Article pubs.acs.org/jmc

Haloemodin as Novel Antibacterial Agent Inhibiting DNA Gyrase and Bacterial Topoisomerase I Feixia Duan,†,‡,∇ Xiaohong Li,§,∇ Suping Cai,∥,∇ Guang Xin,† Yanyan Wang,† Dan Du,† Shiliang He,† Baozhan Huang,† Xiurong Guo,† Hang Zhao,†,⊥ Rui Zhang,† Limei Ma,† Yan Liu,† Qigen Du,† Zeliang Wei,† Zhihua Xing,† Yong Liang,† Xiaohua Wu,† Chengzhong Fan,# Chengjie Ji,† Dequan Zeng,⊥ Qianming Chen,*,⊥ Yang He,*,† Xuyang Liu,*,∥ and Wen Huang*,† †

Laboratory of Ethnopharmacology, Institute for Nanobiomedical Technology and Membrane Biology, Regenerative Medicine Research Center, West China Hospital, West China Medical School, Sichuan University Keyuan 4 Road No. 1, Gaopeng Avenue, Gaoxinqu, Chengdu, Sichuan 610041 China ‡ College of Light Industry, Textile and Food Engineering, Sichuan University, Chengdu, Sichuan 610065, China § Department of Biopharmaceutics, Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Section 3 Southern Renmin Road, Chengdu 610041, China ∥ Shenzhen Eye Hospital, Jinan University, No. 18, Zetian Road, Futian District, Shenzhen 518040, China ⊥ State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University Chengdu, Sichuan 610041, China # Department of Nuclear Medicine, West China Hospital, Sichuan University, No. 37 Guoxue Alley, Chengdu, Sichuan 610041, China S Supporting Information *

ABSTRACT: Drug-resistant bacterial infections and lack of available antibacterial agents in clinical practice are becoming serious risks to public health. We synthesized a new class of haloemodins by modifying a traditional Chinese medicine component, emodin. The novel haloemodin exerts strong inhibitory activity on bacterial topoisomerase I and DNA gyrase, and not on the topoisomerases of human origin. In principle, it shows remarkable antibacterial activities against laboratory and clinically isolated Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus. We further expanded its antibacterial spectrum into against Gram-negative bacteria with the assistance of polymyxin B nonapeptide, which helps haloemodin to penetrate through the bacterial outer membrane. Finally, the therapeutic effect of haloemodin in vivo was confirmed in curing S. aureus-induced keratitis on rabbit model. With distinctive structural difference from the antibiotics we used, the haloemodins are of value as promising antibacterial pharmacophore, especially for combat the infections caused by drug-resistant pathogens.

■

INTRODUCTION

The emergence of multidrug-resistant bacteria and the shortage of clinically available antibacterial agents is becoming a serious threat to the public health nowadays. Methicillin-resistant Staphylococcus aureus (MRSA) currently causes worldwide infections both in hospital and community, 1−9 while vancomycin-resistant Enterococcus faecium (VRE), vancomycin-resistant Staphylococcus aureus, and carbapenem-resistant Enterobacteriaceae, become new risks for deadly infections.10−18 So, new classes of antibacterial agents are urgently in need to combat the attack of emerging drug-resistant bacteria. Emodin (1; Figure 1a), 6-methyl-1, 3, 8-trihydroxyanthraquinone, a major bioactive constituent of Chinese herbal medicine Polygonum cuspidatum roots and Rheum palmatum L., has received attention due to its wide range of potential pharmacological activities, although its antibacterial potency and mechanisms remain controversial.19−21 During our researches concerning the bioactivities of emodin derivatives, we synthesized a class of novel haloemodins (Figure 1b,c) and © 2014 American Chemical Society

Figure 1. Chemical structures of emodin and haloemodins. (a−c) Chemical structures of emodin (1, (a) 2,4-diiodoemodin (2 (b), and haloemodins (3−7 (c). 3: R1 = H, R2 = R3 = R4 = Cl. 4: R1 = R2 = Br, R3 = R4 = H. 5′′ R1 = I, R2 = R3 = R4 = H. 6: R1 = R2 = R4 = I, R3 = H. 7: R1 = I, R2 = Cl, R3 = R4 = H.

investigated the bioactivities of the haloemodin in vitro and in vivo. DNA gyrase and bacterial topoisomerase (Topo) I, as enzymes essential for DNA replication and cell division, are Received: October 31, 2013 Published: March 3, 2014 3707

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

important targets for developing antibacterial agents.22−27 We found that the novel haloemodin remarkably inhibits the catalytic activities of both DNA gyrase and bacterial Topo I while it shows weak inhibitory effects on human Topo IIα or human Topo I. We next confirmed the considerable antibacterial activities of the haloemodin against Gram-positive bacteria in vitro, especially against lots of drug-resistant clinical isolates, such as VRE, MRSA, and methicillin-resistant Staphylococcus epidermidis (MRSE). Gram-positive bacteria strains, such as S. aureus, are prevalent pathogens which cause purulent infection of the eye,13,28 while the haloemodin shows the in vivo therapeutic effect on rabbit model of S. aureus induced keratitis.

■

RESULTS Haloemodin Inhibits DNA Gyrase. Our group has focused on the bioactivities of emodin derivatives for a long time. During our process to modify the benzene rings of emodin, we have serendipitously obtained an intermediate, 2,4diiodoemodin (2; Figure 1b). This compound has never been synthesized before, and we have identified its structure thoroughly by HRMS, NMR (Supporting Information), and X-ray analysis of its single crystal (CDCC no. 919828; Figure 2).

