Content Alerts

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2009, p. 1427–1433 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00887-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Vol. 53, No. 4 Mode of Action of Ranbezolid against Staphylococci and Structural Modeling Studies of Its Interaction with Ribosomes䌤 Vandana Kalia,1 Rajni Miglani,2 Kedar P. Purnapatre,1 Tarun Mathur,1 Smita Singhal,1 Seema Khan,1 Sreedhara R. Voleti,2† Dilip J. Upadhyay,1 Kulvinder Singh Saini,3 Ashok Rattan,4 and V. Samuel Raj1* Department of Infectious Diseases,1 Department of Molecular Modeling,2 and Department of Biotechnology,3 New Drug Discovery Research, Ranbaxy Research Laboratories, R & D III, Sector-18, Gurgaon 122 015, India, and SRL Ranbaxy, Sector-18, Gurgaon 122 015, India4 Received 6 July 2008/Returned for modification 18 September 2008/Accepted 9 December 2008 Staphylococci are important causes of infection of the bloodstream, cardiac valves, implanted devices, and skin, with repercussions on mortality and morbidity. Treatment of staphylococcal infections is becoming difficult due to the increasing emergence of resistance to beta-lactams and other antimicrobials, including reduced susceptibility to glycopeptides (3). In recent years, there has been a worldwide increase in infection caused by multiple pathogens. The problem is more severe in the hospital setting, where methicillin-resistant Staphylococcus aureus (MRSA) has emerged. The situation has become quite alarming, with the recent emergence of vancomycin-intermediate S. aureus. The experts raise the possibility of returning to the preantibiotic era (24). At present, pharmaceutical companies are looking at various classes of antibiotics to tackle the emergence of drug-resistant pathogens as well as to create better cures for the infections caused by susceptible bacterial strains. The oxazolidinones are a new class of antimicrobial agents that act against a wide spectrum of bacteria, primarily grampositive pathogens (4, 9, 22). Earlier studies of the mechanism of action of oxazolidinones have shown effects on protein synthesis and no effect on DNA and RNA synthesis (8). The oxazolidinone binds to the 50S ribosomal subunit and competes with chloramphenicol and lincomycin (16). The oxazolidinones bind to the 50S subunit first and then interact with the 30S subunit to inhibit the formation of the initiation complex during bacterial translation by preventing the formation of the N-formyl-methionyl-tRNA-ribosome-mRNA ternary complex (28, 30). The nuclear magnetic resonance data presented by Zhou et al. suggest that only the 50S subunit is absolutely required for binding and that any interaction with the 30S subunit is probably indirect (30). Recently, Leach et al. reported that oxazolidinones interact with the A site of the bacterial ribosome, where they interfere with the placement of the aminoacyl-tRNA (15). Linezolid is the first antibacterial drug in the oxazolidinone class used for the treatment of complicated skin and skin structure infections caused by S. aureus (MRSA and methicillinsusceptible S. aureus), Streptococcus pyogenes, or Streptococcus agalactiae; uncomplicated skin and soft tissue infections caused by S. aureus or S. pyogenes; hospital-acquired pneumonia caused by S. aureus; community-acquired pneumonia caused by Streptococcus pneumoniae or S. aureus; and vancomycinresistant Enterococcus faecium infections (22). Further, many investigational oxazolidinones are reported across the globe and have shown a good spectrum of activity against grampositive pathogens (1, 2, 4, 7, 9, 17, 27). Ranbezolid, an investigational oxazolidinone, showed excellent in vitro activity against gram-positive pathogens, especially methicillin-susceptible S. aureus and MRSA, and also against methicillin-susceptible Staphylococcus epidermidis and methicillin-resistant S. epidermidis (MRSE) (5, 11, 19, 20). In addition, ranbezolid is the first oxazolidinone which showed similar activities against both gram-negative and gram-positive anaerobes (6, 11). Further, ranbezolid exhibited significant in vitro activity against slime-producing staphylococci and mycobacteria (18, 26). In this study, we have analyzed the properties of * Corresponding author. Mailing address: Department of Infectious Diseases, New Drug Discovery Research, Ranbaxy Research Laboratories, R & D III, Sector-18, Gurgaon 122 015, India. Phone: 91 124 2342001, ext. 5237. Fax: 91 124 2343544. E-mail: samuel.raj @ranbaxy.com. † Present address: Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 040, India. 