Discovery of small-molecule modulators of the RNA-binding proteins LIN28 and RNase L

Abstract

RNA-binding proteins (RBPs) regulate all aspects of RNA biology and metabolism and are emerging targets for the development of molecular probe compounds and therapeutics for various diseases. While oligonucleotide-based and peptide-based strategies have been applied to modulate RNAs and protein–RNA interactions, efficient RBP-targeting strategies utilizing small molecules, which bear inherent merits in comparison with other chemotypes, remain obscure. In this thesis, I studied small-molecule-based strategies to target three different RBPs, the oncogenic miRNA-binding protein LIN28, the antiviral ssRNA-cleaving protein RNase L and the associated dsRNA-binding protein OAS. LIN28, which negatively regulates let-7 miRNAs that downregulate the translation of numerous oncogenic proteins, is overexpressed in many human cancers and is a driver of tumor progression and metastasis. Thus, LIN28 inhibition via small molecules is a promising strategy for the development of cancer therapeutics. Reported LIN28 inhibitors suffer from poor inhibitory potency, insufficient characterization of mechanism of action, limited structure-activity relationship, and poor cellular activity. This work employed a screening-based and a scaffold-based approach for the identification of LIN28 inhibitors with new scaffolds and improved potency. In the former approach, trisubstituted pyrrolinones were identified as LIN28 inhibitors via a fluorescence polarization assay-based screening. The most active pyrrolinone 41 increased the expression levels of mature let-7 in LIN28-expressing cells. A following structure-activity relationship study revealed biphenyl compounds, such as 85, that showed a more potent effect in inducing let-7 maturation. In the latter approach, a spirocyclization strategy was applied based on the chromenopyrazole scaffold of reported LIN28 inhibitors to increase the LIN28-inhibitory potency induced by the rigidity of the spirocyclic scaffold. The identified and well-characterized inhibitors are worthy starting points for the development of anticancer drugs and LIN28-targeting chemical probes. The dsRNA-cleaving protein RNase L and the ssRNA-binding protein OAS are key enzymes in the human antiviral innate immune response. OAS detects foreign dsRNA upon viral infection, leading to OAS activation and production of the second messenger 2’-5’A. The binding of 2’-5’A to RNase L leads to RNase L dimerization and activation. The RNase L dimer then cleaves ssRNAs resulting in a global translational arrest and a cellular antiviral state. Small-molecule activators of OAS and RNase L are thus promising candidates for the development of broad-spectrum antiviral therapeutics. Furthermore, RNase L activators are useful components to build bifunctional RNase L recruiters to achieve proximity-induced targeted degradation of RNAs. In this work, robust assays for the screening and validation of OAS and RNase L activators were developed and used for the discovery of small-molecule modulators. While the identification of OAS and RNase L activators proved to be challenging, potential RNase L binders were identified. Additionally, a rational design approach led to the discovery of thiophenones as RNase L inhibitors that showed more potent inhibitory potency than reported RNase L inhibitors that target the nucleotide-binding pocket of RNase L Collectively, the results of this thesis demonstrate the feasibility targeting RBPs using a variety of small-molecule-based strategies. Screening- and scaffold-based approaches enabled the identification of modulators with new scaffolds and improved potency in targeting RBPs. The established assays, the identified compounds, and the optimization strategies presented in this work will be useful for the future development of small molecules targeting RBPs.