Figure 3. Inhibition on DNA gyrase and Topo IIα by 2 and emodin (a−e). (a−c) The inhibition on kDNA decatenation induced by S. aureus DNA gyrase (a), E. coli DNA gyrase (b), and human Topo IIα (c). (d−e) The inhibition on pHOT-1 supercoiling catalyzed by S. aureus DNA gyrase (d), and E. coli DNA gyrase (e). Ciprofloxacin and etoposide were used as positive controls.

Figure 2. Crystal structure of 2. Iodine atom is highlighted in purple, carbon atom in gray, oxygen in red, and hydrogen in white.

The inhibitory activities of 2 on S. aureus and Escherichia coli DNA gyrase were characterized by kinetoplast DNA (kDNA) decatenation assay and pHOT-1 supercoiling assay in the presence of ATP29,30 and were compared with that of emodin. Compound 2 inhibits the activities of DNA gyrase more effectively than emodin (Figure 3a,b,d,e). Compound 2 completely inhibited double-stranded catenated kDNA decatenation mediated by DNA gyrase at the concentration of 90 μM (Figure 3a,b) and totally suppressed pHOT-1 supercoiling at the concentration of 30 μM (Figure 3d,e). Emodin just exerts partial inhibition at the concentration of 90 μM on these two reactions induced by DNA gyrase. We also assayed the inhibitory activities of 2 and emodin on human Topo IIα by a specific kDNA decatenation assay. It is worth noting that unlike emodin which strongly inhibits the activity of human Topo IIα, 2 has little inhibitory effect against human Topo IIα (Figure 3c). This indicates its additional benefit from the safety point of view compared with its parent nucleus. Haloemodin Inhibits Bacterial Topo I. We determined whether 2 inhibits the catalytic activity of Topo I (both in human and E. coli form) or it causes single-stranded breaks under the catalytic effects of Topo I by Topo I-mediated DNA

relaxation assay. We also investigated the interaction of 2 with DNA helix by determining whether 2 induces DNA unwinding characteristic of DNA intercalators. Unwinding of the double strands of the DNA helix is a hallmark feature of DNA intercalators, and this can be tested by examining the distribution of topoisomers generated by relaxation of closed circular DNA with Topo I. As negative supercoiled plasmids are transformed to relaxed plasmid, and to positive supercoils under the catalytic effects of Topo I, the typical up-and-down profile can be observed. Positive supercoiled DNA with bound intercalators also induces an upward gel shift, thereby a distance between positive supercoiled DNA and negative supercoiled DNA can be also observed. Neither typical up-and-down distribution of topoisomers nor gel shift of supercoiled pHOT-1 was observed when negative supercoiled pHOT-1 were incubated with Topo I of E. coli and human origin separately in the presence of 2 at gradient concentrations (Figure 4a,b), illustrating that 2 is not a DNA intercalator. Instead, 2 selectively inhibits the catalytic activity 3708

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

Figure 4. The electrophoresis photos of supercoiled pHOT-1 incubated with Topo I in the presence of 2 and camptothecin. (a) Electrophoresis photos of supercoiled pHOT-1 incubated with E. coli Topo I. (b) Electrophoresis photos of supercoiled pHOT-1 incubated with human Topo I. Camptothecin was used as positive control.

Table 1. The MIC Values of 2 against Gram-Positive Bacteria Stainsa MIC (mg/mL)

a

organism (number of isolates)

2

emodin

S. aureus ATCC 6538 S. aureus ATCC 29213 MSSAb (15) MSSEc (15) MRSA (15) MRSE (15) E. faecalis AfCC 29212 VS-Enterococcus faecalisd (15) VS-Enterococcus faeciume (15) VRE (15) B. cereus ATCC 10231 B. laterosporus ATCC 64 S. pneumoniae ATCC 49619 Streptococcus pneumoniae (18)

0.002 0.008 0.008 0.001−0.032 0.004−0.032 0.004−0.008 0.016 0.008−0.016 0.016 0.008 0.004 0.004 0.064 0.032−0.128

0.256 0.256

VAN

FOX

PEN

0.002 0.002 0.0005−0.002 0.001−0.002 0.128 0.128

>0.256

0.002 0.0005−0.002 0.0005 >0.256 0.002 0.002

0.256 0.256 >0.256

0.00025−0.001 0.008−1

MIC values were determined by ager dilution assay. bMethicillin-sensitive Staphylococcus aureus. cMethicillin-sensitive Staphylococcus epidermidis. vancomycin-sensitive Enterococcus faecalis. evancomycin-sensitive Enterococcus faecium.

d

Figure 5. Inhibition on the growth of S. aureus ATCC 6538 by agents. (a−c) Inhibition on the growth of S. aureus ATCC 6538 by 2 (a), emodin (b), and vancomycin (c). ODi and ODu, optical density of inoculated and uninoculated medium at 600 nm. Plots show means of triplicates with SD. MIC, the minimum inhibitory concentration value.

of Topo I of E. coli origin without producing single-stranded breaks (Figure 4a), while emodin possesses no inhibitory effect on Topo I of E. coli origin (Supporting Information Figure S1). The E. coli Topo I-mediated pHOT-1 relaxation was sup-

pressed gradually as the concentration of 2 increased and was completely inhibited by 2 at the concentrations above 90 μM (Figure 4a). However, unlike the samples containing camptothecin, no nicked open circular pHOT-1 induced by 2 3709

