20] and JFCR39 [21] to the compounds has been used to identify their molecular target(s) [22–24]. Cancer cells, either with upregulated or downregulated characteristic biological pathway induced by gene mutations, will differentially respond to target compound. In the case of yeast, target gene expression can be modulated experimentally due to accessibility of genome sequence, and of many libraries of deletion mutants or over expressing transformants [25,26]. Nishimura et al. identified the non-protein target and novel mechanism of action of theonellamides, a marine antifungal compound, by using chemical genomic profiling of S. pombe [27]. DNA micro-array became an important tool in the analysis of global gene expression analysis of the cells, providing an opportunity to monitor the cellular responses following exposure to a drug under physiological condition. Recently, Golub and co-workers developed a more robust system, the connectivity map, which uses gene expression signature for profiling in mammalian cells with a novel data analysis method [28,29]. The latest updating of the connectivity map containing more than 700 expression profiles representing more than 1000 compounds can be accessed at the web (http://www. broadinstitute.org/cmap/).

2D-DIGE profiling as a strategy drug target identification The development of two-dimensional electrophoresis (2DE) supported by powerful identification tools, introduced proteomics into the area of pharmacological drug evaluation [30–32]. In most instances, the spots representing individual proteins in cellular extracts are already identified and quantifiable by specific labeling. The drug exposure may cause a downregulation or upregulation of a protein, or may induce the appearance of a new protein spot when compared with untreated cells. Depending on the test compound the induced protein patterns differ to a great extent, and this feature is used for target identification. Proteome profiling of LAF389, a synthetic analogue of antitumor bengamides derived from marine sponges, revealed the changes in mobility on 2DE of a subset of proteins isolated from the treated cells. Detailed analysis of one of the proteins, 14-3-3-g, showed that LAF389 treatment resulted in the retention of the aminoterminal methionine, suggesting that LAF389 inhibited Current Opinion in Chemical Biology 2011, 15:57–65

methionine aminopeptidases (MetAp). In vitro assay confirmed that bengamide directly inhibited both MetAp1 and MetAp2. LAF389 demonstrated promising antitumor activity during preclinical studies, however, phase I clinical trial has been terminated due to unpredictable cardiovascular toxicity [33]. The 2DE has certain limitations, such as low resolutions and bias caused by membrane proteins. Furthermore, 2DE can only visualize the most abundant proteins, which may not play any role in drug activity. In recent years, a stable isotope labeling has been applied for protein quantification in MS-based proteomics, such as isotope-coded affinity tag (ICAT), isobaric tag for relative and absolute quantification (iTRAQ), and the application of a stable isotope labeled amino acids in cell culture (SILAC) [34]. The MS can identify a large number of proteins bypassing the gel-visualizing step, the peptide digestion with site-specific protease before quantification made protein detection easier. As a result, 2DE gel-based methods have largely been superseded by MS-based proteomics. However, protein profiling of cell lysate sometimes requires both, 2DE and MS, as complementary methods when one of them fails the identification [35]. An example of utilization of MS-based proteomics for comprehensive target profiling is a successful application of a mixed broad-specificity kinase inhibitor matrix (‘kinobeads’), which capture a large proportion of the proteins [36,37]. In combination with iTRAQ technology, a quantitative expression data of cell ‘kinome’ are obtained and such approach was used for the analysis of the BCR-ABL tyrosine kinase inhibitor, imatinib and other second-generation drugs. As the result dasatinib and bosutinib were found to be less selective than imatinib and nilotinib, and new targets for imatinib and nilotinib (the receptor tyrosine kinase DDR1 and quinone-oxidoreductase NQO2) were identified. Also, they predicted that dasatinib and bosutinib may have a potential to induce side effects related to immunosuppression. The 2DE based proteomic techniques were further improved by the introduction of a two-dimensional difference gel electrophoresis (2D-DIGE), enabling the measurement of the abundance of each protein spot between different gels with a high accuracy due to introduction of an internal standard [38]. Recently, we developed a proteomic profiling system for target analysis of compounds based on cancer cells proteome analysis by a 2D-DIGE (Figure 2) [39]. The expression data presented by 300 spots, reproducibly detected in all images of HeLa cells treated with 19 compounds, were successfully classified by cluster analysis according to their mechanism of action. Iejimalide, isolated as anticancer natural small molecule compound, has been reported to inhibit vacuolar type ATPase (V-ATPase), but its cellular www.sciencedirect.com

