Target identification of bioactive compounds

To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets.     

Computational analysis of ligand relationships within target families

Computational tools for the large-scale analysis and prediction of ligand-target interactions and the identification of small molecules having different selectivity profiles within target protein families complement research in chemical genetics and chemogenomics. For computational analysis and design, such tasks require a departure from the traditional focus on single targets, hit identification, and lead optimization. Recently, studies have been reported that profile compounds in silico against arrays of targets or systematically map ligand-target space. In order to identify small molecular probes that are suitable for chemical genetics applications, molecular diversity needs to be viewed in a way that partly differs from principles guiding conventional library design.     

Identification and validation of protein targets of bioactive small molecules

Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.     

Iodine scanning of a phenazine inhibitor of vacuolar sorting

Affinity reagents are often used to address the target identification problem in chemical genetics. The design of such reagents so that the linker does not occlude interactions with protein targets is an ongoing challenge. This work describes a systematic approach to synthesize derivatives of a bioactive that should avoid interference with binding to targets and be readily converted to affinity reagents.     

Expanding applications of chemical genetics in signal transduction

Chemical genetics represents an expanding collection of techniques applied to a variety of signaling processes. These techniques use a combination of chemical reporters and protein engineering to identify targets of a signaling enzyme in a global and non-directed manner without resorting to hypothesis-driven candidate approaches. In the last year, chemical genetics has been applied to a variety of kinases, revealing a much broader spectrum of substrates than had been appreciated. Here, we discuss recent developments in chemical genetics, including insights from our own proteomic screen for substrates of the kinase ERK2. These studies have revealed that many kinases have overlapping substrate specificity, and they often target several proteins in any particular downstream pathway. It remains to be determined whether this configuration exists to provide redundant control, or whether each target contributes a fraction of the total regulatory effect. From a general perspective, chemical genetics is applicable in principle to a broad range of posttranslational modifications (PTMs), most notably methylation and acetylation, although many challenges remain in implementing this approach. Recent developments in chemical reporters and protein engineering suggest that chemical genetics will soon be a powerful tool for mapping signal transduction through these and other PTMs.     

Expanding applications of chemical genetics in signal transduction

Chemical genetics represents an expanding collection of techniques applied to a variety of signaling processes. These techniques use a combination of chemical reporters and protein engineering to identify targets of a signaling enzyme in a global and non-directed manner without resorting to hypothesis-driven candidate approaches. In the last year, chemical genetics has been applied to a variety of kinases, revealing a much broader spectrum of substrates than had been appreciated. Here, we discuss recent developments in chemical genetics, including insights from our own proteomic screen for substrates of the kinase ERK2. These studies have revealed that many kinases have overlapping substrate specificity, and they often target several proteins in any particular downstream pathway. It remains to be determined whether this configuration exists to provide redundant control, or whether each target contributes a fraction of the total regulatory effect. From a general perspective, chemical genetics is applicable in principle to a broad range of posttranslational modifications (PTMs), most notably methylation and acetylation, although many challenges remain in implementing this approach. Recent developments in chemical reporters and protein engineering suggest that chemical genetics will soon be a powerful tool for mapping signal transduction through these and other PTMs.     

Combining functional genomics and chemical biology to identify targets of bioactive compounds

Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification.     

Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography

Chemical proteomics is an effective approach to focused proteomics, having the potential to find specific interactors in moderate-scale comprehensive analysis. Unlike chemical genetics, chemical proteomics directly and comprehensively identifies proteins that bind specifically to candidate compounds by means of affinity chromatographic purification using the immobilized candidate, combined with mass spectrometric identification of interacting proteins. This is an effective approach for discovering unknown protein functions, identifying the molecular mechanisms of drug action, and obtaining information for optimization of lead compounds. However, immobilized-small molecule affinity chromatography always suffers from the problem of non-specific binders. Although several approaches were reported to reduce non-specific binding proteins, these are mainly focused on the use of low-binding-affinity beads or insertion of a spacer between the bead and the compound. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of true interactors from many non-specific binders, including carrier proteins, such as serum albumin, and are expected to be valuable for drug discovery.     

Chemoproteomic approaches to drug target identification and drug profiling

Chemoproteomics represents a new research discipline at the interface of medicinal chemistry, biochemistry, and cell biology focused on studying the molecular mechanisms of action of drugs and other bioactive small molecules. Research strategies frequently combine phenotypic screening with subsequent target identification, and aim at a proteome-wide characterization of drug-induced changes in cellular protein expression and post-translational modifications. In recent years quantitative mass spectrometry has taken center stage in many of these approaches. This review describes experimental strategies in current chemical proteomics research, discusses recent examples of successful applications, and highlights areas in drug discovery where chemical proteomics-based assays using native endogenous proteins are expected to have substantial impact.     

