Qiangzhe Zhang et al. npg Cell Research (2008) 18:1077-1079. 1077 © 2008 IBCB, SIBS, CAS All rights reserved 1001-0602/08 $ 30.00 npg LETTER TO THE EDITOR www.nature.com/cr Enhancement of RNAi by a small molecule antibiotic enoxacin Qiangzhe Zhang 1, *, Caihong Zhang 1, *, Zhen Xi 1 1 State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, Nankai University, Tianjin 300071, China Cell Research (2008) 18:1077-1079. doi: 10.1038/cr.2008.287; published online 23 September 2008 Dear Editor, RNAi. We hypothesized that inhibitors of RNA helicases may increase the stability of double-stranded siRNA, so RNAi has become a mainstream molecular tool for as to enhance RNAi efficiency. Since a large family of assessing the functions of genes in mammalian cells [1]. fluoroquinolone antibiotics target bacterial DNA gyrase Large-scale RNA interference-based analyses are often complexed with the targeted DNA possibly in A-form complicated by false positive and negative hits due to (similar to RNA) [6] and since they also exhibit antiviral off-target effects [2] and interferon response [3], which activity through interference with Tat-TAR interaction can be attributed at least in part to the use of high con- [7], we decided to screen a library of commercially avail- centrations of siRNA. Lowering the amounts of siRNAs able fluoroquinolone antibiotics, with the hope that some and shRNAs can effectively and expediently mitigate the of the analogs may cross-inhibit relevant human RNA off-target effect and interferon response [4]. However, in helicases. Herein, we report that enoxacin, one of the RNAi experiments, lowering the concentration of siRNA fluoroquinolone antibiotics known to inhibit bacterial gy- is often accompanied by a lower knockdown efficiency. rase and topoisomerase IV with minimal effects on their One of the key factors affecting RNAi efficiency is the mammalian counterparts, can increase RNAi efficiency. stability of double-stranded siRNA. We reasoned that We have found that enoxacin can reduce the concentra- measures that could stabilize double-stranded RNA may tions of siRNA by 2~5-fold for the same RNAi knock- lead to increased RNAi efficiency. Given that RNAi down efficiency. requires a number of cellular proteins, it should be possi- A dual-luciferase reporter assay system was used to ble, at least in theory, to regulate the efficiency of RNAi screen small organic compounds that were capable of en- using small organic molecules. To stabilize ds-RNA used hancing RNAi efficiency. The siRNA used in our screen in RNAi, we therefore envisioned the following meth- is siFL867-885, which can effectively suppress the firefly ods: (1) to design more stable siRNA (screening for cer- luciferase reporter activity at 10 nM siRNA duplexes tain sequences, modification of RNA); (2) to increase the [8]. This system has been reported to be a robust siRNA activity of ds-RNA protective proteins (such as ds-RNA- screening system [8]. As a starting point, we diluted binding proteins, or simply binding domains); (3) to in- siFL867-885 to a concentration of 8.4×10–10 M, and at hibit the activity of ds-RNA-dissolving proteins (such as this concentration the RNAi knockdown was partial. RNA helicases); (4) to stabilize ds-RNA and/or its pro- A small library of fifteen widely used fluoroquinolone tein complex with small organic compounds. Our long- antibiotics (Figure 1B) was screened using the dual-lu- standing interest in drug-nucleic acids interaction [5] led ciferase reporter system. By statistical analysis of experi- us to search for potential small molecular regulators of mental data (Figure 1A), seven compounds were found to increase RNAi efficiency (Figure 1B, the compounds shown with asterisk (*)). Among these 7 fluoroquino- lones exhibiting statistically significant RNAi-enhancing *These two authors contributed equally to this work. activities, enoxacin and norfloxacin were more active Correspondence: Zhen Xi Tel/Fax: +86-22-23504782 than others at the same concentration. We then chose E-mail: [email protected] enoxacin and norfloxacin for further characterization. It www.cell-research.com | Cell Research npg Enhancement of RNAi by a small molecule antibiotic enoxacin 1078 1200000 A control compound siRNA siRNA+compoud 1000000 800000 600000 120000 100000 Firefly luciferase activity 80000 60000 40000 20000 0 Compound Enoxacin Fleroxacin Ofloxacin DifloxacinPefloxacin NorfloxacinGatifloxacin Balofloxacin Pazufloxacin Levofloxacin PrulifloxacinSparfloxacinNadifloxacinCiprofloxacinLomefloxacin B C 100 Balofloxacin Fleroxacin Pazufloxacin Ofloxacin Levofloxacin enoxacin + enoxacin – 16.7 nM 95 Norfloxacin Gatifloxacin Enoxacin Prulifloxacin Sparfloxacin 1.67 nM 90 0.84 nM 85 0.42 nM Nadifloxacin Ciprfloxacin Lomefloxacin Difloxacin Pefloxacin RNAi knockdown percentage 80 0.21 nM 1×10–10 1×10–9 1×10–8 siRNA concentration (M) Figure 1 (A) Screening of 15 fluoroquinolones for RNAi enhancing activity in cultured HEK-293 cells. Statistically significant difference of RNAi efficiency was observed in