Enhancement of RNAi by a small molecule antibiotic enoxacin

Cell Research, Sep 2008

Qiangzhe Zhang, Caihong Zhang, Zhen Xi

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Enhancement of RNAi by a small molecule antibiotic enoxacin

Cell Research | Enhancement of RNAi by a small molecule antibiotic enoxacin Qiangzhe Zhang 0 Caihong Zhang 0 Zhen Xi 0 Dear Editor 0 State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University , Tianjin 300071, China Cell Research (2008) 18:1077-1079 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 statistical analysis of experimental data (Figure 1A), seven compounds were found to increase RNAi efficiency (Figure 1B, the compounds shown with asterisk (*)). Among these 7 fluoroquinolones exhibiting statistically significant RNAi-enhancing activities, enoxacin and norfloxacin were more active than others at the same concentration. We then chose enoxacin and norfloxacin for further characterization. It npg Enhancement of RNAi by a small molecule antibiotic enoxacin 1078 control compound siRNA siRNA+compoud Balofloxacin Fleroxacin Pazufloxacin Ofloxacin Levofloxacin Norfloxacin Gatifloxacin Enoxacin Prulifloxacin Sparfloxacin 1.67 nM 16.7 nM Nadifloxacin Ciprfloxacin Lomefloxacin Difloxacin Pefloxacin C 100 e g a t en 95 c r e p nw90 o d k con 85 k i A RN80 enoxacin + enoxacin ? 0.84 nM 0.42 nM 0.21 nM was found that the RNAi-enhancing activity of enoxacin was dose-dependent (see Supplementary information, Figure S1). The EC50 of enoxacin as an RNAi enhancer is about 30 mM. This concentration is far below the IC50 of enoxacin for inhibition of topoisomerase II in human macrophages (IC50 = 1 485 mM) (see Supplementary information, Figure S2) [ 9 ]. At 120 mM, enoxacin started to cause non-specific reduction of the firefly luciferase activity (see Supplementary information, Figure S3). The RNAi efficiency of different concentrations of siRNA (siFL867-885) in the presence or absence of enoxacin was also examined. At 12 h post-transfection, HEK-293 cells were treated with or without 50 mM enoxacin. An optimum RNAi-enhancing effect by enoxacin was observed when the siRNA concentration was at 8.4?10?10 M. Generally, enoxacin can reduce the amount of siRNA by 2~5-fold to achieve the same RNAi knockdown efficiency (Figure 1C). In summary, we have demonstrated that certain fluoroquinolone antibiotics such as enoxacin, in addition to their powerful clinic use for the treatment of infections in humans and animals [ 10 ], can be used to increase RNAi efficiency. Enoxacin can significantly reduce the amount of siRNA (by 2~5-fold) to achieve the same RNAi efficacy. The precise mechanism of this RNAi enhancement remains unclear at present. While our manuscript was in preparation, a similar finding with more detailed analysis was reported by Jin and colleagues [ 11 ], who proposed that enoxacin acts by potentially increasing RISC loading efficiency through a mechanism depending on the protein factor TRBP. Nevertheless, one cannot rule out the possibility that the effect of enoxacin on RNAi is due to the cross-interaction with human RNA helicases and the stabilization of RNAi molecules, especially in view of the finding that human RNA helicase A (RHA) is an active RISC component and functions in RISC as an siRNA loading factor [ 12 ]. Further mechanistic study (such as QSAR, photolabelling and affinity purification) is being actively pursued in our lab to elucidate the molecular basis of the observation reported here. Other dsRNA stabilizing agents are still waiting to be explored for the regulation of RNAi. We believe that the finding of small molecular RNAi enhancers could potentially be used as a tool in genome-scale loss-of-function screening with RNAi, especially in cases that are highly dependent on the use of relatively low concentrations of siRNA and shRNA. It may also be a useful molecular probe to shed new light on the RNAi process. (Experimental materials and methods are depicted in the Supplementary information, Data S1) Acknowledgments We thank Jun O Liu from Johns Hopkins School of Medicine for critical discussions and proof-reading of the manuscript. This work was supported by the National Key Project for Basic Research of China (Supplementary information is linked to the online version of the paper on the Cell Research website.) 1 Cullen LM , Arndt GM . Genome-wide screening for gene function using RNAi in mammalian cells . Immunol Cell Biol 2005 ; 83 : 217 - 223 . 2 MacDonald ML , Lamerdin J , Owens S , et al. Identifying offtarget effects and hidden phenotypes of drugs in human cells . Nat Chem Biol 2006 ; 2 : 329 - 337 . 3 Bridge AJ , Pebernard S , Ducraux A , Nicoulaz AL , Iggo R . Induction of an interferon response by RNAi vectors in mammalian cells . Nat Genet 2003 ; 34 : 263 - 264 . 4 Martin SE , Caplen NJ . Applications of RNA interference in mammalian systems . Annu Rev Genomics Hum Genet 2007 ; 8 : 81 - 108 . 5 Xi Z , Zhang R , Yu Z , Ouyang D. The interaction between tylophorine B and TMV RNA . Bioorg Med Chem Lett 2006 ; 16 : 4300 - 4304 . 6 Noble CG , Barnard FM , Maxwell A . Quinolone-DNA interaction: sequence-dependent binding to single-stranded DNA reflects the interaction within the gyrase-DNA complex . Antimicrob Agents Chemother 2003 ; 47 : 854 - 862 . 7 Richter S , Parolin C , Palumbo M , Pal? G . Antiviral properties of quinolone-based drugs . Curr Drug Targets Infect Disord 2004 ; 4 : 111 - 116 . 8 Xu Y , Zhang HY , Thormeyer D , et al. Effective small interfering RNAs and phosphorothioate antisense DNAs have different preferences for target sites in the luciferase mRNAs . Biochem Biophys Res Commun 2003 ; 306 : 712 - 717 . 9 Cort?zar TM , Coombs GH , Walker J . Leishmania panamensis: comparative inhibition of nuclear DNA topoisomerase II enzymes from promastigotes and human macrophages reveals anti-parasite selectivity of fluoroquinolones, flavonoids and pentamidine . Exp Parasitol 2007 ; 116 : 475 - 482 . 10 Sukul P , Spiteller M. Fluoroquinolone antibiotics in the environment . Rev Environ Contam Toxicol 2007 ; 191 : 131 - 162 . 11 Shan G , Li Y , Zhang J , et al. A small molecule enhances RNA interference and promotes microRNA processing . Nat Biotechnol 2008 ; 26 : 933 - 940 . 12 Robb GB , Rana TM. RNA Helicase A interacts with RISC in human cells and functions in RISC loading . Mol Cell 2007 ; 26 : 523 - 537 .

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Qiangzhe Zhang, Caihong Zhang, Zhen Xi. Enhancement of RNAi by a small molecule antibiotic enoxacin, Cell Research, 2008, 1077-1079, DOI: 10.1038/cr.2008.287