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

strains leads to the diversity in antibacterial potencies of 2. We further assayed the MIC of 2 against E. coli PQ 37, which has a mutational defect in the OM permeability barrier. As we expected, 2 inhibits the growth of E. coli PQ 37 at the concentration of 0.008 mg/mL, while 2 shows no inhibitory potency against E. coli (Figure 6a). This result shows that the LPS layer outside of the Gram-negative bacteria prevents 2 from accessing the bacterial cell interior and weakens its antibacterial activities. This suggests that once the LPS barrier outside of the Gram-negative bacteria was diminished, 2 should penetrate into bacterial cell and in principle inhibit the growth of E. coli by inhibiting bacterial DNA gyrase. There are several compounds that can help small molecules to go through the LPS barriers, and one of these compounds is polymyxin B nonapeptide (PMBN). PMBN can effectively increase the permeability of bacterial outer membrane (OM) to small-molecule agents by binding to LPS at low concentrations and has almost no impact on the growth of bacteria.31−33 Therefore, we chose PMBN to assist 2 in spreading into the cell interior of E. coli to see whether or not 2 inhibits the growth of E. coli in this case. As we expected, neither PMBN nor 2 completely inhibited the growth of E. coli when used alone at the concentrations we used. The viability of E. coli maintained 70% after being treated with PMBN at the concentration of 0.002 mg/mL for 24 h (Figure 6b), and compound 2 has no inhibiting effect against E. coli at the concentrations bellow 0.256 mg/mL (Figure 6c). However, PMBN magnificently improves the antibacterial activity of 2 against E. coli, and in the presence of 0.002 mg/ mL PMBN, the MIC value of 2 against E. coli was assayed to be 0.008 mg/mL (Figure 6c). This means that, with the help of PMBN, the MIC values of 2 decrease to the value equivalent to that against S. aureus (Table 1). These results show that with the assistance of the agents which can break the barrier of LPS, such as PMBN, the antibacterial spectrum of 2 can be expanded into Gram-negative bacteria. According to the discovery on antibacterial evaluation against both laboratory strains and clinical isolates, we confirmed 2 as a very efficient antibacterial agent, especially against certain clinical drug-resistant isolates. This novel antibacterial compound inhibits the bacterial DNA gyrase and has a totally different chemical structure from the known antibiotics which target bacterial DNA gyrase, such as the quinolones. It even has a structural difference from the main types of antibiotics currently used, including β-lactams, glycopeptides, aminoglycosides, lipopeptides, and quinolones, etc. Therefore, this novel haloemodin could offer a new way to combat the problem of infectious diseases induced by drug-resistant bacteria. Consequently, we further synthesized more emodin derivatives (Figure 1c) by introducing different halogen substituents on different positions and evaluated their antibacterial effects. In general, all these haloemodins can inhibit the growth of Grampositive bacteria more effectively than their parent nucleus emodin (Table 3). In case of iodoemodins, diiodoemodin (2) is the most efficient, while triiodoemodin (7) is better than monoiodoemodin (5). Among the haloemodins with two halogen substituents, diiodoemodin (2) is also the most efficacious compared with dibromoemodin (4), dichloroemodin, and 2-iodo-4-chloroemodin (7). We have also tried several methods to introduce fluorine substituents to emodin but unfortunately failed to obtain desirable fluoroemodin. Compound 2 shows the most efficient antibacterial activity among all these compounds.

was observed (Figure 4a), illustrating that compound 2 just inhibits the catalytic activity of E. coli Topo I, does not produce nicked intermediate by stabilizing covalent DNA/Topo I complexes. More important, 2 does not inhibit the human Topo I-mediated relaxation at the all concentrations we tested (Figure 4b). Antibacterial Activity Evaluation. Because bacterial DNA gyrase is an important antibacterial target, we assayed the minimum concentration (MIC) of 2 against multiple laboratory and clinically isolated bacterial strains to evaluate whether it possesses antibacterial activities. Initially, we examined the antibacterial activity of 2 against several laboratory bacterial strains and found that it inhibits the growth of Gram-positive bacteria such as Staphylococcus aureus, Enterococcus faecalis, Bacillus laterosporus, Bacillus cereus, and Streptococcus pneumoniae (Table 1). Impressively, its antibacterial activity is more than a hundred-fold stronger than its parent nucleus emodin. The MIC of 2 against S. aureus ATCC 6538 was assayed to be 0.002 mg/mL, less than 1% of that of emodin (0.256 mg/mL) and equal to that of vancomycin (Figure 5a−c). On the basis of these results, we next screened its antibacterial effects against 121 strains of clinically isolated Gram-positive bacteria, including 45 drug-resistant isolates. As we expected, 2 inhibits the growth of all the 121 strains of Gram-positive bacteria (Table 1) and, especially, it exhibits remarkable antibacterial effect against the drug-resistant bacteria MRSA, MRSE, and VRE (Table 1). The MIC values of 2 against MRSA and MRSE were assayed to be 0.004−0.032 and 0.004−0.008 mg/mL, respectively, while that of cefoxitin was shown to be 0.128 and 0.004−0.128 mg/mL (Table 1). Meanwhile, its MIC values against VRE were tested to be 0.008−0.016 mg/mL, and vancomycin shows no inhibitory activity on the growth of VRE at concentrations bellow 0.256 mg/mL (Table 1). Interestingly, among these Gram-positive bacteria strains we have found that 2 shows relatively weaker efficiency against S. pneumoniae for both laboratory and clinically isolated strains, with MIC values fluctuating from 0.064 to 0.128 mg/mL (Table 1). Next, we investigated the antibacterial effect of 2 against Gram-negative bacteria. Compound 2 exhibits much weaker inhibiting effect against the growth of these Gram-negative bacteria for both laboratory and clinically isolated strains such as E. coli and Pseudomonas aeruginosa (Table 2). It is worth mentioning that among the tested Gram-negative strains, 2 shows relatively better effect against Bacteroides fragilis than the other Gram-negative strains. We suppose that the difference in LPS structure between B. fragilis and other Gram-negative Table 2. The MIC Values of 2 against Gram-Negative Bacterial Strainsa MIC range (mg/mL) organism (number of isolate) E. coli ATCC 25922 E. coli PQ 37 P. aeruginosa ATCC 27853 P. aeruginosa (13) B. fragilis ATCC 25285 B. fragilis (18) a