the presence of 7 compounds as shown with asterisk. The Y-axis denotes the chemiluminescence data of firefly luciferase activities. The final concentrations of siRNA duplexes (siFL867-885) and com- pounds are 8.4×10–10 M and 50 mM, respectively. The plotted data represent the mean±SE. The asterisk (*) denotes statisti- cally significant difference (P < 0.05). (B) Chemical structures of 15 fluoroquinolones. The compounds showing statistically significant RNAi enhancing effect are labeled with asterisk (P < 0.05). (C) RNAi efficiency of different concentrations of siRNA (siFL867-885) in the presence or absence of enoxacin. At 12 h post-transfection, HEK-293 cells were treated without or with enoxacin at the final concentration of 50 mM. The Y-axis denotes the RNAi knockdown percentage of firefly luciferase activity after correction by renilla luciferase activity (using normalized expression ratios between the firefly luciferase avtivity and the renilla luciferase activity in the dual-luciferase reporter assay system) [6]. The RNAi-enhancing activity by enoxacin reaches the maximum at the siRNA concentration of 8.4×10–10 M. All experiments with siRNAs were performed in triplicates and re- peated at least twice. Each value represents the mean±SE. was found that the RNAi-enhancing activity of enoxacin to cause non-specific reduction of the firefly luciferase was dose-dependent (see Supplementary information, activity (see Supplementary information, Figure S3). The Figure S1). The EC50 of enoxacin as an RNAi enhancer RNAi efficiency of different concentrations of siRNA is about 30 mM. This concentration is far below the IC50 (siFL867-885) in the presence or absence of enoxacin of enoxacin for inhibition of topoisomerase II in human was also examined. At 12 h post-transfection, HEK-293 macrophages (IC50 = 1 485 mM) (see Supplementary in- cells were treated with or without 50 mM enoxacin. An formation, Figure S2) [9]. At 120 mM, enoxacin started optimum RNAi-enhancing effect by enoxacin was ob- Cell Research | Vol 18 No 10 | October 2008 Qiangzhe Zhang et al. npg 1079 served when the siRNA concentration was at 8.4×10–10 (2003CB114403), National Natural Science Foundation M. Generally, enoxacin can reduce the amount of siRNA of China (20272029, 20572053, 20421202, 20432010), by 2~5-fold to achieve the same RNAi knockdown effi- Ministry of Education of China (104189) and Nankai ciency (Figure 1C). University. In summary, we have demonstrated that certain fluo- roquinolone antibiotics such as enoxacin, in addition to References their powerful clinic use for the treatment of infections in humans and animals [10], can be used to increase RNAi 1 Cullen LM, Arndt GM. Genome-wide screening for gene func- efficiency. Enoxacin can significantly reduce the amount tion using RNAi in mammalian cells. Immunol Cell Biol 2005; of siRNA (by 2~5-fold) to achieve the same RNAi effi- 83:217-223. 2 MacDonald ML, Lamerdin J, Owens S, et al. Identifying off- cacy. The precise mechanism of this RNAi enhancement target effects and hidden phenotypes of drugs in human cells. remains unclear at present. While our manuscript was in Nat Chem Biol 2006; 2:329-337. preparation, a similar finding with more detailed analysis 3 Bridge AJ, Pebernard S, Ducraux A, Nicoulaz AL, Iggo R. was reported by Jin and colleagues [11], who proposed Induction of an interferon response by RNAi vectors in mam- that enoxacin acts by potentially increasing RISC load- malian cells. Nat Genet 2003; 34:263-264. ing efficiency through a mechanism depending on the 4 Martin SE, Caplen NJ. Applications of RNA interference in protein factor TRBP. Nevertheless, one cannot rule out mammalian systems. Annu Rev Genomics Hum Genet 2007; 8:81-108. the possibility that the effect of enoxacin on RNAi is 5 Xi Z, Zhang R, Yu Z, Ouyang D. The interaction between due to the cross-interaction with human RNA helicases tylophorine B and TMV RNA. Bioorg Med Chem Lett 2006; and the stabilization of RNAi molecules, especially in 16:4300-4304. view of the finding that human RNA helicase A (RHA) 6 Noble CG, Barnard FM, Maxwell A. Quinolone-DNA inter- is an active RISC component and functions in RISC as action: sequence-dependent binding to single-stranded DNA an siRNA loading factor [12]. Further mechanistic study reflects the interaction within the gyrase-DNA complex. Anti- (such as QSAR, photolabelling and affinity purification) microb Agents Chemother 2003; 47:854-862. is being actively pursued in our lab to elucidate the mo- 7 Richter S, Parolin C, Palumbo M, Palù G. Antiviral properties of quinolone-based drugs. Curr Drug Targets Infect Disord lecular basis of the observation reported here. Other ds- 2004; 4:111-116. RNA stabilizing agents are still waiting to be explored 8 Xu Y, Zhang HY, Thormeyer D, et al. Effective small interfer- for the regulation of RNAi. We believe that the finding ing RNAs and phosphorothioate antisense DNAs have dif- of small molecular RNAi enhancers could potentially be ferent preferences for target sites in the luciferase mRNAs.