䌤 Published ahead of print on 15 December 2008. 1427 Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen. KALIA ET AL. ranbezolid against staphylococci, and we report the mode of action and its interaction with ribosomes. MATERIALS AND METHODS Bacterial strains and antibiotics. The following ATCC strains were used for the mode-of-action studies: S. aureus 25923 and S. epidermidis 23760 (MRSE). Ranbezolid was synthesized in house, and other antibiotics were procured from commercial sources. The radiolabeled compounds [3H]thymidine, [3H]uridine, [14C]isoleucine, [14C]acetate, and [3H]N-acetylglucosamine were procured either from GE Healthcare (United States) or from the Board of Radiation and Isotope Technology (India). Macromolecular synthesis in S. aureus and S. epidermidis. Macromolecular biosynthesis inhibition in S. aureus and S. epidermidis was studied as described by Oliva et al., with some modifications (23). The following radiolabeled precursors were added to cells for the macromolecular synthesis inhibition assays: protein assay, [14C]isoleucine; DNA assay, [3H]thymidine; RNA assay, [3H]uridine; cell wall assay, [3H]N-acetylglucosamine; and fatty acid assay, [14C]acetate. In brief, S. aureus 25923 and S. epidermidis 23760 were grown in Mueller-Hinton broth (MHB) medium, and radioactive precursors (1 ␮Ci/ml for 3H-labeled and 0.1 ␮Ci/ml for 14C-labeled compounds) were added during the early logarithmic phase (optical density at 600 nm [OD600] of 0.3). After 5 min, the inhibitors were added and their MICs determined by the microdilution method. The macromolecules (DNA, RNA, protein, cell wall, and fatty acid) were precipitated with ice-cold trichloroacetic acid (final concentration of 5% [wt/vol]) and filtered on glass fiber filters (1.0 ␮M A/B glass multiwell filter plates, Pall Corporation). The plates were dried overnight at 37°C, and quantification of radioactivity was done by using OptiPhase safe scintillation fluid and counting with a scintillation counter (Wallac Ltd.). For each set of experiments, an antibiotic known to specifically inhibit the macromolecule was included as a positive control. Time-kill kinetic study against S. aureus and S. epidermidis. A time-kill kinetics assay was performed as per Hoellman et al. (11). S. aureus 25923 was exposed to ranbezolid and linezolid at concentrations ranging from 1 ␮g/ml to 16 ␮g/ml. S. epidermidis 23760 was exposed to ranbezolid and linezolid at concentrations ranging from 0.25 to 8 ␮g/ml. Time-kill assays were analyzed by determining the reductions in viable count (log10 CFU/ml) at 2, 4, 8, 24, and 30 h and comparing those with that at 0 h. Antibiotics were considered bactericidal at the lowest concentration that reduced the original inoculum by ⱖ3 log10 CFU/ml (99.9% killing) at each time point and bacteriostatic if reduced by ⬍3 log10 CFU/ml. Cell membrane permeability study. The BacLight kit from Molecular Probes was used to assess membrane damage with drugs, as described by Hilliard et al. (10). S. aureus 25923 as well as S. epidermidis 23760 was grown overnight in MHB medium at 37°C. The culture was diluted 1:100 in fresh MHB and grown up to an OD600 of 0.5 to 0.6. The bacterial suspension was centrifuged at 10,000 ⫻ g for 15 min, and the cell pellet was washed once in sterilized distilled water. The cell pellet was resuspended to one-tenth of the original volume and then diluted 1:20 into either water or water containing ranbezolid and linezolid at