2

PIP

>0.256 0.008 >0.256

0.004

>0.256 0.256

0.002−0.256

CLI

0.004

0.064−0.256

0.0005−0.002 0.00003→0.256

MIC values were determined by ager dilution assay. 3710

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

Figure 6. Inhibition on the growth of E. coli and E. coli PQ37 by agents. (a) Inhibition on the growth of E. coli and E. coli PQ37 by 2. (b) Inhibition on the growth of E. coli by PMBN independently. (c) The inhibition on the growth of E. coli by 2 in the presence of PMBN (0, 0.001, and 0.002 mg/ mL). ODi and ODu, optical density of inoculated and uninoculated medium at 600 nm; plots show mean values of triplicates with SD.

Table 3. The MIC Values of Emodin and the Haloemodins against Laboratory Strainsa MIC (mg/mL)b organism

1

3

4

5

6

7

VAN

S. aureus ATCC 6538 S. aureus ATCC 29213 B. cereus ATCC 10231 B. laterosporus ATCC 64 E. coli ATCC 25922 P. aeruginosa ATCC 27853

0.256 0.256 0.256 0.256 >0.256 >0.256

0.008 0.016 0.008 0.008 >0.256 >0.256

0.008 0.016 0.008 0.008 >0.256 >0.256

0.016 0.032 0.016 0.016 >0.256 >0.256

0.008 0.016 0.008 0.008 >0.256 >0.256

0.032 0.064 0.032 0.032 >0.256 >0.256

0.002

FOX

PIP

0.002 0.002 0.002 0.004 0.004

a

MIC values were determined by ager dilution assay. bCompounds: emodin (1), 4,5,7-trichloroemodin (3), 2,4-dibromoemodin (4), 2-iodoemodion (5), 2,4,7-triiodoemodin (6), 2-iodo-4-chloroemodin (7).

Haloemodin Cures S. aureus-Induced Keratitis in a Rabbit Model. To evaluate the therapeutic action of 2 in vivo, we constructed a rabbit model of S. aureus-induced keratitis. Then 24 h after S. aureus infection, the model eyes manifested as obvious conjunctival congestion, chemosis, and purulent secretions, accompanied by corneal edema and purulent as well as operative ulcer at the 7 o’clock and 11 o’clock positions, respectively (Figure 7a(i),(iii), 7b(i),(iii), and 7c(i),(iii)). After continuous treatment for 10 d, topical application of 2 and ofloxacin both lowered the severity of infection, obviously alleviating conjunctival congestion, conjunctival chemosis, and infiltration, reducing the operative ulcer area and depth (Figure 7b(ii),(iv) and 7c(ii),(iv)). Moreover, continuous treatment with 2 effectively alleviate the corneal edema (Figure 7c(ii), (iv)), while the eyes developed severe corneal edema at a level corresponding to the edge of the pupil and dense opacity involving the corneal over the pupil area (Figure 7b(ii),(iv)). The results above reveal that 2 has an effective therapeutic effect on S. aureus-induced bacterial keratitis in rabbit model. In Vitro Cytotoxicity, Phototoxicity, and Acute Toxicity Evaluation in Mice. The in vitro cytotoxicity study by MTT test showed that 2 has no obvious influence on CHL cell viability at concentrations of 0.064 mg/mL (treatment time, 24 h), and the IC90 of 2 was assayed to be 0.256 mg/mL (Supporting Information Figure S2). The cytotoxicity of 3−7 is also shown in Supporting Information Figure S2. In general, the cell viability of CHL maintains over 75% after being treated with haloemodins (3−7) at a concentration of 0.256 mg/mL for 24 h (Supporting Information Figure S2). The IC90 was 0.256 mg/mL for 3 and was 0.128 mg/mL for 4 and 6. The phototoxicity of 2 was estimated by in vitro 3T3 NRU phototoxicity test in accordance with OECD Testing Guideline 432, and the PIF value and MPE value of 2 were estimated to be 2.095 and 0.148 (Supporting Information Figures S3 and S4, Table S1), showing that 2 could be probable phototoxic in vitro to

cultured cells. For intravenous injection, the LD50 for 2 in this study for mice was 109.6 mg/kg, and no mortality occurred within 7 days after a single intravenous injection of 100 mg/kg (Supporting Information Figure S5).

■

DISCUSSION AND CONCLUSIONS

In this work, we reported a new class of antimicrobial agents, haloemodins, which have distinctive structural difference from the antibiotics we currently use. These novel haloemodins are modified from a traditional Chinese medicine component emodin and have much stronger antibacterial potency against Gram-positive bacteria than emodin itself. More importantly, 2 was confirmed to possess remarkable antibacterial activities against clinically isolated drug-resistant Gram-positive strains VRE, MRSA, and MRSE. To the best of our knowledge, this is the first report of antimicrobial activities and in vivo curative effect of haloemodin. Additionally, 2 is more efficacious than ofloxacin in treating S. Aureus-induced keratitis in a rabbit model. The remarkable antibacterial activity of 2 is probably due to its strong inhibitory activity on the bacterial topoisomerases. The halogen substituents on the benzene rings may increases the affinity of haloemodin to the bacterial topoisomerases, and this is the first time for an anthraquinone derivative to be reported as bacterial DNA gyrase inhibitors DNA gyrase and bacterial Topo I, as essential enzymes for bacterial viability involved in DNA replication, are important antibacterial targets.22−27 During our research, 2 was found very effective in inhibiting the bacterial DNA gyrase-catalyzed kDNA decatenation and DNA supercoiling and exert nearly no inhibitory effect on human Topo IIα. Interestingly, we also found that 2 is much more effective than emodin in inhibiting the catalytic activity of E. coli Topo I and it selectively inhibits the Topo I of E. coli origin and shows no inhibitory effect on human Topo I. In contrast, emodin, as the parent nucleus of 2, shows slight inhibitory effects against bacterial DNA gyrase and 3711

