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Biotransformation of Chinese Herbs and Their Ingredients
WORLD SCIENCE AND TECHNOLOGY MODERNIZATION OF TRADITIONAL CHINESE MEDICINE AND MATERIA MEDICA
Volume 12, Issue 4, August 2010 Online English edition of the Chinese language journal Cite this article as: Mode Tradit Chin Med Mater Med, 2010, 12(4): 502–510
REVIEW
Biotransformation of Chinese Herbs and Their Ingredients Yue Rongcai1, Shan Lei1, Yan Shikai1, 2, Zhao Jing1, 3, Zhang Weidong1, 2 * 1
School of Pharmacy, Second Military Medical University, Shanghai 200433, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China 3 Department of Mathematics, Logistical Engineering University, Chongqing 400016, China 2
Abstract: A key task for the traditional Chinese medicine (TCM) modernization is to interpret the scientific connotation of TCM through the development of new theories and modern techniques. Chemical proteomics is a powerful mass spectrometry-based affinity chromatography approach for identifying proteome-wide small molecule–protein interactions. Chemical proteomics is used to study and detect proteomes with specific chemical molecules that interact with target proteins. Here, we review the main techniques of chemical proteomics and their applications in the research of active ingredients, target proteins and synergistic mechanisms and in drug discoveries of TCM. Chemical proteomics study provides new ideas for the TCM modernization, which may help to reveal the essential principles of TCM at the molecular level, and thus facilitates the new drug discovery of TCM and the inheritance and development of TCM theories. Key Words: Chemical biology, chemical proteomics, functional proteomics, Chinese Material Medica
As a precious wealth of Chinese culture, traditional Chinese medicine (TCM) has developed a unique theoretical system and functional application, and there is no doubt in its effectiveness and practicality for more than two thousand years of clinical application. But, the chemical constituents are so complex, and the chemical ingredients improve the efficacy of synergies in an appropriate concentration, which makes the study of its mechanisms very difficult and impede the international promotion of TCM. Therefore, utilizing modern technology and exploring new methods and models may help to clarify the multitarget, multilink, multilevel mechanism of TCM and may reveal its essential principles at the molecular level. This is not only one of the key issues of modernization of TCM but also mandatory for internationalization of TCM. In 1990s, Schreiber, at Harvard University, developed such a discipline called Chemical biology, which provides new ideas for the TCM modernization. Arising from a real (and for that reason synthetic) chemical approach toward unraveling biological problems and systems, chemical biology was initially employed for high-throughput screening of small molecules (synthetic and natural products) using biological (cellular) assays and thus believed to discover and optimize the important physiological and pathological regulations of small molecule drugs. The latest development in chemical proteomics offers opportunities for the modernization of TCM. Chemical proteomics makes use of small molecules that can
be used to covalently modify a set of related enzymes and subsequently allows their purification and identification as valid drug targets. Furthermore, such method enables rapid biochemical analysis and small molecule screening of targets, thereby, accelerating the process of target validation and drug discovery. Chemical proteomics provides new ideas and methods to study the modernization of TCM and is becoming an effective method in the research of active ingredients, target proteins and synergistic mechanisms and in drug discoveries of TCM. Here, we describe the basic theories of chemical proteomics and its application in TCM.
1 Basic theory and technology of chemical proteomics The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better way to treat human disease. Chemical proteomics, a multidisciplinary science, can be used to distill the flood of new information. As an important technical means of chemical biology, chemical proteomics makes use of specific molecules that can interfere with target proteins to interact and detect the proteome, which may systematically reveal the specific protein function and their interaction with chemical molecules at the molecular level. Unlike the identity-based or abundance-based proteomics, chemical
Received date: 16 July 2010 * Corresponding author. Tel: +86-21-81871244; E-mail: [email protected] Foundation item: Supported by the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (2009ZX09311-001, 2009ZX09103-319) Copyright © 2010, World Science and Technology Press. Published by Elsevier BV. All rights reserved.
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proteomics is used to study the proteome from the functional point of view and, therefore, is considered as a new promising function-based proteomics [1]. With the further development of science and technology, application of chemical proteomics in the field of TCM is gradually apparent, and its related molecular docking and network biology provide new ideas and methods for exploring the biological complex system of TCM. 1.1
Experimental techniques of chemical proteomics
Chemical proteomics is a powerful mass spectrometrybased affinity chromatography approach [2] for identifying proteome-wide small molecule–protein interactions. It originates in two different ways [3]: (1.1.1) activity-based probe profiling (ABPP), which focuses on the enzyme activity of a particular protein family and (1.1.2) a compound-centric chemical proteomics (CCCP), which focuses on characterizing the molecular mechanism of action of an individual bioactive small molecule (Fig. 1). ABPP and CCCP serve different purposes. ABPP detects members of a class of enzymes that are active under certain conditions. This method can lead to the identification of new proteins or it can be applied to determine the selectivity profile of drugs targeting an enzyme family via pretreatment of the lysate with the drug of interest and subsequent labeling and identification of the remaining enzymes using appropriate reactive probes. In contrast to ABPP, CCCP enables one to characterize the target profile of a bioactive compound. This does not provide direct information about the activation state of identified proteins. However, as the more unbiased approach, CCCP allows the identification of binders of unexpected biochemical classes [4, 5] and can be used to find entirely novel targets. 1.1.1
Activity-based probe profiling
The Cravatt group at Scripps Research Institute, USA, [1, 6-8] developed a chemical proteomics approach—ABPP, which makes use of chemical probes that can modify a set of related enzymes to study protein structure and function. Such probes have been termed activity-based probes (ABPs) to reflect their need for an active enzyme to facilitate covalent modification (the reactive group attaches covalently to the enzyme of interest, and then, the tag allows for identification and purification; therefore, using the fluorescent ABP tag or biotin can “fishing” out target enzymes one by one from the protein group [9]). Other chemical probes have been designed by target noncatalytic residues on proteins and enzymes. These ABPs require highly selective, tight binding to targets to be useful probes for distinct protein or enzyme families. Regardless of their mechanism of action, chemical probes are finding increasing use in the field of proteomics and have great potential to aid in the process of target identification and validation. It combines the advantages of chemistry and biology, and
ABPP
CCCP Drug
Warhead Linker Tag Protein extracts (cells, tissues)
Probe
Af finity chromatography
Proteolytic digest
Beads
Drug matrix
