Enhancement of RNAi by a small molecule antibiotic enoxacin

Abstract

RNAi has become a mainstream molecular tool for assessing the functions of genes in mammalian cells [1]. Large-scale RNA interference-based analyses are often complicated by false positive and negative hits due to off-target effects [2] and interferon response [3], which can be attributed at least in part to the use of high concentrations of siRNA. Lowering the amounts of siRNAs and shRNAs can effectively and expediently mitigate the off-target effect and interferon response [4]. However, in RNAi experiments, lowering the concentration of siRNA is often accompanied by a lower knockdown efficiency. One of the key factors affecting RNAi efficiency is the stability of double-stranded siRNA. We reasoned that measures that could stabilize double-stranded RNA may lead to increased RNAi efficiency. Given that RNAi requires a number of cellular proteins, it should be possible, at least in theory, to regulate the efficiency of RNAi using small organic molecules. To stabilize ds-RNA used in RNAi, we therefore envisioned the following methods: (1) to design more stable siRNA (screening for certain sequences, modification of RNA); (2) to increase the activity of ds-RNA protective proteins (such as ds-RNAbinding proteins, or simply binding domains); (3) to inhibit the activity of ds-RNA-dissolving proteins (such as RNA helicases); (4) to stabilize ds-RNA and/or its protein complex with small organic compounds. Our longstanding interest in drug-nucleic acids interaction [5] led us to search for potential small molecular regulators of RNAi. We hypothesized that inhibitors of RNA helicases may increase the stability of double-stranded siRNA, so as to enhance RNAi efficiency. Since a large family of fluoroquinolone antibiotics target bacterial DNA gyrase complexed with the targeted DNA possibly in A-form (similar to RNA) [6] and since they also exhibit antiviral activity through interference with Tat-TAR interaction [7], we decided to screen a library of commercially available fluoroquinolone antibiotics, with the hope that some of the analogs may cross-inhibit relevant human RNA helicases. Herein, we report that enoxacin, one of the fluoroquinolone antibiotics known to inhibit bacterial gyrase and topoisomerase IV with minimal effects on their mammalian counterparts, can increase RNAi efficiency. We have found that enoxacin can reduce the concentrations of siRNA by 2~5-fold for the same RNAi knockdown efficiency.A dual-luciferase reporter assay system was used to screen small organic compounds that were capable of enhancing RNAi efficiency. The siRNA used in our screen is siFL867-885, which can effectively suppress the firefly luciferase reporter activity at 10 nM siRNA duplexes [8]. This system has been reported to be a robust siRNA screening system [8]. As a starting point, we diluted siFL867-885 to a concentration of 8.4×10-10 M, and at this concentration the RNAi knockdown was partial. A small library of fifteen widely used fluoroquinolone antibiotics ( Figure 1B) was screened using the dual-luciferase reporter system. By s...

Identification of Enoxacin as an Inhibitor of Osteoclast Formation and Bone Resorption by Structure-Based Virtual Screening

“…To explore this, we assayed several fluoroquinolones for their ability to affect the interaction between V-ATPase and F-actin in vitro and to inhibit osteoclast formation. These included norfloxacin, which was reported to stimulate microRNA activity and others (pefloxacin and levofloxacin) that did not 19,21. We found that only pefloxacin had a detectable effect on the interaction between B-subunit and F-actin (Figure 7A).…”

Rational Identification of Enoxacin as a Novel V-ATPase-Directed Osteoclast Inhibitor

“…Concurrent with our identification of enoxacin as an inhibitor of B2-microfilament binding, another group screening for small molecule modulators of microRNA activity identified enoxacin as a stimulator of microRNA processing, and suggested this was through interaction with TAR RNA-binding protein 2 (TRBP2) [108]. This finding could, in principle, explain some or all of the observations with regard to osteoclast formation and function.…”

Probing the microRNA pathway with small molecules

“…We found that it could dramatically enhance the processing of shRNA into siRNAs and promote the loading of siRNAs/miRNAs into the RISC, significantly reducing the dosage required to achieve equal knockdown efficiency in mammalian cells (Shan et al, 2008). Enoxacin was identified as an RNAi enhancer in an independent chemical screening by another group, as well (Zhang et al, 2008). …”