concentrations ranging from 1 to 8 ␮g/ml. For each set of membrane integrity experiments, the known membrane disrupting agent valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as positive controls. Bacteria and compounds were incubated at room temperature (⬃25°C) with continuous shaking for 15 to 30 min. The suspension was centrifuged at 10,000 ⫻ g for 10 min, washed with sterilized water, and resuspended to an OD670 of 0.325. A volume of 100 ␮l of the bacterial suspension was added to a 96-well black color Nunc tissue culture plate. BacLight reagent (3 ␮l/ml bacterial suspension) was then added to each well, and the plates were incubated in the dark for 15 min at room temperature. The plate was read at excitation/emission maxima of 480/500 nm (Syto 9) and 490/635 nm (propidium iodide) in a fluorescence microplate reader. Cell-free transcription/translation assay. In vitro translation of the luciferase gene was done with bacterial or mammalian ribosome using commercially available Escherichia coli S30 and a TNT kit (Promega), as per the manufacturer’s protocol. The percent inhibition of luciferase activity in the presence of various concentrations of drug was determined and compared with that for the control. The 50% inhibitory concentrations were calculated using Graph Pad Prism software. Molecular modeling studies for interaction of ranbezolid with ribosome. Molecular docking studies were carried out using Surflex in the SYBYL7.3 molecular modeling package (version 7.3; Tripos Associates Inc., St. Louis, MO) installed on a Silicon Graphics Fuel workstation running on the IRIX 6.5 operating system (13, 14, 25). Surflex is a new docking module that employs an idealized active-site ligand, called a protomol, as a target to generate putative poses of molecules or molecular fragments. These putative poses are scored using the Hammerhead scoring function, which also serves as an objective func- ANTIMICROB. AGENTS CHEMOTHER. tion for local optimization of poses. The crystal structure of an E. coli 50S large subunit of ribosome (Protein Data Bank identification no. [PDB ID] 2AW4) was taken, and its working model was built using residues within 30 Å of A2541 by using information from Leach et al. (15). Charges were removed, and hydrogen atoms were added to the crystal structure. Amber FF99 charges were loaded on the ribosome crystal structure. Protomol (active site) and the sfxc files (surflex descriptor file) were generated in automatic mode. Threshold and bloat parameters were adjusted to best describe the active site. Linezolid and ranbezolid were drawn in SYBYL7.3, and Gasteiger-Hückel charges were added to them. Linezolid and ranbezolid were docked in the generated sfxc file. RESULTS To elucidate the molecular mechanism of action of a new investigational oxazolidinone, ranbezolid, we examined its inhibitory activity in bacterial macromolecular synthesis. Further, we examined the effect of ranbezolid and linezolid on the membrane integrity of staphylococci and inhibition of the in vitro translation system. Ranbezolid showed better performance than linezolid. Macromolecular synthesis inhibition. The effect of ranbezolid and linezolid on the incorporation of radiolabeled precursors into protein, DNA, RNA, cell wall, and lipid was determined. Ranbezolid (1 ␮g/ml) and linezolid (2 ␮g/ml) were found to be potent inhibitors of protein synthesis in S. aureus 25923 as well as in S. epidermidis 23760 (Fig. 1 and 2). DNA and RNA synthesis was not inhibited by either ranbezolid or linezolid in S. aureus 25923 or in S. epidermidis 23760. Ranbezolid showed inhibition of cell wall and lipid synthesis in S. epidermidis 23760 at 60 min (Fig. 2a). Though there was inhibition of cell wall and lipid synthesis at 60 min by linezolid in S. epidermidis, the effect was less than that of ranbezolid. The control compounds demonstrated specific inhibition of the expected targets at their MICs compared to effects on other biosynthetic pathways (data not shown). Kill kinetics of ranbezolid and