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

strain S. pneumoniae, which has an additional polysaccharide capsule outside the cell wall. So we supposed that the antibacterial activity of 2 was influenced by bacterial superficial structures. PMBN is an OM permeabilizer, which binds to LPS and increases the permeability of bacterial OM to a smallmolecule agent and does not inhibit the growth of bacteria at low concentrations.32,33 With the assistance of PMBN, 2 eventually inhibits the growth of Gram-negative bacterial strains effectively. This proves that in combination with LPS-binding agent PMBN, the antibacterial spectrum of 2 can be expanded into Gram-negative bacteria. In conclusion, 2 is a novel small molecule with substantial antibacterial activities against Gram-positive bacteria, especially the drug-resistant isolates MRSA and VRE in vitro and in vivo. Its specific inhibitory activities against topoisomerases of bacteria origin suggest superiority in safety. Haloemodin, novel bacterial topoisomerase inhibitor, is of value as novel class of pharmacophore for advanced clinical development to combat the attack of emerging drug-resistant bacteria strains.

■

EXPERIMENTAL SECTION

Compounds Synthesis and Identification. Emodin, as a kind of phenolic compound, is a suitable substrate for electrophilic aromatic substitution reactions. In our preliminary investigation of emodin derivatives for their potent antibacterial activity, a number of structural modifications of emodin, mainly the introduction of halogens onto the aromatic rings by electrophilic substitution, was carried out. 2,4Diiodoemodin (2), 2-iodioemodin (5), and 2,4,5-triiodoemodin (6) were prepared by the reaction of emodin with increased mole ratios of elemental iodine in alkali conditions. Chlorination of emodin was subject to the catalysis of acid and MnO2 to afford 2,4,7-trichloro emodin (3). N-Bromosuccinimide (NBS) was employed to catalyze the bromination of emodin which afforded 2,4-dibromoemodin (4). 2Iodine-4-chloroemodin (7) was synthesized by the chlorination of 2iodioemodin (5) under the catalysis of H2O2 in acetic acid. The preparation details of all the compounds are described in the Supporting Information. We used HPLC to confirm the purity of compound 1−7 to be ≥95%. The chromatographic conditions and the specific values of the purity of 1−7 are shown in the Supporting Information. Topo II-Mediated kDNA Decatenation Assay. DNA decatenation assays were performed as TopoGEN instruction in the presence or absence of 2 or emodin. First, a 20 μL of mixture containing 1 U of Topo II (TopoGEN), S. aureus DNA gyrase (TG2000GSA-1), E. coli DNA gyrase (TG2000G-1) or human Topo IIα (TG2000H-1), and 200 ng of catenated kDNA (TopoGEN) in assay buffer was incubated at 37 °C for 30 min. Then the reaction was terminated by the addition of 5 μL of 5× universal stop solution (TG4037). Samples were resolved by electrophoresis on 1% agarose gel which allowed rapid resolution of catenated DNA from minicircles, stained with 0.5 mg/ mL ethidium bromide (EB), and visualized with UV light. DNA Gyrase-Mediated pHOT-1 Supercoiling Assay. The DNA supercoiling assay was performed as TopoGEN instruction in the presence or absence of 2 and emodin. Circular plasmid relaxed DNA pHOT-1 (TopoGEN) was used as DNA substrate. Typically, 20 μL of a mixture containing 100 ng of relaxed pHOT-1, 1 U of S. aureus DNA gyrase (TG2000GSA-1) or E. coli DNA gyrase (TG2000G-1), and the grade concentrations of 2 or emodin were incubated at 37 °C for 30 min. The reaction was terminated by the addition of 2.2 μL of 10% sodium dodecyl sulfate (TG4060). Samples were resolved by electrophoresis on 1% agarose gel for electrophoresis. After electrophoresis, the gels were stained with 0.5 mg/mL ethidium bromide and visualized with UV light. Ciprofloxacin was used as positive control. Topoisomerase I-Mediated pHOT-1 Relaxation Assay and DNA Unwinding Assay. DNA relaxation assay and unwinding assay were performed as TopoGEN instruction in the presence or absence of 2 and emodin. Circular plasmid negative supercoiled DNA pHOT-1

Figure 7. Treatment of S. aureus keratitis in the NZW rabbit by 2. (a) Model group. (b) Ofloxacin group. (c) Compound 2 group. The keratitis induced by intracorneal injection of S. aureus for 24 h is presented in (i) and (iii). Twenty-four h after infection, 30 μL of solvent of 3.0 mg/mL ofloxacin (b) or 4.3 mg/mL compound 2 (c) for injection were topically applied to each eye. After continuous treatment for 10 days, the clinical severity of infected eyes is presented in (ii) and (iv). (i) and (ii), diffuse illumination; (iii) and (iv), direct slit illumination.