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Figure 2

A proteomic profiling system for drug target analysis using 2D-DIGE.

effects have not been elucidated in details. In cluster analysis, iejimalide A clustered in the same tree that included bafilomycin A1 and concanamycin A, wellknown V-ATPase inhibitors (Figure 3), suggesting that 2D-DIGE-based proteomic profiling is applicable for

target analysis of a compound as are other cell-based comparing methods. This method is characterized by a high reproducibility given the experiments are performed under strict condition. Therefore, the results are kept in the form of database and serve for comparison with the

Figure 3

Clustering of iejimalide A and well-known anticancer agents based on the data of proteomic analysis using 2D-DIGE. HeLa cells treated with a compound or with DMSO for 18 h were analyzed by 2D-DIGE and hierarchical cluster analysis was performed using the quantitative data of spots. Compounds were classified according to the mechanism of action such as V-ATPase inhibitors (bafilomycin A1 and concanamycin A), HSP90 inhibitors (geldanamycin and radicicol), and tubulin inhibitors (vinblastine and nocodazole). Iejimalide A clustered in the same tree that included VATPase inhibitors. This figure was modified based on the previously published data [39]. www.sciencedirect.com

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expression data of separated experiments. There is no need to identify the spots by using expensive and highly accurate MS system; the mechanism of action of a novel compound can be established with a high accuracy if it induces similar proteomic profile to that induced by wellcharacterized reference compound and the profile is contained in the database. Compared with gene expression profiling, which can simultaneously measure the expression of more than 20 000 genes, proteome analysis provides us only with the opportunity to trace at most 1000 proteins. Furthermore, it is relatively difficult to obtain reproducibility of protein detection because of instability, heterogeneity and wide range of abundance of proteins. Global proteomic expression profiling of the compounds has not been performed actively on a large scale. However, proteome analysis has an advantage in analyzing protein directly, and may include information on protein modification; profiling results would be expected to have different aspect and may be informative for target identification in some cases.

Figure 4

Compound

Metabolic Activation “Activated”

Cytoplasm Nucleus

Sensing

Nrf2

Keap1

Nrf2

Nrf2

Nrf2

ARE

Keap1 GSHs UGTs SULTs

Conjugation

Activated metabolite

Neutralized conjugate

Phase 2 enzymes

Phase 3 transporter

MRPs OATPs

Membrane transporter

Current Opinion in Chemical Biology

A schematic presentation of the role of Keap1/Nrf2 axis in living cells. Good guys sometimes turn our good job into disaster.

Defined molecular target and clarified mechanism of action as a condition for beneficial cancer chemotherapy The perfect matching of a highly expressed target with the exactly fitting drug would be an ideal case. One may observe such cases in the in vitro condition, however, not often in vivo. In most instances, cancer cells try to escape from harmful effects of a drug by employing several mechanisms, for example upregulation of the transporters, drug-damaged DNA repair, drug metabolizing enzymes, to name a few, or epigenetic apparatus, to switch suppressor genes off. Therefore, the a priori recognition of those effects may prevent applying an ineffective treatment or may help selection of the drug combination overcoming the above-mentioned problems. Future developments in the field of proteome studies should include the molecular mechanisms behind the defense system being built up by the treated cancer. Also host factors determine the effectiveness of anticancer drugs; particularly, the activity of drug metabolizing enzymes controlling the retention of drugs in the treated organism. Many anticancer drugs induce the oxidative stress in cancer cells due to generation of reactive oxygen species (ROS), which if not scavenged will cause a damage to subcellular fractions, particularly to DNA. To combat the ROS the cells employ the Keap1-Nrf2 defense axis [40]. Under physiological condition the nuclear factor erythroid-2-related factor 2 (Nrf2) forms a complex with Keap1, which rapidly dissociates in the presence of ROS, releasing Nrf2. Free Nrf2 translocates to the nucleus and binds the antioxidant response element (ARE) present in several genes controlling intracellular Current Opinion in Chemical Biology 2011, 15:57–65