Af finity chromatography
Gel electrophoresis
"Shotgun"
LC-MS/MS
Bioinf ormatic analysis
Fig. 1 Comparison of ABPP and CCCP
it studies the complex interactions between small molecules and biological macromolecules at the molecular level so as to reveal the key regulatory mechanism of small molecules under physiological or pathological conditions. ABPP is an important method for finding drug target protein and its function. At present, the method has been successfully used for studying the functions of target enzymes, such as serine protease [10, 11], hydrosulfide protease [12], cysteine protease [13, 14] and ubiquitin protease [15-18]. Some new targets of small molecules in the disease process have been found through the research [19]. Recently, the Scripps Research Institute described a robust and versatile proteomic platform that enables direct visualization of the topography and magnitude of proteolytic events on a global scale. They used this method to generate a proteome-wide map of proteolytic events induced by the intrinsic apoptotic pathway [20]. In addition, Li X et al [21] designed, synthesized, characterized, and applied a trimodular ABP to photoaf¿nity labeling of the Ȗ-aminobutyric acid type B GABA(B) receptors transiently expressed in Chinese hamster ovary cells. The probe exhibits high speci¿city to photoaf¿nity labeling, which makes the probe valuable for studying the localization and function of GABA (B) receptors in living cells. As a tool for detection, probe can specifically interact with targets and can be detected by special methods. Chemical probe is applied to detect changes in chemical molecules to obtain the information of macromolecules, which is the tool used in chemical proteomics research. In their most basic form, chemical probes consist of three specific functional elements: a reactive group for covalent attachment to the enzyme; a linker region that can modulate reactivity and specificity of the reactive group; and a tag for identification and purification of modified enzymes [22]. Reactive groups must be both reactive toward a specific residue on a protein and
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inert toward other reactive species within cell or cell extract. Generally, the reactive group of most successful chemical probes is designed by the chemistries of covalent. Different inhibitor mechanisms of various enzyme families serve the role as selective inhibiting effect of chemical probe. Such small molecule inhibitors rely on mechanistic differences of individual enzyme classes as a means for selective targeting. So far, there are many successful examples to design the chemical probes based on the three-dimensional structure of target proteins [23-25]. The links region usually accomplished using a long chain alkyl, polyethylene glycol or peptide to connect reactive group and the tag. Its primary function is to provide enough space between the response group and the tag to prevent steric hindrance that could block access of the reactive group or accessibility of the tag for the purpose of purification [19, 26]. The most commonly used tags are biotin, fluorescent and radioactive tags to facilitate the identification and separation of probe-modified proteins. 1.1.2
Compound-centric chemical proteomics
CCCP includes drug affinity chromatography that is a “classical” method for the purification of proteins, but nowadays, this method is performed in combination with modern highresolution mass spectrometry (MS) and statistics or bioinformatics for subsequent identification of binding proteins. Chemical proteomics study starts with immobilizing a bioactive compound on a matrix in a way that does not interfere with its activity. The small molecule ligand is modified by introducing an appropriate functional group (referred to as a linker), through which it can be immobilized by getting attached with the affinity matrix—a step that is important for later phase separation. There are various commercially available activated resins that allow for the attachment of specific chemical groups (for example, sulfhydryl, amino, hydroxyl or carboxyl groups). After the immobilized small molecule ligand being incubated with protein extracts, any unbound proteins can subsequently be removed in a series of washing steps, and specifically bound proteins are separated by solid-phase elution using buffer conditions that disrupt the interaction between the target protein and the immobilized small molecule ligand. Finally, the protein is typically identified by mass spectrometry. The results are searched against an appropriate protein database (for example, Swiss-Prot or the US National Center for Biotechnology Information) with a search engine (for example, Mascot or Sequest) before being submitted to a more in-depth bioinformatic analysis. Predicting or inferring the protein targets needs to be validated in the biological method, such as surface plasmon resonance (SPR) [27, 28] and isothermal titration calorimetry (ITC) [29]. The former especially applies for molecule interactions, which can maintain proteins in a natural state to provide some functional information, such as real-time distribution of target proteins in the cells, combination with dynamic analysis and changes of concentration. The latter is a
biophysical technique that can be used to detect the combination of small molecules and biological macromolecules thermodynamic parameters (n, K, ǻH and ǻS). There are many other methods for validating the interactions between compounds and proteins, such as cocrystallization of proteins with compounds to determine the binding sites [26, 29]; sitedirected mutagenesis of protein binding site sequence (and then verify whether proteins bind); and GST-pulldown experiments. 1.2 Calculation of chemical proteomics —molecular docking After the completion of the human genome project in 2001, the focus of research has shifted to establishing the functions of the numerous gene products, i.e., proteins; on the other hand, with the rapid development of computer technology and an unprecedented increase of computing power, structure-based drug design and screening can be feasible. In the field of molecular modeling, docking is a method that predicts the preferred orientation of one molecule to other when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions. Molecular docking can reveal small molecule interaction between target proteins; hence, it can not only make up for deficiencies in animal pharmacological experimental methods but also reduce the human and material inputs. As the calculation of chemical proteomics, molecular docking can make use of active small molecules (natural products or synthetic compounds) as a probe to search protein structure database to discover their binding protein and use it as hints to find the target drugs. The core of computer-aided drug design is the simulation of drug molecules interacting with target proteins [30]. Molecular docking can be believed as a problem of “lock and key,” where one is interested in finding the correct relative orientation of the “key” which will open up the “lock” (where on the surface of the lock is the key hole, which direction to turn the key after it is inserted, etc.). Here, the protein is considered a “lock,” and the ligand is considered a “key.” Molecular docking may be defined as an optimization problem, which would describe the “best-fit” orientation of a ligand that binds to a particular protein of interest. During the process, the ligand and the protein adjust their conformation to achieve an overall “best-fit,” and this kind of conformational adjustments resulting in the overall binding is referred to as “induced-fit” [31]. The focus of molecular docking is to computationally simulate the molecular recognition process. The aim of molecular docking is to achieve an optimized conformation for both the protein and the ligand and a relative orientation between protein and ligand such that the free energy of the overall system is minimized. Al-
