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Plant roots anchor the plant to the soil and absorb water and nutrients for growth. Understanding the molecular mechanisms regulating root development is essential for improving plant survival and agricultural productivity. Extensive molecular genetic studies have provided important information on crucial components for the root development control over the last few decades. However, it is becoming difficult to identify new regulatory components in root development due to the functional redundancy and lethality of genes involved in root development. In this study, we performed a chemical genetic screen to identify novel synthetic compounds that regulate root development in Arabidopsis seedlings. The screen yielded a root growth inhibitor designated as ‘rootin’, which inhibited Arabidopsis root development by modulating cell division and elongation, but did not significantly affect shoot development. Transcript analysis of phytohormone marker genes revealed that rootin preferentially altered the expression of auxin-regulated genes. Furthermore, rootin reduced the accumulation of PIN1, PIN3, and PIN7 proteins, and affected the auxin distribution in roots, which consequently may lead to the observed defects in root development. Our results suggest that rootin could be utilized to unravel the mechanisms underlying root development and to investigate dynamic changes in PIN-mediated auxin distribution.

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.

Chemical genetics utilizes small molecules to perturb biological processes. Unlike conventional genetics methods, which involve the alteration of genetic information mostly with lasting effects, chemical genetics allows temporary and reversible alterations of biological processes. Furthermore, it enables the alteration of biological processes in a dose-dependent manner, providing an advantage over conventional genetics. In the present chapter, the general procedures of forward chemical genetic screening are described. Forward chemical genetic screening can be performed in three steps. The first step involves the identification of small molecules that induce phenotypic or physiological changes in a biological system from a chemical library. In the second step, cellular targets that interact with the isolated chemical, which are mostly proteins, are identified. Although several methods can be applied in the second step, the most common one is affinity pull-down assay using a target protein that binds to the isolated compound. However, affinity pull-down of a target protein is a formidable barrier in forward chemical genetics. We introduced a tagged chemical library approach that significantly facilitates the identification of target proteins. The third step consists of the validation of the target protein, which should include the assessment of target specificity. This step is critical because small molecules often show pleiotropic effects due to low specificity. The specificity test may include a competition assay using cold competitors and a genetic study using mutants or transgenic lines modified for the cellular target.

The development of a relatively simple, reliant and cost-effective animal test will greatly facilitate drug development. In this study, our goal was the establishment of a rapid, simple, sensitive and reproducible zebrafish xenograft model for anti-cancer drug screening. We optimized the conditions for the cancer cell xenograft in terms of injected cell numbers, incubation temperature and time. A range of human carcinoma cell types were stained with a fluorescent dye prior to injection into the fish larvae. Subsequent cancer cell dissemination was observed under fluorescent microscopy. Differences in injected cell numbers were reflected in the rate of dissemination from the xenograft site. Paclitaxel, known as a microtubule stabilizer, dose-dependently inhibited cancer cell dissemination in our zebrafish xenograft model. An anti-migratory drug, LY294002 (phosphatidylinositol 3-kinase inhibitor) also decreased the cancer cell dissemination. Chemical modifications to increase cancer drug pharmacokinetics, such as increased solubility (17-DMAG compared to geldanamycin) could also be assessed in our xenograft model. In addition to testing our new model using known anti-cancer drugs, we carried out further validation by screening a tagged triazine library. Two novel anti-cancer drug candidates were discovered. Therefore, our zebrafish xenograft model provides a vertebrate animal system for the rapid screening and pre-clinical testing of novel anti-cancer agents, prior to the requirement for testing in mammals. Our model system should greatly facilitate drug development for cancer therapy because of its speed, simplicity and reproducibility.

With the successful results of the Human Genome Project, we are now faced with the problem of handling numerous gene targets whose functions remain to be studied; this challenge is being undertaken by the field known as functional genomics. Chemical genetics is an emerging new paradigm to attack this challenging problem, of which there are two approaches: forward and reverse. Although reverse chemical genetics uses a “cause-to-effect” approach, forward chemical genetics uses an “effect-to-cause” approach. As opposed to conventional genetics where gene knock-outs or overexpression, forward chemical genetics uses a small-molecule library to produce a novel phenotype that eventually is employed in the elucidation of gene function. Compared with conventional genetics, chemical genetics holds several unique advantages. A successful forward genetic study provides not only knowledge about a novel gene's function, but also it provides a small molecule on/off switch that can regulate biologic processes. These small molecules will be extremely useful biologic probes as well as potential new drug candidates. Three main components make up forward chemical genetics: 1) chemical toolbox generation, 2) phenotypic screening, and 3) target identification. Although all three components require additional refinement, target identification poses the greatest challenge. Herein, the general concepts of chemical genetics, with a focus on the forward approach, and the technical overviews for each component are described.