linezold against S. aureus and S. epidermidis. The results of time-kill analysis for ranbezolid and linezolid are presented in Fig. 3. Ranbezolid and linezolid were mainly bacteriostatic against S. aureus 25923 (up to 8⫻ MIC for 30 h). But, ranbezolid showed bactericidal activity at the concentration of 16 ␮g/ml against S. aureus 25923 (Fig. 3a). Ranbezolid showed bactericidal (99.9% killing) activity against S. epidermidis 23760, whereas linezolid did not show any bactericidal effect (Fig. 3b). Bactericidal effect of ranbezolid at 4⫻ MIC (4 ␮g/ml) against S. epidermidis 23760 was observed at 6 h. Linezolid showed bacteriostatic effect at 4⫻ MIC (8 ␮g/ml) at 24 h. Thus, effective killing was observed with ranbezolid compared to linezolid. In fact, ranbezolid showed superior killing activity at 4 h (8 ␮g/ml) as well as at 6 h (4 ␮g/ml). Membrane permeability assay. Bacterial membrane damage was examined by using the BacLight assay (Fig. 4). The known membrane-disrupting agents valinomycin (100 ␮M) and CCCP (15 ␮M) damaged the S. aureus membrane as well as the S. epidermidis membrane, with BacLight assay values resulting in ⬍40% of the control value. Linezolid (4 ␮g/ml) and ranbezolid (4 ␮g/ml) had no effect on the membrane integrity of S. aureus (Fig. 4a). Cell membrane integrity damage was observed with S. epidermidis 23760 (MRSE) by ranbezolid at 4 ␮g/ml, whereas no damage was observed with linezolid at 4 ␮g/ml. Since there was an indication of membrane damage by ranbezolid (4 ␮g/ml) with S. epidermidis, the effect of ranbezolid on Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. 1428 VOL. 53, 2009 MODE OF ACTION OF RANBEZOLID 1429 S. epidermidis was determined at various concentrations (1 to 8 ␮g/ml). As shown in Fig. 4b, the membrane integrity damage was observed at 2 to 8 ␮g/ml of ranbezolid. A dose-dependent effect of ranbezolid against S. epidermidis was observed. Ranbezolid exhibited a mean BacLight value, 50% of the control (Fig. 4b), indicating membrane-damaging activity in this assay at 2⫻ MIC (2 ␮g/ml). However, ranbezolid displayed no effect on the membrane at 1⫻ MIC (1 ␮g/ml), compared to the known membrane-disrupting agents valinomycin and CCCP. In vitro cell-free transcription and translation assay (S30 and TNT assay). An in vitro cell-free transcription and translation assay was used to find out the mode of action of ranbezolid and its specificity toward bacterial ribosomes. Ranbezolid was found to be a more potent inhibitor of the cell-free in vitro transcription and translation system than linezolid (Table 1). The 50% inhibitory concentration (IC50) of linezolid was 100 ␮M for bacterial ribosomes, and that of ranbezolid was 17 ␮M. Ranbezolid showed at least five- to sixfold better potency than linezolid as a bacterial ribosome inhibitor. The safety index was found to be sevenfold higher for ranbezolid than for linezolid. Thus, our data clearly indicate that ranbezolid is a potent bacterial protein synthesis inhibitor, not a mammalian protein synthesis inhibitor. Interaction of ranbezolid with ribosomes. The linezolid binding site (or active site) of E. coli 50S ribosomes was found to lie within the important residues G2053, A2054, C2055, G2056, G2057, A2058, A2059, G2061, A2062, C2452, A2451, A2503, U2504, G2505, U2506, G2576, A2577, U2585, C2611, C2612, etc. From the overlay of the docked conformation of linezolid with the recently cocrystallized linezolid structures with 50S ribosome subunits of Haloarcula marismortui (PDB ID 3CPW) and Deinococcus radiodurans (PDB ID 3DLL), as shown in Fig. 5a, it is evident that linezolid has identical orientations and interacts with the residues in similar manners (12, 29). Moreover, ranbezolid has a FIG. 2. Macromolecular synthesis inhibition in S. epidermidis ATCC 23760 (MRSE) by (a) ranbezolid and (b) linezolid. The MICs were 1 ␮g/ml for ranbezolid and 2 ␮g/ml for linezolid. Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. FIG. 1. Macromolecular synthesis inhibition in S. aureus ATCC 25923 by (a) ranbezolid and (b) linezolid. The MICs determined by the microdilution method were 1 ␮g/ml for ranbezolid and 2 ␮g/ml for linezolid. KALIA ET AL. FIG. 3. (a) Time-kill kinetics of ranbezolid and linezolid against S. aureus ATCC 25923 (䡬, control; ●, ranbezolid at 4 ␮g/ml; 䡺, ranbezolid at 8 ␮g/ml; f, ranbezolid at 16 ␮g/ml; ‚, linezolid at 4 ␮g/ml; Œ, linezolid at 8 ␮g/ml; ƒ, linezolid at 16 ␮g/ml). (b) Time-kill kinetics of ranbezolid and linezolid against S. epidermidis ATCC 23760 (䡬, control; ●, ranbezolid at 2 ␮g/ml; 䡺, ranbezolid at 4 ␮g/ml; f, ranbezolid at 8 ␮g/ml; ‚, linezolid at 4 ␮g/ml; Œ, linezolid at 8 ␮g/ml). structure almost identical to that of linezolid, in which the morpholine ring has been replaced with a piperazine ring attached with a methyl nitrofuran ring. The chemical structures of these two ligands are shown in Fig. 5b. Docking studies carried out on ranbezolid thus showed similar orientations and interactions within the active site of E. coli (Fig. 6a). It was observed that ranbezolid has a higher total score after consensus than linezolid (6.9 and 5.2, respectively). The oxazolidinone rings of both inhibitors stack with the face of the uracil base of U2504. Residues present on the sides of the oxazolidinone ring are the sugar moiety of G2505 and the base of G2061. In the preferred conformation after the consensus scoring, the amide side-chain moiety of ranbezolid is in a folded position over the oxazolidinone ring. The N-H of amide on the oxazolidinone ring of ranbezolid makes a hydrogen bond from the 5⬘-oxygen of G2505, as shown in Fig. 6a. The central aromatic ring is stabilized by the bases A2451 and C2452. The piperazine and morpholine rings are stabilized by van der Waals interactions with the sugar residues A2451, C2452, and U2506. The nitrofuran ring is extended toward the residues C2507, G2583, and U2584. The furan ring is also stabilized through van der Waals interactions by U2584 and U2506, and the nitro group on the ANTIMICROB. AGENTS CHEMOTHER. FIG. 4. Effect of ranbezolid (RAN) and linezolid (LNZ) on cell membrane integrity of (a) S. aureus 25923 and (b) S. epidermidis 23760. The BacLight bacterial viability kit (Molecular Probes) is composed of the green-fluorescent nucleic acid stain (Syto 9) and the red-fluorescent nucleic acid stain (propidium iodide). In this assay, the Syto 9 and propidium iodide stains compete for binding to the bacterial nucleic acid. Syto 9 labels cells with both damaged and intact membranes, whereas propidium iodide penetrates only cells with damaged membranes. The fluorescence ratio of Syto 9 (480/500 nm) to propidium iodide (490/635) was calculated and compared with that for controls. furan ring forms a hydrogen bond with the ⫺NH2 moiety on the base of G2583 (Fig. 6a). In other docked conformations of ranbezolid, the methyl nitrofuran adopts different orientations within the active site, as shown in Fig. 6b. This could be due to the flexibility provided by the methylene group in ranbezolid, and the nitro group has possible H-bonding interactions with different residues, U2584 or G2505, of the 50S ribosome subunit. TABLE 1. IC50s for ranbezolid and linezolid in an in vitro transcription/translation system against bacterial and mammalian ribosomes Oxazolidinone Ranbezolid Linezolid a IC50 (mM)a S30 TNT Safety index (TNT IC50/S30 IC50) 0.017 ⫾ 0.005 0.1 ⫾ 0.002 14.7 ⫾ 2.6 11.9 ⫾ 1.1 865 118 Values are means ⫾ standard deviations. Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. 