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though it is only from the perspective of computer simulation, it has strong theoretical and scientific basis in screening small molecule to clarify the interaction with the target protein and to optimize molecular structure of important guiding significance. Ruo Cheng et al [32] used molecular modeling to predict that histone deacetylase (HDAC) may be the target of sphingosine-1-phosphate (S1P), and predicted the key combining models and residues between them, then accurately forecast their ability to affinity equal to existing HDAC inhibitor SAHA. These theoretical predictions provide a crucial hint for the role of S1P, and this was confirmed by other experiments. The method of molecular docking and experimental verification provides a reliable way to find target proteins of active ingredients of TCM. 1.3
Network biology
Network biology provides a quantifiable description of the networks that characterize various biological systems, which was proposed in 2004 [33]. Advances in network biology indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biology and pathology of disease. Network biology provides a new perspective to explore the complex components of TCM. In essence, TCM takes effect through active ingredients binding target protein in vivo, affects biological processes and then plays a therapeutic effect. Therefore, for investigating the mechanism of small molecules, one must understand biological processes and their interaction network. At the protein level, interactions between proteins form a protein complex that participates in a biological process. Such relationships can be represented by protein–protein interaction network. Protein–protein interaction is the foundation of all life activities, and in vivo studies of protein interactions formed in complex network are the important means for complete awareness toward their physiological functions and understanding their molecular mechanisms. Now, with the development of protein–protein interaction research techniques (including yeast two-hybrid, surface plasmon resonance, affinity chromatography, and immunoprecipitation), the information in protein interaction database can construct target protein network for a particular disease or a drug and further enable to research the mechanism of TCM. For example, Yue QX et al [28] constructed the possible network associated with ganoderic acid D (GAD) target-related proteins and discussed the possible contribution of these proteins to the cytotoxicity of GAD. Drawing target network method is especially suitable for the study of TCM; this is because TCM characterizes multiple targets and plays effective role in a combined result of many active ingredients regulating on the extensive body of biological molecular networks. Therefore, from a biological network point of view, we can get access to a more comprehensive understanding
of the mechanism.
2 Application of chemical proteomics in the modernization of TCM Chemical compositions of TCM are very complex, with the multitarget, multilink, multilevel features. The majority of various diseases and drug targets are at the protein (enzyme, receptor and signal transduction protein) levels, while the subjects of chemical proteomics are all the proteins in the cells. Therefore, chemical proteomics is applied to search for drug targets. By comparing protein expression profiles before and after administration to find different proteins as the main objective, chemical proteomics brings opportunities in the modernization of TCM. The significance of chemical proteomics in the modernization of TCM is not only to discover new drugs but also, more importantly, to reduce the blindness in the detection of targets and to speed up the detection rate. Finally, TCM can be adopted into clinical trials and introduced in the market [34]. Chemical proteomics provides new methods and technologies in its research at the level of organs, cells and molecules. The chemical proteomics can be used for protein expression and identification in the cells and regulation of all aspects of proteomics research. Specifically, it can filter the effective ingredients of TCM, verify possible targets, screen small molecule ligand and the binding protein to describe the new protein function, further explain the mechanism of TCM and develop new drugs (Fig. 2). Chemical proteomics is gradually highlighting in the modernization of TCM, which shows great potential in the research of active ingredients, target proteins, synergistic mechanisms and drugs of TCM. 2.1
Screening active ingredients
Screening active ingredients is an important step in the drug discovery of TCM. We consider the TCM system as a natural combinatorial chemical library. Compared with a synthetic combinatorial chemical library, it has more abundant diversities in chemical structures with potent pharmacological activities and it is easy and inexpensive to make. But it is very difficult to isolate and purify the active compounds through TCM. The traditional methods for separation and identification of active compounds from herbs are timeconsuming and expensive. Classical approaches of a onecompound, one-assay variety are often untenable because of time and resource constraints. Chemical proteomics addresses a novel approach to screen and identify the active compounds from TCM in single-step frontal immunoaffinity chromatography with mass spectrometry detection. A binding assay must be performed against a target at some point and this usually means assaying either a large number of beads or a mixture of compounds in solution. Most of the approaches to combining molecular recognition with mass spectrometry have incorporated a capture and release method-
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Fig. 2 Application of chemical proteomics in
the modernization of TCM ology, where compound mixtures are applied to immobilized receptors, and the high-affinity ligands stripped with a denaturing solution and presented to the mass spectrometer [35]. Luo H used immobilized polyclone antibodies of a compound coupled with mass spectrometry to screen active compounds from an extract of Phyllanthus urinaria L. The screening results of antihepatitis C virus showed that five compounds in the column could be retained compared with others, and further experiments confirmed that these compounds have a better inhibitory activity to hepatitis C virus [36]. In addition, flexible docking, depending on its fast and accurate calculation and simulation, enables prediction of the affinity between small molecules and proteins. A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. As the calculation of chemical proteomics, molecular docking may be effective for screening the ingredients from TCM complex system and then utilizing selective chemical probes to identify and verify the target protein in cell and animal models. Docking may be applied to hit identification and lead optimization. Based on the crystallographic structure of human topoisomerase I (Topo I)-DNA covalent complex, a general model for the ternary drug-DNA-Topo I complex for camptothecin (CPT) derivatives has been developed using flexible docking techniques and thus elucidated the mode of action of CPT compounds interacting with Topo I and DNA from the atomic level for further designing of novel potent CPT derivatives [37]. 2.2
Finding target proteins
Target discovery plays an important role in clinical application and in drug discovery of TCM. Chemical proteomics selects small molecules that inhibit protein as drug targets, which shift the focus of researchers from identifying new targets to finding targets that are more easily recognized, thereby increases the chance of success. So far, there are many examples of chemical proteomics approaches to identify and validate target proteins.
