S-Benproperine, an Active Stereoisomer of Benproperine, Suppresses Cancer Migration and Tumor Metastasis by Targeting ARPC2.

Metastasis, in which cancer cells migrate to other tissues and form new tumors, is a major cause of both cancer death and treatment failure. In a previous study, benproperine (Benp) was identified as a cancer cell migration inhibitor and an inhibitor of actin-related protein 2/3 complex subunit 2 (ARPC2). However, Benp is a racemic mixture, and which stereoisomer is the active isomer remains unclear. In this study, we found that S-Benp is an active isomer and inhibits the migration and invasion of cancer cells much more strongly than R-Benp, with no effect on normal cells. The metastasis inhibitory effect of S-Benp was also verified in an animal model. Validating that inhibitors bind to their targets in cells and tissues has been a very challenging task in drug discovery. The direct interactions between ARPC2 and S-Benp were verified by surface plasmon resonance analysis (SPR), a cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS). In the mutant study with ARPC2F225A cells, S-Benp did not bind to ARPC2F225A according to CETSA and DARTS. Furthermore, we validated that S-Benp colocalized with ARPC2 in cancer cells and directly bound to ARPC2 in tumor tissues using Cy3-conjugated S-Benp according to CETSA. Finally, actin polymerization assays and immunocytochemistry showed that S-Benp suppressed actin remodeling such as lamellipodium formation. Taken together, our data suggest that S-Benp is an active stereoisomer of Benp and a potential metastasis inhibitor via ARPC2 binding.

Small-molecule modulators of protein-RNA interactions.

Protein-RNA interactions (PRIs) play crucial roles in diverse cellular pathways, from transcription to liquid-liquid phase separation, and its dysregulation is associated with a wide range of human disorders. Therefore, there is great emphasis on discovering small-molecule modulators that target unexplored PRIs by developing robust PRI assays. In particular, targeting PRIs could offer innovative solutions to expand the druggable genome, as only a small portion of protein-coding genes are currently targeted by drugs. This review describes the therapeutic potential of targeting PRIs using small molecules, biochemical and cell-based experimental tools for observing PRIs, and several PRI modulators. We also highlight emerging technologies and the challenges in developing PRI modulators.

Harnessing stress granule formation by small molecules to inhibit the cellular replication of SARS-CoV-2.

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 the IRF3-mediated antiviral mechanism in the presence of poly(I:C) and inhibited the replication of SARS-CoV-2 by inducing stress granule formation. Thus, modulating multimolecular crowding can be a promising strategy against SARS-CoV-2.

Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay.

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.

Restoring Let-7 microRNA Biogenesis Using a Small-Molecule Inhibitor of the Protein-RNA Interaction.

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 Förster 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.

Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28.

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.