redox balance (e.g., glutamate cysteine ligase, heme oxygenase-1), drug detoxification (e.g., glutathione Stransferase and NAD(P)H quinone oxidoreductase-1, UDP-glucuronosyltransferase), and transporters (multidrug resistant proteins) (Figure 4). The upregulation of Nrf2 activates drug detoxifying enzymes and excessively generated aggressive metabolic species are easily scavenged, thus preventing DNA damage and the initiation of carcinogenic events. As demonstrated by Ohnuma et al., falcarindiol, a diacetylene natural compound, prevented cytotoxicity of menadione against rat normal liver cells, that was accompanied by upregulation of catalase, glutathione S-transferase and NAD(P)H:quinone oxidoreductase [41]. Furthermore, falcarindiol formed an adduct with a cysteine SH function of Keap1 that led to the release of free Nfr2 ready for activation of ARE controlled genes, and, in consequence, to neutralization of toxic effects [42]. The impact of Nrf2 upregulation in cancer tissue, however, is rather negative, since the delivered active drug is subjected to extensive neutralization and only a tiny fraction is able to reach the targets, thus resulting in apparent chemoresistance [43]. Jiang et al., have shown that endometrial cancer cell lines expressing increased levels of Nrf2 were more resistant to cisplatin and paclitaxel than those with low levels [44]. The most convincing data on the role Nrf2 expression in the modulation chemotherapeutic outcome were recently published by Solis et al. [45]. They showed that increased Nrf2 expression and decreased expression of Keap1 were common in non-small cell lung cancers and were associated with worse recurrence-free survival in squamous cell www.sciencedirect.com

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carcinoma patients who had received adjuvant platinumbased chemotherapy. This double-edge sword phenomenon, high Nrf2 expression levels in healthy, and low in cancer tissues, may contribute to the development of attractive anticancer drugs, and proteomic analysis should be of great importance. The groundbreaking studies presented by Wu et al. [46], showed that ethacrynic acid and parthenolide were able to activate Nrf2 in normal peripheral blood mononuclear cells as a toxicity preventing measure, but were less potent in chronic lymphocytic leukemia (CLL) cells expressing high levels of ROS, thus providing selective cytotoxicity in this disease. The importance of Nrf2 expression in healthy tissues is recently reported by the group of Tohoku University [47]; it was revealed that Nrf2 / mice are more prone to metastatic dissemination of 3LL cancer. Thus, keeping at least status quo of Nrf2 in healthy tissues, while carefully decreasing in cancer is an important challenge in cancer therapy. Our group, applying proteomics, found certain measures counteracting cancer cells defense mechanism against ROS. Kawatani et al. [14] showed the inhibitory effect of methyl-gerfelin on glyoxalase 1 activity, the enzyme controlling the levels of reactive and cytotoxic 2-oxo-aldehydes [48]. This case illustrates how important is good tuning of proteomic profiling system in identification of the targets directly responsible for anticancer activity as well as those hits indicating significant off-target effects.

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Conclusion

10. Skelton LA, Ormerod MG, Titley JC, Jackman AL: Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action. Cytometry 1998, 33:56-66.

Recent anticancer drug development, focused on new molecular targets, requires the signature called ‘proof of concept’, usually a selective biomarker mimicking the activity of a drug. On the contrary, clinical studies, on classical and new drugs, provide a great deal of information on biomarkers of anticancer activity, its absence, or on toxic effects, too. It is reasonable to translate that information from patient’s level to a single cell level enabling researchers to perform a global evaluation of new compound within short time, getting a full spectrum of biological activity. Proteome profiling seems to be suitable approach when we are able to identify the spots of clinically relevant proteins.

Acknowledgements This study was supported partly by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Basic Science Research Project of RIKEN.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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