1430 VOL. 53, 2009 MODE OF ACTION OF RANBEZOLID 1431 DISCUSSION The oxazolidinones are inhibitors of bacterial ribosomal protein synthesis, but unlike other antimicrobial agents that target the ribosome, the oxazolidinones appear to have a unique mechanism of action (4, 16, 28). Oxazolidinones inhibit protein synthesis by binding to the 23S rRNA of the 50S ribosomal subunit and interfering with the initiator fMet-tRNA binding to the P site of the ribosomal peptidyltransferase center (28). As oxazolidinones, both ranbezolid and linezolid clearly inhibited protein synthesis in S. aureus as well as S. epidermidis. Our data are consistent with other studies, as these oxazolidinones inhibit protein synthesis in similar manners (4, 16). The clear inhibitions of protein synthesis by both ranbezolid and linezolid are oxazolidione class-specific effects, as they bind to 50S ribosomes and inhibit protein synthesis (28, 30). Ranbezolid showed specificity toward bacterial ribosomes but not toward mammalian ribosomes and was found to be a potent inhibitor of bacterial ribosomes. This potency may be due to the tight binding of ranbezolid to bacterial ribosomes as well as additional interaction sites between ranbezolid and ribosomes. The additional inhibition of cell wall and lipid synthesis by ranbezolid in S. epidermidis was observed. This could be one of the reasons for the better in vitro as well as bactericidal activity of ranbezolid against S. epidermidis. Such additional or nonspecific modes of action, like cell wall and lipid synthesis inhibition by oxazolidinones, have not yet been reported by others. In a BacLight assay, agents which produce permeability val- ues of ⬍40% of the control are considered to be membranedamaging agents (10). The positive controls valinomycin (100 ␮M) and CCCP (15 ␮M) severely damaged the S. aureus membrane as well as the S. epidermidis membrane, with a BacLight assay value of ⬍40% of the control. Ranbezolid and linezolid at 4 ␮g/ml had no effect on membrane integrity in S. aureus. So far, no reports regarding the bacterial membrane damage activity by oxazolidinones are available. However, ranbezolid showed additional membrane damage activity in S. epidermidis. The data suggest that certain structural features of ranbezolid may be affecting membrane integrity. Ranbezolid has a structure almost identical to that of linezolid, but the morpholine ring has been replaced with a piperazine ring and has an additional nitrofuran ring. The nitrofuran ring may contribute to the damage of membrane integrity in S. epidermidis. Since membrane integrity is damaged by ranbezolid even at 2 ␮g/ml (2⫻ MIC) in S. epidermidis, it could be one of the reasons for its bactericidal activity. Linezolid did not show any membrane damage effect up to 8 ␮g/ml (4⫻ MIC) against S. epidermidis and also showed a bacteriostatic effect against S. epidermidis in time-kill kinetics studies. So, membrane damage activity may be the reason for the bactericidal effect of ranbezolid against S. epidermidis. Since ranbezolid specifically inhibits bacterial protein synthesis, the next foremost question to be answered was the binding mode of ranbezolid to bacterial ribosomes. As shown in Fig. 5b, ranbezolid is structurally similar to linezolid, whose Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. FIG. 5. Active site residues of 50S ribosomes of E. coli for the interaction of linezolid and ranbezolid. (a) Overlay of docked conformation of linezolid in E. coli with the cocrystal structures of linezolid with bacterial 50S ribosomes. Critical active site residues of E. coli are shown in white-stick model; labels for E. coli, H. marismortui, and D. radiodurans are shown in red, orange, and green, respectively. Linezolid is shown in capped-stick model (magenta, E. coli; green, H. marismortui; and orange, D. radiodurans). (b) Two-dimensional chemical structures of linezolid (LNZ) and ranbezolid (RAN). 1432 KALIA ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 6. Binding conformation of ranbezolid and interactions with ribosome residues. (a) Putative binding conformation and preferred conformation of ranbezolid (yellow, capped-stick model) and its interactions with the 50S ribosome residues (atom type, stick model); putative binding conformation of linezolid is shown in magenta, line model. (b) Other low-scoring conformations of ranbezolid (violet, orange, cyan; stick model) in which methyl nitrofuran ring adopts different orientations within the active site. H bonds are shown in dotted black lines, with their corresponding distances given. ACKNOWLEDGMENTS We thank Pradip Kumar Bhatnagar for helpful discussions and critical comments. The financial support for research from Ranbaxy Research Laboratories is acknowledged. REFERENCES 1. Barry, A. L. 1988. In vitro evaluation of DuP 105 and DuP 721, two new oxazolidinone antimicrobial agents. Antimicrob. Agents Chemother. 