exerts no inhibition on E. coli Topo I but possesses relatively strong inhibitory activity upon the mammalian Topo IIα of human origin. The poor inhibitory activities against topoisomerases of human origin suggest a superiority of 2 in safety. Unexpectedly, 2 exerts inhibitory activity against E. coli (Gram-negative) DNA gyrase but shows no independent inhibitory potency against E. coli. However, 2 effectively inhibits the growth of E. coli PQ 37, which have a mutational defect in the OM permeability barrier. As we know, the Gram-negative bacteria, such as E. coli, has an OM outside of the cell wall, and the LPS distributing on the outer leaf of the OM can prevent a small-molecule agent from getting into cells. We also noticed that 2 shows relatively weaker efficiency against Gram-positive 3712

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

approximately 7.5 × 106 CFU. Rabbit eyes were randomly divided into three groups: compound 2 group, model group, and ofloxacin group. Compound 2 was diluted in polyethylene glycol−water solvent (v/v, 1:1). Then 24 h after infection, 30 μL of 2 (4.3 mg/mL) or ofloxacin (3.0 mg/mL) for injection was topically applied to each eye respectively every 4 h for four doses from 8:00 a.m. to 8:00 p.m. every day. The treatment lasted for 10 days in a masked fashion. Eyes were examined before and after treatment by slit lamp biomicroscopy with a YZ5G slit lamp microscope (66 Vision-Tech, China). Acute Toxicity Evaluation in Mice. Male and female adult Kunming mice (18−22 g) were obtained from the Experimental Animal Center of Sichuan University (Sichuan, China) and housed under pathogen-free conditions with a 12 h light/dark cycle. Food and water were given ad libitum throughout the experiment. All procedures, care, and handling of the animals were approved by the Sichuan Animal Care and Use Committee. The in vivo acute toxicity of 2 in Kunming mice was assessed by a single intravenous injection at various doses (n = 10, mice/group, 200 μL per mouse). Compound 2 was dissolved in polyethylene glycol−water solvent (v/v, 1:1) and the controls received injections of vehicle alone. The mortality was recorded for 7 days after administration. The 50% lethal dose (LD50) was estimated by probit analysis using SPSS version 19.0 (SPSS, Inc., Chicago, IL).

(TopoGEN) was used as DNA substrate. Briefly, 100 ng of negative supercoiled pHOT-1 (TopoGEN) was incubated with grade concentrations of 2 and emodin for 15 min in ambient temperature prior to being relaxed using Topo I. Then the mixture added with 1 U of Topo I of E. coli (NEB) or human origin (TopoGEN) was incubated at 37 °C for 30 min. The reaction was terminated by the addition of 2.2 μL of 10% SDS solution (TopoGEN). After digestion with proteinase K (50 μg/mL, 60 min at 37 °C), samples were loaded onto a 1% agarose gel without ethidium bromide, stained with 0.5 mg/ mL ethidium bromide, and visualized with UV light. Camptothecin, which stabilize the nicked intermediate, was used as positive control to indicate the formation of open circular plasmid. MIC Determination. Staphylococcus aureus ATCC6538, Staphylococcus aureus ATCC 29213, Brevibacillus laterosporus ATCC 64, Bacillus cereus ATCC10231, Escherichia coli ATCC 25922, Enterococcus faecalis AfCC 29212, Streptococcus pneumoniae ATCC 49619, Pseudomonas aeruginosa ATCC 27853, and Bacteroides fragilis ATCC 25285 were purchased from China Center of Industrial Culture Collection (CICC). Escherichia coli strain PQ 37 [F-thr leu his-4 pyrD thi galE galK or galT lacΔU169 srl300::Tn10 rpoB rpsL uvrA rfa trp::Muc+ sfiA::Mud(Ap, lac)ts] were gained from the College of Food Science and Nutritional Engineering, China Agricultural University. All the clinical isolates were obtained from the Institute of Clinical Pharmacology, Peking University. Emodin and haloemodins were resolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Vancomycin hydrochloride, cefoxitin sodium salt, penicillin G sodium salt, piperacillin sodium salt, clindamycin 2-phosphate, and ampicillin were resolved in sterile distilled water. MIC values were determined using the CLSI agar dilution method and broth microdilution assay for aerobic and anaerobic bacteria.34−36 E. coli PQ 37 were cultured in LA medium. In the microdilution assay, the bacterial growth was calculated as the different values of ODi and ODu, where ODi and ODu are optical density of inoculated mediums and their corresponding uninoculated wells. The MIC values were recorded as the lowest concentrations of compounds showing no growth of bacteria. MIC values were determined at least twice on separate days, with the higher value used to represent the MIC value. The MIC50 and MIC90 values of compounds were evaluated using SPSS version 19.0 (SPSS, Inc., Chicago, IL). In Vitro Cytotoxicity Measurements and Phototoxicity Evaluation. Chinese hamster lung (CHL) fibroblast cells (inoculated concentration approximately 5 × 104 cells/mL) were cultured on 96well plate and incubated at 37 °C with 5% CO2 overnight and then were treated with compounds (2−7) at different concentrations for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cell viability.31 Absorbance of each well was measured at 570 nm in a Microplate reader (Versa 800). The viability index of untreated cells (cells plus medium) was tested as negative control. Cells incubated with medium alone represented 100% viability. Experiments were performed in triplicate. Phototoxicity evaluation of 2 was estimated by in vitro 3T3 NRU phototoxicity test in accordance with OECD Testing Guideline 432, and the detailed procedure is shown in Supporting Information. Compound Evaluation in Rabbit Model of S. aureus Keratitis. Fifteen New Zealand White (NZW) rabbits (2.0−3.0 kg) were obtained from the Experimental Animal Center of Sichuan University (Sichuan, China) and housed under pathogen-free conditions with a 12 h light/dark cycle. Food and water were given ad libitum throughout the experiment. All animal treatments were strictly in accordance with the tenets of the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research. Before in vivo studies, all eyes of rabbits were examined routinely to exclude any disorders of the cornea, iris, pupil, lens, vitreous, and retina. S. aureus strains were incubated overnight in Luria−Bertani medium (pH 7.0) at 37 °C. This culture was diluted in physiological saline to approximately 1.5 × 108 CFU/mL. Rabbits were anesthetized by injection of sodium pentobarbital solution via auricular vein with the dosage of 30 mg/kg. Proparacaine hydrochloride (0.5% Alcaine, w/v; Alcon) was topically applied to each eye before intrastromal injection. Each cornea was intrastromally injected with a 50 μL suspension containing

