As the development of multidisciplinary science proceeded over the past decades, the combination of structural biology and chemical genetics screening has been constantly combined to identify potent inhibitors of proteins of interest and meanwhile fully illustrate their modes of actions. Two types of proteins, hormone receptors & metabolic enzymes have been taken as popular targets of chemical screening. The underlying reason is rather obvious and clear, receptors often possess a ligand-binding pocket that enables synthetic chemical molecules to occupy and thereby work as competitive inhibitors (Vaidya et al., 2019). Apart from that, certain enzymes catalyzing critical metabolic pathways could possibly be inhibited by either chemical mimics of their original substrate which cannot be catalyzed by the protein, or certain compound that are able to bind to the other sites of the enzyme and trigger conformational changes of the protein, thereby preventing the normal ligand-protein binding activity (Sun et al., 2017; He et al., 2012).

Figure 1. Chemical genetics approach to develop ABA receptor agonists

(Vaidya et al., 2019)

Being proven effective and productive by multiple successful examples, the aforementioned approaches target a broad hormone signaling pathway or certain critical metabolic process, thus, once the small molecule effector is applied, it would trigger a wide range of biological changes and affect the growth and development exponentially. However, under certain circumstances when researchers only intend to affect a specific biological process or even the expression of a certain set of genes, these approaches failed to provide an applicable solution.

In this study, we focused on another type of protein, the transcription factors that often act as the final executors of certain signaling pathways by either promoting or inhibiting the expression of certain sets of genes. Since changing the transcription of a set of genes could reflect faintly on the phenotypes of organisms, it is often not feasible to conduct Phenotype-Directed Screening over certain transcription factors. To settle this problem, we developed a fluorescence-based screening system in E. coli that could be used in both Target-directed virtual screening and high-throughput experimental screening since the bacterium could be easily cultured within hours and the green fluorescence could be conveniently detected. Here, we utilized a reporter plasmid containing eGFP driven by certain promoters whose transcription could be inhibited by the binding of an inhibitory transcription factor, which resides on another plasmid and its intracellular level could be manipulated via the concentration of IPTG. Through this reporter system, when the inhibitory TF is induced, it would repress the expression of eGFP. If an effective molecule is added into the solution, it could inhibit the function of the transcription factor and thereby induce the expression of eGFP, whose fluorescence could be detected via multiple strategies. This approach could also be applied to the stimulatory transcription factors following the same route.

Figure 2. The screening system without effective molecule

Figure 3. The screening system with effective molecule

In all, using a two plasmid fluorescence-based reporter system, we are able to perform easy and convenient high-throughput chemical screening for inhibitors of transcription factors. And the corresponding small effector molecules could be utilized to exert precise control over the expression of certain sets of genes.

1. Vaidya, Aditya S, Helander, Jonathan D. M, Peterson, Francis C, Elzinga, Dezi, Dejonghe, Wim, Kaundal, Amita, . . . Cutler, Sean R. (2019). Dynamic control of plant water use using designed ABA receptor agonists. Science (American Association for the Advancement of Science), 366(6464), Eaaw8848.
2. Sun, Xiangzhong, Li, Yaxin, He, Wenrong, Ji, Chenggong, Xia, Peixue, Wang, Yichuan, . . . Guo, Hongwei. (2017). Pyrazinamide and derivatives block ethylene biosynthesis by inhibiting ACC oxidase. Nature Communications, 8(1), 15758.
3. He, Wenrong, Brumos, Javier, Li, Hongjiang, Ji, Yusi, Ke, Meng, Gong, Xinqi, . . . Guo, Hongwei. (2011). A Small-Molecule Screen Identifies l-Kynurenine as a Competitive Inhibitor of TAA1/TAR Activity in Ethylene-Directed Auxin Biosynthesis and Root Growth in Arabidopsis. The Plant Cell, 23(11), 3944-3960.