32:150– 152. 2. Bozdogan, B., and P. C. Appelbaum. 2004. Oxazolidinones: activity, mode of action, and mechanism of resistance. Int. J. Antimicrob. Agents 23:113–119. 3. Chopra, I. 2003. Antibiotic resistance in Staphylococcus aureus: concerns, causes and cures. Expert Rev. Anti Infect. Ther. 1:45–55. 4. Daly, J. S., G. M. Eliopoulos, S. Willey, and R. C. Moellering, Jr. 1988. Mechanism of action and in vitro and in vivo activities of S-6123, a new oxazolidinone compound. Antimicrob. Agents Chemother. 32:1341–1346. 5. Das, B., S. Rudra, A. Yadav, A. Ray, A. V. Rao, A. S. Srinivas, A. Soni, S. Saini, S. Shukla, M. Pandya, P. Bhateja, S. Malhotra, T. Mathur, S. K. Arora, A. Rattan, and A. Mehta. 2005. Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid. Bioorg. Med. Chem. Lett. 15:4261–4267. 6. Ednie, L. M., A. Rattan, M. R. Jacobs, and P. C. Appelbaum. 2003. Antianaerobe activity of RBX 7644 (ranbezolid), a new oxazolidinone, compared with those of eight other agents. Antimicrob. Agents Chemother. 47:1143– 1147. 7. Eliopoulos, G. M., C. B. Wennersten, H. S. Gold, and R. C. Moellering, Jr. 1996. In vitro activities in new oxazolidinone antimicrobial agents against enterococci. Antimicrob. Agents Chemother. 40:1745–1747. 8. Eustice, D. C., P. A. Feldman, I. Zajac, and A. M. Slee. 1988. Mechanism of action of DuP 721: inhibition of an early event during initiation of protein synthesis. Antimicrob. Agents Chemother. 32:1218–1222. 9. Ford, C. W., J. C. Hamel, D. M. Wilson, J. K. Moerman, D. Stapert, R. J. Yancey, Jr., D. K. Hutchinson, M. R. Barbachyn, and S. J. Brickner. 1996. In vivo activities of U-100592 and U-100766, novel oxazolidinone antimicrobial agents, against experimental bacterial infections. Antimicrob. Agents Chemother. 40:1508–1513. 10. Hilliard, J. J., R. M. Goldschmidt, L. Licata, E. Z. Baum, and K. Bush. 1999. Multiple mechanisms of action for inhibitors of histidine protein kinases Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. piperazine ring makes van der Waals contacts with the sugar residues of A2451, C2452, and U2506. Figures 6a and 6b show the various conformations of ranbezolid generated through docking studies that indicate a possible hydrogen-bonding interaction between the nitrofuran moiety with the residues G2505, U2584, and G2583, indicating a dynamic H-bonding linkage, although one cannot fix one orientation through rigid body docking. The preferred and top-ranked conformation indicates the H-bonding interaction with G2583. Miller et al. reported that the conformations of the oxazolidinone rings were similar in both E. coli and S. aureus models, which have an aromatic ring stacked with the uracil base of U2504 and also feature a hydrogen bond between the amide NH and the 5⬘ oxygen of G2505 (21). Overall, both ranbezolid and linezolid interact in similar fashions with ribosomes, while the additional van der Waals and H-bonding interactions made by the nitrofuran moiety of ranbezolid may offer a plausible explanation for its better antibacterial activity over that of linezolid. Thus, ranbezolid has many advantages over linezolid in the oxazolidinone class of antibacterial drugs. Ranbezolid is a potent protein synthesis inhibitor with selectivity toward bacterial but not mammalian protein synthesis. Membrane integrity damage and additional inhibition of cell wall and lipid synthesis by ranbezolid against S. epidermidis could make ranbezolid potent as well as bactericidal. Additional binding-site interactions of ranbezolid with bacterial ribosomes explain its stronger interactions with the target pathogen. Thus, ranbezolid is a potent inhibitor against staphylococcus infection and has added advantages over linezolid. 