In a recent study, a serial affinity chromatography approach was introduced, which identified the protein targets of FK 506 (FKBP12), benzenesulfonamide (carbonic anhydrase II) and methotrexate (dihydrofolate reductase) [38]. Recently, a biotinylated form of diazonamide A (an inhibitor of spindle assembly in mammalian cells) was immobilized on avidinagarose beads, whereas a comparison variant that used an inactive analogue with a similar structure was carried out in parallel. Bound proteins were eluted from the matrices and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then, the resultant protein patterns (obtained after the gels had been stained) were compared, resulting in the identification of ornithine-įaminotransferase (OAT), which may play an unanticipated role in tumorigenesis [39]. A recent discovery is the identification of the purine derivative Qs11 as a synergist of Wnt signaling pathway; this synergist acts through inhibition of the guanosine triphosphatase activating protein of adenosine diphosphateribosylation factor 1(ARFGAP1) and is the only inhibitor of this protein till date [40]. A recent report described the investigation of the mechanism of action of the antitumor macrolide pladienolide, which was originally identified in a cell-based reporter gene assay. Kotake et al [41] successfully confirmed the splicing factor SF3b complex as the target of pladienolide, which was further validated by RNAi and colocalization experiments. Moreover, they demonstrated competition of the radioactively labeled compound with unmodified pladienolide upon immunoprecipitation of the SF3b complex, as well as inhibition of the splicing activity. The exact target protein, however, remained elusive. A related concurrent study by Kaida et al [42] also identified the SF3b complex as a target. The SF3b complex is the target of both FR901464, the natural product, and Spliceostatin A, its methylated analog. Successful validation through competition of the original active compound versus the acetylated inactive analog, along with splicing assays and RNAi experiments, confirmed the interaction. Chemical proteomics also has broad prospects in TCM. In 2009, Dal Piaz et al [27] isolated twenty-four new sesterterpenes from the aerial parts of SalVia dominica. The evaluation of the biological activity of sesterterpenes by means of a chemical proteomics approach was realized. Obtained results showed that 18 of 24 sesterterpene lactones interact with tubulin, a tyrosine ligase (TTL), an enzyme involved in the tyrosination cycle of the C-terminal of tubulin, and inhibit TTL activity in cancer cells. Thus, chemical proteomics provides great potential for target discovery of active ingredients of TCM. 2.3
Explaining synergistic mechanism
TCM often plays a role in multiple targets, although the effects of each target are not very strong. After accessing the
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information on possible protein targets, these low affinity, multitarget characteristics can be explained by comparing the active ingredient between different target proteins and their biological function at the molecular level. Specifically, the synergistic mechanisms are divided into two types: one is different active ingredients bind the same protein targets, which have a synergistic effect in combination with the protein, and the other is different active ingredients bind different protein targets, which have a synergistic effect on the same signaling pathway. Here, network biology may provide a more comprehensive understanding of the synergistic mechanism of TCM. In trying to address the possible bene¿cial effects of TCM formula with current biomedical approaches, Chen Zhu and Chen Saijuan group use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model [43]. They showed that the dissection of the mode of action of clinically well-established TCM formula, such as RIF, should be possible by combined application of both analytical and synthetic research approaches at the molecular, cellular, and organismal levels. This study may be considered a useful pilot trial in exploring the value of traditional formula on a larger scale and in helping to bridge western and Chinese medicine in the era of systems biology. To learn more about the network level and synthetic mechanism, we searched OMIM database to identify APL treatment gene based on above literature and found that six genes encoding proteins, as well as proteins regulated by the RIF, can be mapped to the human protein interaction network and KEGG pathway; and we constructed the protein interaction network and protein-signaling relationship network. These results provide a theoretical basis to further reveal the RIF in the treatment of APL (Fig. 3).
2.4
Discovering new Drugs
TCM provides an extensive foundation for implementing a strategically focused pharmacological research program that aimed at the development of new drugs. Chemical proteomics can reveal the role of TCM and target processes with different protein expression profile analyses, thus reveal the complex system of molecular regulatory mechanism. In addition, it is also able to analyze single chemical composition extended to a wide range of chemical composition, analyze single drugs and formula to study the mechanism of TCM comparatively, and understand multitarget, multilink, multilevel mechanism that is different from western medicine and protein expression in different organs and organizations; thus, it can interpret the scientific connotation of TCM in detail. This is a new way for developing new drugs of TCM. According to the characteristics of TCM and its chemical composition, combined with ABPP/CCCP, molecular docking and related network biology, we explore the idea of drug discovery of TCM (Fig. 4) and conduct a preliminary study under the guidance of chemical proteomics.
3
Conclusion
An interdisciplinary approach to TCM may provide a platform for the discovery of novel therapeutics composed of multiple chemical compounds. The introduction of chemical biology in the field of TCM has begun to show its feasibility and rationality. As a product of the latest developments in chemical biology and a new generation of functional proteomics, chemical proteomics has a good prospect. It provides new ideas for the TCM modernization, which may help to reveal the essential principles at the molecular level, and thus facilitates the new drug discovery of TCM and the inheritance and development of TCM
Fig. 3 The protein interaction network (I) and protein-signaling relationship network (II) of RIF in the treatment of APL Notes: Box nodes: RIF-regulated proteins; circular nodes: other proteins; triangle nodes: pathways; gray nodes: APL disease gene encoding proteins; white node (NCOA6, SMAD2): neither RIF-regulated proteins nor APL disease gene encoding proteins, but a bridge to connect these proteins.
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Ef fective TCM
Separation and identification of compounds
Active ingredients
Chemical Proteomics
Target discovery
Disease-related network and proteins interaction network construction
Synergistic mechanism interpretation
Drug discovery of TCM
Fig. 4 Idea of drug discovery of TCM
theories.