Forward chemical genetics: progress and obstacles on the path to a new pharmacopoeia

Forward chemical genetics is a new method to systematize the discovery and use of small molecules as tools for basic biological research. This approach requires three basic components: a library of compounds; an assay, in which the library is screened for a cellular or organismal phenotype; and a method to trace an active compound to its biological target. Bioactive compounds have traditionally been isolated from natural product extracts, although 'diversity-oriented synthesis' and commercial compound collections are gaining in prominence. New techniques, such as image-based screening and the cytoblot method, have increased the throughput of phenotypic assays. Strategies are also being developed to streamline target identification using molecular biological approaches.     

Tools and methodologies capable of isolating and identifying a target molecule for a bioactive compound

Elucidating the mechanism of action of bioactive compounds, such as commonly used pharmaceutical drugs and biologically active natural products, in the cells and the living body is important in drug discovery research. To this end, isolation and identification of target protein(s) for the bioactive compound are essential in understanding its function fully. And, development of reliable tools and methodologies capable of addressing efficiently identification and characterization of the target proteins based on the bioactive compounds accelerates drug discovery research. Affinity-based isolation and identification of target molecules for the bioactive compounds is a classic, but still powerful approach. This paper introduces recent progress on affinity chromatography system, focusing on development of practical affinity matrices and useful affinity-based methodologies on target identification. Beneficial affinity chromatography systems with using practical tools and useful methodologies facilitate chemical biology and drug discovery research.     

Profiling the Protein Targets of Unmodified Bio‐Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry

Identifying the target proteins of bioactive small molecules is a key step in understanding mode‐of‐action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand‐modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity‐based protein profiling. Target proteins are then enriched by performing a pull‐down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read‐out). This review highlights recent proteomic approaches utilizing data‐dependent analysis and data‐independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio‐active molecules can be further validated.     

大肠杆菌热休克或冷休克启动子调控的基因表达载体

基因重组技术已经成为获得各种酶和生物活性蛋白的主要手段。虽然很多基因已在大肠杆菌中得到高效表达,但是当人们认定某种蛋白对科学研究或生产应用极为重要时,却常常因为其基因表达水平很低或产生包涵体而感到束手无策。表达载体pHsh和pEXC通过激活热休克或冷休克转录调控机制提高分子伴侣的表达水平,从而降低目标蛋白的细胞毒性并减少包涵体形成。应用于生物合成、分子修饰或生物降解的高温酶可以通过pHsh系统表达获得高产,而科研和诊疗所需要的来源于动植物和常温微生物的基因可以通过pEXC系统获得高效表达。这些新载体的发展为重组蛋白的小规模制备和大规模生产提供了新策略和有效途径。     

Human monogenic disorders - a source of novel drug targets

The decrease in new drug applications and approvals over the past several years results from an underlying crisis in drug target identification and validation. Model organisms are being used to address this problem, in combination with novel approaches such as the International HapMap Project. What has been underappreciated is that discovery of new drug targets can also be revived by traditional Mendelian genetics. A large fraction of the human gene repertoire remains phenotypically uncharacterized, and is likely to encode many unanticipated and novel phenotypes that will be of interest to pharmaceutical and biotechnological drug developers.     

Tagged library approach facilitates forward chemical genetics

Forward chemical genetics has been highlighted as a new method for the study of various biological pathways using exogenous ligands. However, limited success in the field has demonstrated that, in many cases, it is not feasible to determine the protein targets of small-molecule probes. Identifying protein targets is an integral part of forward chemical genetics and is also the most challenging. Over the past decade, several biochemical and genetic methods have been developed to facilitate target identification processes. Even so, one of the major difficulties is that these methods require the chemical modification of active compounds, with a significant amount of structure-activity relationship (SAR) study to ensure that the small-molecule tags do not compromise bioactivity. In this article, we will highlight a new strategy for small molecule libraries that have built-in linkers in order to avoid this well-known problem and demonstrate their successful use in forward chemical genetics.     

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.     

Xenopus: an ideal system for chemical genetics

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.     

Neuroprotective strategies targeting apoptotic and necrotic cell death for stroke

It has been a major challenge to develop effective therapeutics for stroke, a leading cause of death and serious debilitation. Intensive research in the past 15 years have implicated many regulators and the related mechanisms by which neuronal cell death is regulated. It is now clear that even a brief ischemic stroke may trigger complex cellular events that lead to both apoptotic and necrotic neuronal cell death in a progressive manner. Although efforts at developing specific chemical inhibitors for validated targets have been successful for in vitro enzymatic assays, the development of some of such inhibitors into human therapy has been often hindered by their in vivo bioavailability profile. Considerations for the ability to chemically target a cellular mechanism in manner compatible with disease targets in vivo might be emphasized early in the development process by putting a priority on identifying key targets that can be effectively targeted chemically. Thorough interrogation of cellular pathways by saturation chemical genetics may provide a novel strategy to identify multiple key molecular entities that can be targeted chemically in order to select a target suitable for the treatment of intended human diseases such as stroke.