MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells. Lin28 protein binds to let-7 miRNA precursors to inhibit their maturation. Herein, we developed a FRET-based, high-throughput screening system to identify small-molecule inhibitors of the Lin28-let-7 interaction. We employed unnatural amino acid mutagenesis and bioorthogonal chemistry for the site-specific fluorescent labeling of Lin28, which ensures the robustness and reliability of the FRET-based protein-miRNA binding assay. Using this direct binding assay, we identified an inhibitor of the oncogenic Lin28-let-7 interaction. The inhibitor enhanced the production of let-7 miRNAs in Lin28-expressing cancer cells and reduced the level of let-7 target oncogene products.

Protein–RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein–RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity‐based protein–RNA binding assays by testing multiple environment‐sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let‐7 microRNA and the important immunomodulatory Roquin–Tnf CDE interaction as representative targets. We adapted this assay to high‐throughput screening for the identification of pyrazolyl thiazolidinedione‐type molecules as potent small‐molecule inhibitors of protein–microRNA interactions. We clearly showed the structure–activity relationships of this new class of Lin28–let‐7 interaction inhibitors, and confirmed that cellular mature let‐7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione‐type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein–RNA interactions.

Abnormal function of RNA-binding proteins can lead to dysregulation of RNA function, causing a variety of disease states. Thus, developing small-molecule modulators of protein−RNA interactions is one of the key challenges in chemical biology. Herein, we performed a high-throughput screening of chemical libraries using a Forster resonance energy transfer-based Lin28−let-7 interaction assay to identify a potent small-molecule inhibitor of the protein− microRNA interaction, as it is an important target implicated in stem cell-like phenotypes in cancer cells. The new inhibitor KCB3602 selectively restored cellular let-7 microRNA levels, decreased the expression of a panel of oncogenes responsible for cancer stem cell maintenance, and showed potential anticancer activities. We expect that our Lin28−let-7 interaction inhibitor will provide a good starting point for pharmacological eradication of cancer stem cells.

Peroxisome proliferator‐activated receptor gamma (PPARγ) has been a major therapeutic target for the treatment of type 2 diabetes. However, the use of PPARγ‐targeting drugs such as rosiglitazone and pioglitazone has significantly declined due to adverse effects caused by their classical transcriptional agonism. Meanwhile, blocking the obesity‐induced phosphorylation of PPARγ at Ser273 by Cdk5 has been proposed as the key to developing insulin‐sensitizing effects of PPARγ‐targeting drugs. In this study, we rationally designed and synthesized selective PPARγ phosphorylation inhibitor through a crystal structure‐based approach. During this process, we observed a distinct degradation pattern of the anilinic cyanoacrylamide moiety via the spontaneous retro‐aldol reaction. Thus, we developed a novel reversible covalent inhibitor of PPARγ phosphorylation, SB1495, containing aliphatic cyano‐acrylamide, through systematic structural modification, in silico docking studies, time‐dependent monitoring of stability in aqueous media, and in vitro kinase assay. We also demonstrated its inhibitory activity on PPARγ phosphorylation without classical transactivation in a cellular system as well as in an animal model.

We identified small-molecule enhancers of cellular stress granules by observing molecular crowding of proteins and RNAs in a time-dependent manner. Hit molecules sensitized IRF3-mediated antiviral mechanism in the presence of...