Development and evaluation of small molecule- and bifunctional modulators targeting the ribonuclease L for RNA degradation

Abstract

Ribonucleases are one of the critical regulatory enzymes of RNA-involved metabolism in cells and are becoming promising therapeutic targets due to the importance of RNA in many disease mechanisms. In particular, ribonuclease L (RNase L) has attracted great attention as a regulatory enzyme engaged in innate immune responses against viral infections. RNase L plays a vital role in the antiviral response by degrading RNAs, which is suppressed by viruses over evolution. Therefore, activating the RNase L and associated innate immunity by using small molecule-based strategies are one of the promising therapeutic approaches for not only antiviral effect but also RNase L-related anticancer effect. Meanwhile, RNase L-inhibition associates therapeutic potential in treating autoimmune and inflammatory disorders. Although a few small molecule-based RNase L modulators have been reported, the scarcity of potency and selectivity of reported modulators highly requires a thorough optimization and discovery of new scaffolds as well as development of new approaches. In this study, we pursued our efforts in acquiring potent small-molecule modulators of RNase L via two different strategies. The first approach based on small molecule-ligand pursued improved potency of ligands. The second approach was to introduce new chemical modalities to modulate RNase L activity via bifunctional molecules. In the small molecule-ligand approach, we assessed whether scaffold-based design, structure-activity relationship (SAR) study, and rational design could facilitate the generation of a more potent modulator. The scaffold-based design yielded chemically intriguing scaffolds but with no RNase L activity change. The SAR approach yielded compounds which revealed improved binding to RNase L and showed RNase L-mediated cellular downstream effects. The rational design led to a promising fusion scaffold of RNase L modulators, 2-((pyrrol-2-yl)methylene)thiophen-4-one, and compounds which exhibited 30-fold improved inhibitory effect than reported compound in in vitro biochemical evaluation and a potent cellular inhibitory effect. In our second approach, the three bifunctional molecules consist of a homodimerizer, a heterobivalent molecule, and the PROTAC molecule. The homodimerizer resulted in limited improvement of activator potency while heterobivalent molecule did not lead to activity change. Lastly, PROTAC molecules to degrade an RNase L-inhibiting protein have been synthesized. The effect of the PROTAC molecules on the RNase L activation will be evaluated in a future study. We focused on multiple approaches to modulate the challenging but promising target RNase L. This study will contribute to the development of new chemical entities for a potent RNase L modulator and thus for an innate immunity modulator.