■

ASSOCIATED CONTENT

S Supporting Information *

Complete list of the method for synthesis of 2−7. 1H NMR and 13 C NMR spectra for 2−7. Purity determination of 2−7. Inhibitory activity of emodin against bacterial Topo I. Cell viabilities of CHL treated with 2−7. Method for determination of the phototoxicity of 2. Phototoxicity of 2 in vitro. Acute toxicity of 2 in Kunming mice via intravenous injection. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-028-85164073. Fax: +86-028-85164075. E-mail: (W.H.) [email protected]. E-mail: (X.L.) xliu1213@126. com. E-mail: (Y.H.) [email protected]. E-mail: (Q.C.) [email protected]. Author Contributions ∇

F.D., X.L., and S.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The project was supported by the China National “12.5” Foundation (no. 2011BAJ07B04) and Open Foundations (SKLODSCUKF2012-03 and SKLODSCUKF2012-04) from the State Key Laboratory of Oral Diseases Sichuan University and National Natural Science Foundation of China (no. 20972105).

■

ABBREVIATIONS USED ATCC, American Type Culture Collection; CICC, China Center of Industrial Culture Collection; CHL, Chinese hamster lung; CLI, clindamycin; DMSO, dimethylsulfoxide; EB, ethidium bromide; FOX, cefoxitin; HPLC, high-performance liquid chromatography; HRMS, high-resolution mass spectrometry; kDNA, kinetoplast DNA; MIC, minimum concentration; MRSA, Methicillin-resistant Staphylococcus aureus; MRSE, methicillin-resistant Staphylococcus epidermidis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 3713

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714

Journal of Medicinal Chemistry

Article

Enterobacteriaceae. MMWR Morbidity Mortality Wkly. Rep. 2013, 62, 165−170. (16) Perez, F.; Van Duin, D. Carbapenem-resistant Enterobacteriaceae: a menace to our most vulnerable patients. Cleveland Clin. J. Med. 2013, 80, 225−233. (17) Kim, S. Y.; Shin, J.; Shin, S. Y.; Ko, K. S. Characteristics of carbapenem-resistant Enterobacteriaceae isolates from Korea. Diagn. Microbiol. Infect. Dis. 2013, 76, 486−490. (18) Wang, S.-J.; Chiu, S.-H.; Lin, Y.-C.; Tsai, Y.-C.; Mu, J.-J. Carbapenem resistant Enterobacteriaceae carrying New Delhi Metalloβ-lactamase gene (NDM-1) in Taiwan. Diagn. Microbiol. Infect. Dis. 2013, 76, 248−249. (19) Peng, W.; Qin, R.; Li, X.; Zhou, H. Botany, phytochemistry, pharmacology, and potential application of Polygonum cuspidatum Sieb.et Zucc: a review. J. Ethnopharmacol. 2013, 148, 729−745. (20) Oi, H.; Matsuura, D.; Miyake, M.; Ueno, M.; Takai, I.; Yamamoto, T.; Kubo, M.; Moss, J.; Noda, M. Identification in traditional herbal medications and confirmation by synthesis of factors that inhibit cholera toxin-induced fluid accumulation. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 3042−3046. (21) Farnet, C. M.; Wang, B.; Lipford, J. R.; Bushman, F. D. Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 9742−9747. (22) Kohanski, M. A.; Dwyer, D. J.; Collins, J. J. How antibiotics kill bacteria: from targets to networks. Nature Rev. Microbiol. 2010, 8, 423−435. (23) Bradbury, B. J; Pucci, M. J. Recent advances in bacterial topoisomerase inhibitors. Curr. Opin. Pharmacol. 2008, 8, 574−581. (24) Zhao, X.; Xu, C.; Domagala, J.; Drlica, K. DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 13991−13996. (25) Drlica, K.; Zhao, X. DNA gyrase, topoisomerase IV, and the 4quinolones. Microbiol. Mol. Biol. Rev. 1997, 61, 377−392. (26) Yuk-Ching; Tse-Dinh. Bacterial topoisomerase I as a target for discovery of antibacterial compounds. Nucleic Acids Res. 2009, 37, 731−737. (27) Scudiero, O.; Galdiero, S.; Nigro, E.; Del Vecchio, L.; Di Noto, R.; Cantisani, M.; Colavita, I.; Galdiero, M.; Cassiman, J.-J.s; Daniele, A.; Pedoned, C.; Salvatore, F. Chimeric beta-Defensin Analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines. Antimicrob. Agents Chemother. 2012, 57, 1701−1708. (28) Williams, S. C.; Schmaltz, S. P.; Morton, D. J.; Koss, R. G.; Loeb, J. M. Quality of care in U.S. hospitals as reflected by standardized measures, 2002−2004. N. Engl. J. Med. 2005, 353, 255−264. (29) Tanaka, M.; Onodera, Y.; Uchida, Y.; Sato, K.; Hayakawa, I. Inhibitory Activities of Quinolones against DNA Gyrase and Topoisomerase IV Purified from Staphylococcus aureus. Antimicrob. Agents Chemother. 1997, 41, 2362−2366. (30) Mittra, B.; Saha, A.; Chowdhury, A. R.; Pal, C.; Mandal, S.; Mukhopadhyay, S.; Bandyopadhyay, S.; Majumder, H. K. Luteolin, an abundant dietary component is a potent anti-leishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis. Mol. Med. 2000, 6, 527−541. (31) Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 2009, 1794, 808−816. (32) Hancock, R. E.; Wong, P. G. Compounds which increase the permeability of the Pseudomonas aeruginosa outer membrane. Antimicrob. Agents Chemother. 1984, 26, 48−52. (33) Vaara, M. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 1992, 56, 395−411. (34) Performance Standards for Antimicrobial Disk Susceptibility Tests, 11thed.; CLSI: Wayne, PA, 2006. (35) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 9th ed.; CLSI: Wayne, PA, 2006. (36) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 7th ed.; CLSI: Wayne, PA, 2007.