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Volume 12, Issue 4, August 2010 Online English edition of the Chinese language journal Cite this article as: Mode Tradit Chin Med Mater Med, 2010, 12(4): 502–510
REVIEW
Biotransformation of Chinese Herbs and Their Ingredients Yue Rongcai1, Shan Lei1, Yan Shikai1, 2, Zhao Jing1, 3, Zhang Weidong1, 2 * 1
School of Pharmacy, Second Military Medical University, Shanghai 200433, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China 3 Department of Mathematics, Logistical Engineering University, Chongqing 400016, China 2
Abstract: A key task for the traditional Chinese medicine (TCM) modernization is to interpret the scientific connotation of TCM through the development of new theories and modern techniques. Chemical proteomics is a powerful mass spectrometry-based affinity chromatography approach for identifying proteome-wide small molecule–protein interactions. Chemical proteomics is used to study and detect proteomes with specific chemical molecules that interact with target proteins. Here, we review the main techniques of chemical proteomics and their applications in the research of active ingredients, target proteins and synergistic mechanisms and in drug discoveries of TCM. Chemical proteomics study provides new ideas for the TCM modernization, which may help to reveal the essential principles of TCM at the molecular level, and thus facilitates the new drug discovery of TCM and the inheritance and development of TCM theories. Key Words: Chemical biology, chemical proteomics, functional proteomics, Chinese Material Medica
As a precious wealth of Chinese culture, traditional Chinese medicine (TCM) has developed a unique theoretical system and functional application, and there is no doubt in its effectiveness and practicality for more than two thousand years of clinical application. But, the chemical constituents are so complex, and the chemical ingredients improve the efficacy of synergies in an appropriate concentration, which makes the study of its mechanisms very difficult and impede the international promotion of TCM. Therefore, utilizing modern technology and exploring new methods and models may help to clarify the multitarget, multilink, multilevel mechanism of TCM and may reveal its essential principles at the molecular level. This is not only one of the key issues of modernization of TCM but also mandatory for internationalization of TCM. In 1990s, Schreiber, at Harvard University, developed such a discipline called Chemical biology, which provides new ideas for the TCM modernization. Arising from a real (and for that reason synthetic) chemical approach toward unraveling biological problems and systems, chemical biology was initially employed for high-throughput screening of small molecules (synthetic and natural products) using biological (cellular) assays and thus believed to discover and optimize the important physiological and pathological regulations of small molecule drugs. The latest development in chemical proteomics offers opportunities for the modernization of TCM. Chemical proteomics makes use of small molecules that can
be used to covalently modify a set of related enzymes and subsequently allows their purification and identification as valid drug targets. Furthermore, such method enables rapid biochemical analysis and small molecule screening of targets, thereby, accelerating the process of target validation and drug discovery. Chemical proteomics provides new ideas and methods to study the modernization of TCM and is becoming an effective method in the research of active ingredients, target proteins and synergistic mechanisms and in drug discoveries of TCM. Here, we describe the basic theories of chemical proteomics and its application in TCM.
1 Basic theory and technology of chemical proteomics The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better way to treat human disease. Chemical proteomics, a multidisciplinary science, can be used to distill the flood of new information. As an important technical means of chemical biology, chemical proteomics makes use of specific molecules that can interfere with target proteins to interact and detect the proteome, which may systematically reveal the specific protein function and their interaction with chemical molecules at the molecular level. Unlike the identity-based or abundance-based proteomics, chemical
Received date: 16 July 2010 * Corresponding author. Tel: +86-21-81871244; E-mail: [email protected] Foundation item: Supported by the Special Program for New Drug Innovation of the Ministry of Science and Technology, China (2009ZX09311-001, 2009ZX09103-319) Copyright © 2010, World Science and Technology Press. Published by Elsevier BV. All rights reserved.
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proteomics is used to study the proteome from the functional point of view and, therefore, is considered as a new promising function-based proteomics [1]. With the further development of science and technology, application of chemical proteomics in the field of TCM is gradually apparent, and its related molecular docking and network biology provide new ideas and methods for exploring the biological complex system of TCM. 1.1
Experimental techniques of chemical proteomics
Chemical proteomics is a powerful mass spectrometrybased affinity chromatography approach [2] for identifying proteome-wide small molecule–protein interactions. It originates in two different ways [3]: (1.1.1) activity-based probe profiling (ABPP), which focuses on the enzyme activity of a particular protein family and (1.1.2) a compound-centric chemical proteomics (CCCP), which focuses on characterizing the molecular mechanism of action of an individual bioactive small molecule (Fig. 1). ABPP and CCCP serve different purposes. ABPP detects members of a class of enzymes that are active under certain conditions. This method can lead to the identification of new proteins or it can be applied to determine the selectivity profile of drugs targeting an enzyme family via pretreatment of the lysate with the drug of interest and subsequent labeling and identification of the remaining enzymes using appropriate reactive probes. In contrast to ABPP, CCCP enables one to characterize the target profile of a bioactive compound. This does not provide direct information about the activation state of identified proteins. However, as the more unbiased approach, CCCP allows the identification of binders of unexpected biochemical classes [4, 5] and can be used to find entirely novel targets. 1.1.1
Activity-based probe profiling
The Cravatt group at Scripps Research Institute, USA, [1, 6-8] developed a chemical proteomics approach—ABPP, which makes use of chemical probes that can modify a set of related enzymes to study protein structure and function. Such probes have been termed activity-based probes (ABPs) to reflect their need for an active enzyme to facilitate covalent modification (the reactive group attaches covalently to the enzyme of interest, and then, the tag allows for identification and purification; therefore, using the fluorescent ABP tag or biotin can “fishing” out target enzymes one by one from the protein group [9]). Other chemical probes have been designed by target noncatalytic residues on proteins and enzymes. These ABPs require highly selective, tight binding to targets to be useful probes for distinct protein or enzyme families. Regardless of their mechanism of action, chemical probes are finding increasing use in the field of proteomics and have great potential to aid in the process of target identification and validation. It combines the advantages of chemistry and biology, and
ABPP
CCCP Drug
Warhead Linker Tag Protein extracts (cells, tissues)
Probe
Af finity chromatography
Proteolytic digest
Beads
Drug matrix
Af finity chromatography
Gel electrophoresis
"Shotgun"
LC-MS/MS
Bioinf ormatic analysis
Fig. 1 Comparison of ABPP and CCCP