Chemical genetics is a powerful method which utilizes small molecule regulators to reveal the molecular basis of diverse biological processes. However, the current chemical genetic approach sometimes meets a serious bottleneck during the process of target identification. One faces difficulty in conjugating the active compound to an affinity matrix without losing or reducing its activity that leads to laborious structure-activity relationship (SAR) studies. To facilitate this process, we have developed a tagged triazine library containing a built-in linker that provides a straightforward transition from phenotypic screening to target identification. A strategy for constructing a tagged library and applications with a streamlined target identification and subsequent mechanistic study are discussed in this Account.

With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.

Completion of the Human Genome Project has provided the scientific community with a wealth of information to further our knowledge of the gene function. Currently, an enormous effort is being applied to studying the relationship between the mapped genes and human disease. In the past, this enormous endeavor has been mainly left to classical geneticists. However, since the gene products, proteins, are dynamic and multifunctional, it has become clear that the study of genes alone may not be sufficient to see the whole picture. This has stimulated the field of chemical genetics at the interface between chemistry and biology. Chemical genetics, inspired by classical genetics, makes use of small molecules as mutation-inducing agents to study protein function. One approach of chemical genetics, forward chemical genetics, is gaining recognition as a powerful strategy. Forward chemical genetics is a three-step process (Figure 1). First, a collection of small molecules is designed and synthesized. The small molecules are then screened in a model organism for the ability to perturb/change a biological process. Once an interesting phenotype is found, the small molecule responsible for it is used to isolate the target protein(s). Finally, upon identification of the target protein, its identity can be linked with the previously observed phenotype, thus gaining better understanding of that particular biological process. In addition, biologically active small molecule can provide important structural information for further development of novel therapeutic agents. Even though the concept of using small molecules to study cellular processes is not new, in practice, the forward chemical genetics approach is still not systematic. Consequently, systematic strategies at every step of the forward chemical genetic process would greatly accelerate the study of protein function and development of novel therapeutic agents. In this review, we will discuss the strategies and research tools that have been developed to accelerate and systematize the field of forward chemical genetics.

An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.

Investigation of the genetic functions in complex biological systems is a challenging step in recent year. Hence, several valuable and interesting research projects have been developed with novel ideas to find out the unknown functions of genes or proteins. To validate the applicability of their novel ideas, various approaches are built up. To date, the most promising and commonly used approach for discovering the target proteins from biological system using small molecule is well known a forward chemical genetics which is considered to be more convenient than the classical genetics. Although, the forward chemical genetics consists of the three basic components, the target identification is the most challenging step to chemical biology researchers. Hence, the diverse target identification methods have been developed and adopted to disclose the small molecule bound protein. Herein, in this review, we briefly described the first two parts chemical toolbox and screening, and then the target identifications in forward chemical genetics are thoroughly described along with the illustrative real example case study. In the tabular form, the different biological active small molecules which are the successful examples of target identifications are accounted in this research review.

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.

Summary As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long-term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged-triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC50 values in the range of 10 μM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with l-3,4-dihydroxyphenylalanine (l-DOPA) for binding to the DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 μM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation.

Chemical genetics is a powerful method which utilizes small molecule regulators to reveal the molecular basis of diverse biological processes. However, the current chemical genetic approach sometimes meets a serious bottleneck during the process of target identification. One faces difficulty in conjugating the active compound to an affinity matrix without losing or reducing its activity that leads to laborious structure-activity relationship (SAR) studies. To facilitate this process, we have developed a tagged triazine library containing a built-in linker that provides a straightforward transition from phenotypic screening to target identification. A strategy for constructing a tagged library and applications with a streamlined target identification and subsequent mechanistic study are discussed in this Account.

As the human genome sequence is nearly deciphered, it is important to turn the attention to the physiological functions of the genes. Thus, the study of the gene products, the proteins, is the next big challenge. The proteins, however, are not the final gene products in many cases. It has been shown that carbohydrates participate in post-translational modifications and in many other functional regulations, hence the study of the glycome, the entire collection of carbohydrates is essential in order to determine the functions of all genes, and will greatly enhance the field of chemical genetics. Known biological function / targets of carbohydrates and combinatorial synthesis & structural analysis of natural / non-natural carbohydrates are surveyed in this review. Methods to search for new biological targets that include carbohydrate mimetics and carbohydrate scaffolds along with chip technology, are also presented.