Identification and Mechanism of Small Molecule Inhibitors of RNA Interference.

摘要

RNA interference (RNAi) is induced both artificially to knockdown gene expression and naturally during virus infection as a host defense mechanism. Although genetic studies have provided a biochemical framework for RNAi, little is known if key steps in the RNAi pathway can be inhibited by small molecules. This dissertation describes a cell-based small molecule screen to assay for the suppression of naturally occurring RNAi in Drosophila Schneider 2 (S2) cells induced by viral RNA replication. This screen resulted in the identification of twenty lead compounds in the primary screen that were narrowed down to five RNAi inhibitors (RINs) in the secondary screen by Northern blot analysis.;Genetic and biochemical approaches were taken subsequently to determine the targets of these RINs in the RNAi pathway. In Drosophila melanogaster, RNAi begins with the dicing of long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) by the nuclease Dicer-2. This is followed by the assembly of the RNA-induced silencing complex (RISC) that contains a single-stranded siRNA bound to the RNaseH-like Argonaute-2 (Ago2) and cleaves the target mRNA that is complementary to the siRNA. The first set of experiments in this study divided the five RIN compounds into two groups. Although all of the RIN compounds were highly potent in the suppression of RNAi triggered in S2 cells by long dsRNA, artificial initiation of RNAi by synthetic siRNA was also inhibited by RINs 4 and 5, but not by RINs 1-3. These findings indicate that RINs 1-3 target the upstream steps of the RNAi pathway while RINs 4 and 5 target the downstream steps. Further biochemical experiments demonstrated that RINs 1 and 2 inhibited the dicing of long dsRNA into siRNAs and that RIN5 blocked cleavage (slicing) of target mRNA mediated by an siRNA-programmed RISC. These results demonstrate for the first time that both dicing and slicing in the core RNAi pathway can be targeted for inhibition by small molecules.;The last set of experiments determined if the RIN compounds were able to suppress the RNAi-mediated antiviral immunity triggered by infection initiated by virion inoculation. Both Flock house virus (FHV) and Cricket paralysis virus (CrPV) replicated to much higher levels in S2 cells following treatment with each RIN compound and RINs 3, 4 and 5 dramatically enhanced the cytopathic effect of both positive-strand RNA viruses. The B2 protein encoded by FHV is a virus suppressor of RNAi (VSR) indispensable for infection of S2 cells. In the absence of RNAi suppression, the B2-deficient mutant of FHV (FHVDB2) is rapidly cleared in S2 cells following challenge inoculation with viral particles unless RNAi is suppressed in S2 cells, for example, by Ago2 depletion. Treatment of S2 cells with each of the five RIN compounds enhanced the accumulation of FHVDB2. Notably, FHVDB2 replicated to similarly high levels in Ago2-depleted S2 cells and S2 cells treated with RIN5. Moreover, both RINs 2 and 5 enhanced the accumulation and virulence of FHV in adult flies and RIN treatment of Caenorhabditis elegans also partially inhibited RNAi induced by a B2-deficient replicon of FHV or exogenous dsRNA.;In summary, the experiments detailed in this body of work established a cellbased screen for the identification of small molecule antagonists of RNAi. These experiments also provided a pipeline of experiments to establish where and how the identified compounds affect the RNAi pathway.