it studies the complex interactions between small molecules and biological macromolecules at the molecular level so as to reveal the key regulatory mechanism of small molecules under physiological or pathological conditions. ABPP is an important method for finding drug target protein and its function. At present, the method has been successfully used for studying the functions of target enzymes, such as serine protease [10, 11], hydrosulfide protease [12], cysteine protease [13, 14] and ubiquitin protease [15-18]. Some new targets of small molecules in the disease process have been found through the research [19]. Recently, the Scripps Research Institute described a robust and versatile proteomic platform that enables direct visualization of the topography and magnitude of proteolytic events on a global scale. They used this method to generate a proteome-wide map of proteolytic events induced by the intrinsic apoptotic pathway [20]. In addition, Li X et al [21] designed, synthesized, characterized, and applied a trimodular ABP to photoaf¿nity labeling of the Ȗ-aminobutyric acid type B GABA(B) receptors transiently expressed in Chinese hamster ovary cells. The probe exhibits high speci¿city to photoaf¿nity labeling, which makes the probe valuable for studying the localization and function of GABA (B) receptors in living cells. As a tool for detection, probe can specifically interact with targets and can be detected by special methods. Chemical probe is applied to detect changes in chemical molecules to obtain the information of macromolecules, which is the tool used in chemical proteomics research. In their most basic form, chemical probes consist of three specific functional elements: a reactive group for covalent attachment to the enzyme; a linker region that can modulate reactivity and specificity of the reactive group; and a tag for identification and purification of modified enzymes [22]. Reactive groups must be both reactive toward a specific residue on a protein and
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inert toward other reactive species within cell or cell extract. Generally, the reactive group of most successful chemical probes is designed by the chemistries of covalent. Different inhibitor mechanisms of various enzyme families serve the role as selective inhibiting effect of chemical probe. Such small molecule inhibitors rely on mechanistic differences of individual enzyme classes as a means for selective targeting. So far, there are many successful examples to design the chemical probes based on the three-dimensional structure of target proteins [23-25]. The links region usually accomplished using a long chain alkyl, polyethylene glycol or peptide to connect reactive group and the tag. Its primary function is to provide enough space between the response group and the tag to prevent steric hindrance that could block access of the reactive group or accessibility of the tag for the purpose of purification [19, 26]. The most commonly used tags are biotin, fluorescent and radioactive tags to facilitate the identification and separation of probe-modified proteins. 1.1.2
Compound-centric chemical proteomics
CCCP includes drug affinity chromatography that is a “classical” method for the purification of proteins, but nowadays, this method is performed in combination with modern highresolution mass spectrometry (MS) and statistics or bioinformatics for subsequent identification of binding proteins. Chemical proteomics study starts with immobilizing a bioactive compound on a matrix in a way that does not interfere with its activity. The small molecule ligand is modified by introducing an appropriate functional group (referred to as a linker), through which it can be immobilized by getting attached with the affinity matrix—a step that is important for later phase separation. There are various commercially available activated resins that allow for the attachment of specific chemical groups (for example, sulfhydryl, amino, hydroxyl or carboxyl groups). After the immobilized small molecule ligand being incubated with protein extracts, any unbound proteins can subsequently be removed in a series of washing steps, and specifically bound proteins are separated by solid-phase elution using buffer conditions that disrupt the interaction between the target protein and the immobilized small molecule ligand. Finally, the protein is typically identified by mass spectrometry. The results are searched against an appropriate protein database (for example, Swiss-Prot or the US National Center for Biotechnology Information) with a search engine (for example, Mascot or Sequest) before being submitted to a more in-depth bioinformatic analysis. Predicting or inferring the protein targets needs to be validated in the biological method, such as surface plasmon resonance (SPR) [27, 28] and isothermal titration calorimetry (ITC) [29]. The former especially applies for molecule interactions, which can maintain proteins in a natural state to provide some functional information, such as real-time distribution of target proteins in the cells, combination with dynamic analysis and changes of concentration. The latter is a
biophysical technique that can be used to detect the combination of small molecules and biological macromolecules thermodynamic parameters (n, K, ǻH and ǻS). There are many other methods for validating the interactions between compounds and proteins, such as cocrystallization of proteins with compounds to determine the binding sites [26, 29]; sitedirected mutagenesis of protein binding site sequence (and then verify whether proteins bind); and GST-pulldown experiments. 1.2 Calculation of chemical proteomics —molecular docking After the completion of the human genome project in 2001, the focus of research has shifted to establishing the functions of the numerous gene products, i.e., proteins; on the other hand, with the rapid development of computer technology and an unprecedented increase of computing power, structure-based drug design and screening can be feasible. In the field of molecular modeling, docking is a method that predicts the preferred orientation of one molecule to other when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using, for example, scoring functions. Molecular docking can reveal small molecule interaction between target proteins; hence, it can not only make up for deficiencies in animal pharmacological experimental methods but also reduce the human and material inputs. As the calculation of chemical proteomics, molecular docking can make use of active small molecules (natural products or synthetic compounds) as a probe to search protein structure database to discover their binding protein and use it as hints to find the target drugs. The core of computer-aided drug design is the simulation of drug molecules interacting with target proteins [30]. Molecular docking can be believed as a problem of “lock and key,” where one is interested in finding the correct relative orientation of the “key” which will open up the “lock” (where on the surface of the lock is the key hole, which direction to turn the key after it is inserted, etc.). Here, the protein is considered a “lock,” and the ligand is considered a “key.” Molecular docking may be defined as an optimization problem, which would describe the “best-fit” orientation of a ligand that binds to a particular protein of interest. During the process, the ligand and the protein adjust their conformation to achieve an overall “best-fit,” and this kind of conformational adjustments resulting in the overall binding is referred to as “induced-fit” [31]. The focus of molecular docking is to computationally simulate the molecular recognition process. The aim of molecular docking is to achieve an optimized conformation for both the protein and the ligand and a relative orientation between protein and ligand such that the free energy of the overall system is minimized. Al-