11. 12. 13. 14. 15. 16. 17. 18. 19. from bacterial two-component systems. Antimicrob. Agents Chemother. 43: 1693–1699. Hoellman, D. B., G. Lin, L. M. Ednie, A. Rattan, M. R. Jacobs, and P. C. Appelbaum. 2003. Antipneumococcal and antistaphylococcal activities of ranbezolid (RBX 7644), a new oxazolidinone, compared to those of other agents. Antimicrob. Agents Chemother. 47:1148–1150. Ippolito, J. A., Z. K. Kanyo, D. Wang, F. J. Franceschi, P. B. Moore, T. A. Steitz, and E. M. Duffy. 2008. Crystal structure of the oxazolidinone antibiotic linezolid bound to the 50S ribosomal subunit. J. Med. Chem. 51:3353– 3356. Jain, A. N. 1996. Scoring noncovalent protein-ligand interactions: a continuous differentiable function tuned to compute binding affinities. J. Comput. Aided Mol. Des. 10:427–440. Jain, A. N. 2000. Morphological similarity: a 3D molecular similarity method correlated with protein-ligand recognition. J. Comput. Aided Mol. Des. 14:199–213. Leach, K. L., S. M. Swaney, J. R. Colca, W. G. McDonald, J. R. Blinn, L. M. Thomasco, R. C. Gadwood, D. Shinabarger, L. Xiong, and A. S. Mankin. 2007. The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol. Cell 26:393–402. Lin, A. H., R. W. Murray, T. J. Vidmar, and K. R. Marotti. 1997. The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin. Antimicrob. Agents Chemother. 41:2127–2131. Livermore, D. M., M. Warner, S. Mushtaq, S. North, and N. Woodford. 2007. In vitro activity of the oxazolidinone RWJ-416457 against linezolid-resistant and -susceptible staphylococci and enterococci. Antimicrob. Agents Chemother. 51:1112–1114. Mathur, T., P. Bhateja, M. Pandya, T. Fatma, and A. Rattan. 2004. In vitro activity of RBx 7644 (ranbezolid) on biofilm producing bacteria. Int. J. Antimicrob. Agents 24:369–373. Mehta, A., A. V. S. Rao, and A. Rattan. November 2004. World Intellectual Property Organization patent WO 04/099199. MODE OF ACTION OF RANBEZOLID 1433 20. Mehta, A., S. Arora, B. Das, A. Ray, S. Rudra, and A. Rattan. January 2003. World Intellectual Property Organization patent WO 03/007870. 21. Miller, K., C. J. Dunsmore, C. W. Fishwick, and I. Chopra. 2008. Linezolid and tiamulin cross-resistance in Staphylococcus aureus mediated by point mutations in the peptidyl-transferase center. Antimicrob. Agents Chemother. 52:1737–1742. 22. Moellering, R. C. 2003. Linezolid: the first oxazolidinone antimicrobial. Ann. Intern. Med. 138:135–142. 23. Oliva, B., A. O’Neill, J. M. Wilson, P. J. O’Hanlon, and I. Chopra. 2001. Antimicrobial properties and mode of action of the pyrrothine holomycin. Antimicrob. Agents Chemother. 45:532–539. 24. Rattan, A. 2003. RBx-7644. Drugs Future 28:1070–1077. 25. Ruppert, J., W. Welch, and A. N. Jain. 1997. Automatic identification and representation of protein binding sites for molecular docking. Protein Sci. 6:524–533. 26. Sood, R., M. Rao, S. Singhal, and A. Rattan. 2005. Activity of RBx 7644 and RBx 8700, new investigational oxazolidinones, against Mycobacterium tuberculosis infected murine macrophages. Int. J. Antimicrob. Agents 25:464– 468. 27. Sreenivas, K., P. V. Amarnath, A. Mallik, H. Sarnaik, N. S. Kumar, M. Takhi, S. Trehan, M. S. Kumar, J. Iqbal, R. Rajagopalan, and R. Chakrabarti. 2007. In vitro and in vivo antibacterial evaluation of DRF 8417, a new oxazolidinone. J. Antimicrob. Chemother. 60:159–161. 28. Swaney, S. M., H. Aoki, M. C. Ganoza, and D. L. Shinabarger. 1998. The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob. Agents Chemother. 42:3251–3255. 29. Wilson, D. N., F. Schluenzen, J. M. Harms, A. L. Starosta, S. R. Connell, and P. Fucini. 2008. The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning. Proc. Natl. Acad. Sci. USA 105:13339–13344. 30. Zhou, C. C., S. M. Swaney, D. L. Shinabarger, and B. J. Stockman. 2002. 1H nuclear magnetic resonance study of oxazolidinone binding to bacterial ribosomes. Antimicrob. Agents Chemother. 46:625–629. Downloaded from https://journals.asm.org/journal/aac on 16 January 2022 by 3.90.248.229. VOL. 53, 2009