NMR, nuclear magnetic resonance; NZW, New Zealand White; NMR, nuclear magnetic resonance; OM, outer membrane; PEN, penicillin; PIP, piperacillin; PMBN, polymyxin B nonapeptide; Topo, topoisomerase; VAN, vancomycin; VRE, vancomycin-resistant Enterococcus faecium

■

REFERENCES

(1) Enright, M. C.; Robinson, D. A.; Randle, G.; Feil, E. J.; Grundmann, H.; Spratt, B. G. The evolutionary history of methicillinresistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA 2002, 99, 7687−7692. (2) Kuehn, B. MRSA May Move From Livestock to Humans. JAMA, J. Am. Med. Assoc 2012, 308, 1726. (3) Aklilu, E.; Zakaria, Z.; Hassan, L.; Cheng, C. H. Molecular Relatedness of Methicillin-Resistant S. aureus Isolates from Staff, Environment and Pets at University Veterinary Hospital in Malaysia. PloS One 2012, 7, e43329. (4) Ho, P.-L.; Chiu, S. S.; Chan, M. Y.; Gan, Y.; Chow, K.-H.; Lai, E. L.; Lau, Y.-L. Molecular epidemiology and nasal carriage of Staphylococcus aureus and methicillin-resistant S. aureus among young children attending day care centers and kindergartens in Hong Kong. J. Infect. 2012, 64, 500−506. (5) Schneider-Lindner, V.; Quach, C.; Hanley, J. A.; Suissa, S. Antibacterial drugs and the risk of community-associated methicillinresistant Staphylococcus aureus in children. Arch. Pediatr. Adolescent Med. 2011, 165, 1107−1114. (6) García-Á lvarez, L.; Holden, M. T. G.; Lindsay, H.; Webb, C. R; Brown, D. F. J.; Curran, M. D; Walpole, E.; Brooks, K.; Pickard, D. J.; Teale, C.; Parkhill, J.; Bentley, S. D; Edwards, G. F.; Girvan, E. K.; Kearns, A. M.; Pichon, B.; Hill, R. L. R.; Larsen, A. R.; Skov, R. L.; Peacock, S. J.; Maskell, D. J.; Holmes, M. A. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 2011, 11, 595−603. (7) Nickerson, E. K.; West, T. E.; Day, N. P.; Peacock, S. J. Staphylococcus aureus disease and drug resistance in resource-limited countries in south and east Asia. Lancet Infect. Dis. 2009, 9, 130−135. (8) Ke, W.; Huang, S. S.; Hudson, L. O.; Elkins, K. R.; Nguyen, C. C.; Spratt, B. G.; Murphy, C. R.; Avery, T. R.; Lipsitch, M. Patient sharing and population genetic structure of methicillin-resistant Staphylococcus aureus. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 6763−6768. (9) Cooper, B. S.; Medley, G. F.; Stone, S. P.; Kibbler, C. C.; Cookson, B. D.; Roberts, J. A.; Duckworth, G.; Lai, R.; Ebrahim., S. Methicillin-resistant Staphylococcus aureus in hospitals and the community: stealth dynamics and control catastrophes. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 10223−10228. (10) Chemaly, R. F.; Ghantoji, S. S.; Stibich, M. Novel Intervention for Eliminating VRE from the Environment. Arch. Intern. Med. 2011, 171, 1684−1685. (11) Rice, L. B.; Hutton Thomas, R.; Lakticova, V.; Helfand, M. S.; Donskey, C. J. Beta-lactam antibiotics and gastrointestinal colonization with vancomycin-resistant enterococci. J. Infect. Dis. 2004, 189, 1113− 1118. (12) Olivier, C. N.; Blake, R. K.; Steed, L. L.; Salgado, C. D. Risk of Vancomycin-Resistant Enterococcus (VRE) Bloodstream Infection Among Patients Colonized With VRE. Infect. Control Hosp. Epidemiol. 2008, 29, 404−409. (13) Stroh, E. M. Quinupristin/Dalfopristin in Vancomycin-Resistant Staphylococcus aureus Endophthalmitis. Arch. Ophthalmol. 2012, 130, 1323−1324. (14) Hiramatsu, K. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis 2001, 1, 147−155. (15) Jacob, J. T.; Klein, E.; Laxminarayan, R.; Beldavs, Z.; Lynfield, R.; Kallen, A. J.; Edwards, J.; Srinivasan, A.; Fridkin, S.; Rasheed, J. K.; Lonsway, D.; Bulens, S.; Herrera, R.; McDonald, L. C.; Patel, J.; Limbago, B.; Bell, M.; Cardo, D. Vital signs: carbapenem-resistant 3714

dx.doi.org/10.1021/jm401685f | J. Med. Chem. 2014, 57, 3707−3714