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though it is only from the perspective of computer simulation, it has strong theoretical and scientific basis in screening small molecule to clarify the interaction with the target protein and to optimize molecular structure of important guiding significance. Ruo Cheng et al [32] used molecular modeling to predict that histone deacetylase (HDAC) may be the target of sphingosine-1-phosphate (S1P), and predicted the key combining models and residues between them, then accurately forecast their ability to affinity equal to existing HDAC inhibitor SAHA. These theoretical predictions provide a crucial hint for the role of S1P, and this was confirmed by other experiments. The method of molecular docking and experimental verification provides a reliable way to find target proteins of active ingredients of TCM. 1.3
Network biology
Network biology provides a quantifiable description of the networks that characterize various biological systems, which was proposed in 2004 [33]. Advances in network biology indicate that cellular networks are governed by universal laws and offer a new conceptual framework that could potentially revolutionize our view of biology and pathology of disease. Network biology provides a new perspective to explore the complex components of TCM. In essence, TCM takes effect through active ingredients binding target protein in vivo, affects biological processes and then plays a therapeutic effect. Therefore, for investigating the mechanism of small molecules, one must understand biological processes and their interaction network. At the protein level, interactions between proteins form a protein complex that participates in a biological process. Such relationships can be represented by protein–protein interaction network. Protein–protein interaction is the foundation of all life activities, and in vivo studies of protein interactions formed in complex network are the important means for complete awareness toward their physiological functions and understanding their molecular mechanisms. Now, with the development of protein–protein interaction research techniques (including yeast two-hybrid, surface plasmon resonance, affinity chromatography, and immunoprecipitation), the information in protein interaction database can construct target protein network for a particular disease or a drug and further enable to research the mechanism of TCM. For example, Yue QX et al [28] constructed the possible network associated with ganoderic acid D (GAD) target-related proteins and discussed the possible contribution of these proteins to the cytotoxicity of GAD. Drawing target network method is especially suitable for the study of TCM; this is because TCM characterizes multiple targets and plays effective role in a combined result of many active ingredients regulating on the extensive body of biological molecular networks. Therefore, from a biological network point of view, we can get access to a more comprehensive understanding
of the mechanism.
2 Application of chemical proteomics in the modernization of TCM Chemical compositions of TCM are very complex, with the multitarget, multilink, multilevel features. The majority of various diseases and drug targets are at the protein (enzyme, receptor and signal transduction protein) levels, while the subjects of chemical proteomics are all the proteins in the cells. Therefore, chemical proteomics is applied to search for drug targets. By comparing protein expression profiles before and after administration to find different proteins as the main objective, chemical proteomics brings opportunities in the modernization of TCM. The significance of chemical proteomics in the modernization of TCM is not only to discover new drugs but also, more importantly, to reduce the blindness in the detection of targets and to speed up the detection rate. Finally, TCM can be adopted into clinical trials and introduced in the market [34]. Chemical proteomics provides new methods and technologies in its research at the level of organs, cells and molecules. The chemical proteomics can be used for protein expression and identification in the cells and regulation of all aspects of proteomics research. Specifically, it can filter the effective ingredients of TCM, verify possible targets, screen small molecule ligand and the binding protein to describe the new protein function, further explain the mechanism of TCM and develop new drugs (Fig. 2). Chemical proteomics is gradually highlighting in the modernization of TCM, which shows great potential in the research of active ingredients, target proteins, synergistic mechanisms and drugs of TCM. 2.1
Screening active ingredients
Screening active ingredients is an important step in the drug discovery of TCM. We consider the TCM system as a natural combinatorial chemical library. Compared with a synthetic combinatorial chemical library, it has more abundant diversities in chemical structures with potent pharmacological activities and it is easy and inexpensive to make. But it is very difficult to isolate and purify the active compounds through TCM. The traditional methods for separation and identification of active compounds from herbs are timeconsuming and expensive. Classical approaches of a onecompound, one-assay variety are often untenable because of time and resource constraints. Chemical proteomics addresses a novel approach to screen and identify the active compounds from TCM in single-step frontal immunoaffinity chromatography with mass spectrometry detection. A binding assay must be performed against a target at some point and this usually means assaying either a large number of beads or a mixture of compounds in solution. Most of the approaches to combining molecular recognition with mass spectrometry have incorporated a capture and release method-
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Fig. 2 Application of chemical proteomics in
the modernization of TCM ology, where compound mixtures are applied to immobilized receptors, and the high-affinity ligands stripped with a denaturing solution and presented to the mass spectrometer [35]. Luo H used immobilized polyclone antibodies of a compound coupled with mass spectrometry to screen active compounds from an extract of Phyllanthus urinaria L. The screening results of antihepatitis C virus showed that five compounds in the column could be retained compared with others, and further experiments confirmed that these compounds have a better inhibitory activity to hepatitis C virus [36]. In addition, flexible docking, depending on its fast and accurate calculation and simulation, enables prediction of the affinity between small molecules and proteins. A binding interaction between a small molecule ligand and an enzyme protein may result in activation or inhibition of the enzyme. As the calculation of chemical proteomics, molecular docking may be effective for screening the ingredients from TCM complex system and then utilizing selective chemical probes to identify and verify the target protein in cell and animal models. Docking may be applied to hit identification and lead optimization. Based on the crystallographic structure of human topoisomerase I (Topo I)-DNA covalent complex, a general model for the ternary drug-DNA-Topo I complex for camptothecin (CPT) derivatives has been developed using flexible docking techniques and thus elucidated the mode of action of CPT compounds interacting with Topo I and DNA from the atomic level for further designing of novel potent CPT derivatives [37]. 2.2
Finding target proteins
Target discovery plays an important role in clinical application and in drug discovery of TCM. Chemical proteomics selects small molecules that inhibit protein as drug targets, which shift the focus of researchers from identifying new targets to finding targets that are more easily recognized, thereby increases the chance of success. So far, there are many examples of chemical proteomics approaches to identify and validate target proteins.
In a recent study, a serial affinity chromatography approach was introduced, which identified the protein targets of FK 506 (FKBP12), benzenesulfonamide (carbonic anhydrase II) and methotrexate (dihydrofolate reductase) [38]. Recently, a biotinylated form of diazonamide A (an inhibitor of spindle assembly in mammalian cells) was immobilized on avidinagarose beads, whereas a comparison variant that used an inactive analogue with a similar structure was carried out in parallel. Bound proteins were eluted from the matrices and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then, the resultant protein patterns (obtained after the gels had been stained) were compared, resulting in the identification of ornithine-įaminotransferase (OAT), which may play an unanticipated role in tumorigenesis [39]. A recent discovery is the identification of the purine derivative Qs11 as a synergist of Wnt signaling pathway; this synergist acts through inhibition of the guanosine triphosphatase activating protein of adenosine diphosphateribosylation factor 1(ARFGAP1) and is the only inhibitor of this protein till date [40]. A recent report described the investigation of the mechanism of action of the antitumor macrolide pladienolide, which was originally identified in a cell-based reporter gene assay. Kotake et al [41] successfully confirmed the splicing factor SF3b complex as the target of pladienolide, which was further validated by RNAi and colocalization experiments. Moreover, they demonstrated competition of the radioactively labeled compound with unmodified pladienolide upon immunoprecipitation of the SF3b complex, as well as inhibition of the splicing activity. The exact target protein, however, remained elusive. A related concurrent study by Kaida et al [42] also identified the SF3b complex as a target. The SF3b complex is the target of both FR901464, the natural product, and Spliceostatin A, its methylated analog. Successful validation through competition of the original active compound versus the acetylated inactive analog, along with splicing assays and RNAi experiments, confirmed the interaction. Chemical proteomics also has broad prospects in TCM. In 2009, Dal Piaz et al [27] isolated twenty-four new sesterterpenes from the aerial parts of SalVia dominica. The evaluation of the biological activity of sesterterpenes by means of a chemical proteomics approach was realized. Obtained results showed that 18 of 24 sesterterpene lactones interact with tubulin, a tyrosine ligase (TTL), an enzyme involved in the tyrosination cycle of the C-terminal of tubulin, and inhibit TTL activity in cancer cells. Thus, chemical proteomics provides great potential for target discovery of active ingredients of TCM. 2.3
Explaining synergistic mechanism
TCM often plays a role in multiple targets, although the effects of each target are not very strong. After accessing the
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information on possible protein targets, these low affinity, multitarget characteristics can be explained by comparing the active ingredient between different target proteins and their biological function at the molecular level. Specifically, the synergistic mechanisms are divided into two types: one is different active ingredients bind the same protein targets, which have a synergistic effect in combination with the protein, and the other is different active ingredients bind different protein targets, which have a synergistic effect on the same signaling pathway. Here, network biology may provide a more comprehensive understanding of the synergistic mechanism of TCM. In trying to address the possible bene¿cial effects of TCM formula with current biomedical approaches, Chen Zhu and Chen Saijuan group use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model [43]. They showed that the dissection of the mode of action of clinically well-established TCM formula, such as RIF, should be possible by combined application of both analytical and synthetic research approaches at the molecular, cellular, and organismal levels. This study may be considered a useful pilot trial in exploring the value of traditional formula on a larger scale and in helping to bridge western and Chinese medicine in the era of systems biology. To learn more about the network level and synthetic mechanism, we searched OMIM database to identify APL treatment gene based on above literature and found that six genes encoding proteins, as well as proteins regulated by the RIF, can be mapped to the human protein interaction network and KEGG pathway; and we constructed the protein interaction network and protein-signaling relationship network. These results provide a theoretical basis to further reveal the RIF in the treatment of APL (Fig. 3).
2.4
Discovering new Drugs
TCM provides an extensive foundation for implementing a strategically focused pharmacological research program that aimed at the development of new drugs. Chemical proteomics can reveal the role of TCM and target processes with different protein expression profile analyses, thus reveal the complex system of molecular regulatory mechanism. In addition, it is also able to analyze single chemical composition extended to a wide range of chemical composition, analyze single drugs and formula to study the mechanism of TCM comparatively, and understand multitarget, multilink, multilevel mechanism that is different from western medicine and protein expression in different organs and organizations; thus, it can interpret the scientific connotation of TCM in detail. This is a new way for developing new drugs of TCM. According to the characteristics of TCM and its chemical composition, combined with ABPP/CCCP, molecular docking and related network biology, we explore the idea of drug discovery of TCM (Fig. 4) and conduct a preliminary study under the guidance of chemical proteomics.
3
Conclusion
An interdisciplinary approach to TCM may provide a platform for the discovery of novel therapeutics composed of multiple chemical compounds. The introduction of chemical biology in the field of TCM has begun to show its feasibility and rationality. As a product of the latest developments in chemical biology and a new generation of functional proteomics, chemical proteomics has a good prospect. It provides new ideas for the TCM modernization, which may help to reveal the essential principles at the molecular level, and thus facilitates the new drug discovery of TCM and the inheritance and development of TCM
Fig. 3 The protein interaction network (I) and protein-signaling relationship network (II) of RIF in the treatment of APL Notes: Box nodes: RIF-regulated proteins; circular nodes: other proteins; triangle nodes: pathways; gray nodes: APL disease gene encoding proteins; white node (NCOA6, SMAD2): neither RIF-regulated proteins nor APL disease gene encoding proteins, but a bridge to connect these proteins.
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Ef fective TCM
Separation and identification of compounds
Active ingredients
Chemical Proteomics
Target discovery
Disease-related network and proteins interaction network construction
Synergistic mechanism interpretation
Drug discovery of TCM
Fig. 4 Idea of drug discovery of TCM
theories.
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