Determinants of the Species Selectivity of Oxazolidinone Antibiotics Targeting the Large Ribosomal Subunit

DOI 10.1515/hsz-2013-0188 Biol. Chem. 2013; 394(11): 1529–1541

Jagmohan S. Saini, Nadine Homeyer, Simone Fullea and Holger Gohlke* Determinants of the species selectivity of oxazolidinone antibiotics targeting the large ribosomal subunit

Abstract: Oxazolidinone antibiotics bind to the highly Introduction conserved peptidyl transferase center in the ribosome. For developing selective antibiotics, a profound under- Ribosomes are complex nanomachines that perform standing of the selectivity determinants is required. We protein synthesis in all living cells. This pivotal role makes have performed for the first time technically challenging the bacterial ribosome a prominent target for antibiotics molecular dynamics simulations in combination with that exert their antimicrobial effect by interfering with molecular mechanics Poisson-Boltzmann surface area protein synthesis (David-Eden et al., 2010). Several crystal (MM-PBSA) free energy calculations of the oxazolidinones structures of ribosomal subunits bound to different classes linezolid and radezolid bound to the large ribosomal sub- of antibiotics have been published over the last decade units of the eubacterium Deinococcus radiodurans and revealing the binding site and binding mode in atomic the archaeon Haloarcula marismortui. A remarkably good detail as well as the structural basis for antibiotic specific- agreement of the computed relative binding free energy ity (Brodersen et al., 2000; Carter et al., 2000; Bottger et al., with selectivity data available from experiment for lin- 2001; Schlunzen et al., 2001; Hansen et al., 2003; Yonath, ezolid is found. On an atomic level, the analyses reveal an 2005a; Ippolito et al., 2008; Wilson et al., 2008; Belousoff intricate interplay of structural, energetic, and dynamic et al., 2011). These structural insights are complemented determinants of the species selectivity of oxazolidinone by several computational studies addressing the dynamic antibiotics: A structural decomposition of free energy and energetic determinants of antibiotics binding to the components identifies influences that originate from first ribosome (Ma et al., 2002; Trylska et al., 2005; Vaiana et al., and second shell nucleotides of the binding sites and 2006; Aleksandrov and Simonson, 2008; Ge and Roux, lead to (opposing) contributions from interaction ener- 2009, 2010; David-Eden et al., 2010; Romanowska et al., gies, solvation, and entropic factors. These findings add 2011). Overall, these studies have provided crucial insights another layer of complexity to the current knowledge on into the mode of action of several classes of antibiotics that structure-activity relationships of oxazolidinones bind- should facilitate efforts to design new antibiotics in order ing to the ribosome and suggest that selectivity analyses to combat the emerging rise in infections due to antibiotic- solely based on structural information and qualitative resistant bacteria and a related mortality rate. arguments on interactions may not reach far enough. The Oxazolidinones represent one of only two new chemi- computational analyses presented here should be of suf- cal classes of antibiotics that have been introduced in the ficient accuracy to fill this gap. clinics over the past 40 years. Linezolid, the only member of this class approved by the FDA, shows excellent activity Keywords: entropy; eubacterium; free energy; linezolid; against major Gram-positive bacteria and is very effective in MM-PBSA; molecular dynamics simulations. the treatment of infections of the respiratory tract and skin disorders (Moellering, 2003; Leach et al., 2011). The co-crystal structures of linezolid with the large ribosomal subunit aPresent address: BioMed X GmbH, Im Neuenheimer Feld 583, (50S) of Deinococcus radiodurans (D50S), a eubacterium, D-69120 Heidelberg, Germany. *Corresponding author: Holger Gohlke, Institute for Pharmaceutical (Wilson et al., 2008) and Haloarcula marismortui (H50S), and Medicinal Chemistry, Department of Mathematics and Natural an archaeon, (Ippolito et al., 2008) show that the antibi- Sciences, Heinrich Heine University, Universitätsstr. 1, D-40225 otic exerts its action by binding to the A-site of the peptidyl Düsseldorf, Germany, e-mail: [email protected] transferase center (PTC) and, thereby, hinders the proper Jagmohan S. Saini, Nadine Homeyer and Simone Fulle: Institute placement of the incoming aminoacyl-tRNA (Figure 1A). for Pharmaceutical and Medicinal Chemistry, Department of Mathematics and Natural Sciences, Heinrich Heine University, Archaeal ribosomes are considered more ‘eukaryotic-like’ Universitätsstr. 1, D-40225 Düsseldorf, Germany with respect to their antibiotic specificity, i.e., they possess typical eukaryotic elements at the principal antibiotic

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1530 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics

Figure 1 Binding site of oxazolidinone antibiotics in the 50S ribosomal subunit and investigated oxazolidinone derivatives. (A) Global view of the 50S ribosomal subunit showing the location of the binding site of oxazolidinones within the 23S RNA (red). The ribosomal protein chains and the 5S RNA are shown in gray. (B) Close-up view of the binding site with a superposition of binding site nucleotides and linezolid in the H50S (black; PDB code: 3CPW) and D50S (blue; PDB code: 3DLL) structures. Depicted are nucleotides that are part of the first shell of nucleotides around linezolid. Hydrogen bonds are marked by dotted lines: In the H50S structure, a hydrogen bond is formed between the phosphate group of G2505 and the acetamide NH group of linezolid; in the D50S structure, a hydrogen bond is formed between U2585 and the morpholino ring of linezolid. (C) Chemical structures of oxazolidinones investigated in this study: linezolid, radezolid, and rivaroxaban. target sites and require much higher than clinically rel- In order to provide a better understanding of the selec- evant antibiotic concentrations for binding (Mankin and tivity determinants of the oxazolidinone class of antibiotics, Garrett, 1991; Sanz et al., 1993; Hansen et al., 2002; Hansen we have performed for the first time molecular dynamics et al., 2003; Yonath, 2005b). This holds true also for the (MD) simulations in combination with molecular mechan- oxazolidinone class of antibiotics: for co-crystallizing line ics Poisson-Boltzmann surface area (MM-PBSA) free energy zolid with the eubacterial D50S, a concentration of 5 μm calculations of linezolid bound to D50S and H50S. Further- was required, while a 1000-fold higher concentration was more, we investigated the oxazolidinones radezolid and required for co-crystallization with the archaeal H50S rivaroxaban (Figure 1C). Radezolid is a promising member of (Wilson et al., 2008; Wilson, 2011). Furthermore, in a func- the oxazolidinone class of antibiotics, which has completed tional assay using ribosomes isolated from Staphylococcus phase II of clinical trials [(Shaw and Barbachyn, 2011); http:// aureus, a eubacterium, a translation-inhibitory activity of www.rib-x.com, access date: 11th May 2013], requires a 100-

IC50 = 0.9 μm was measured for linezolid (Skripkin et al., times lower concentration than linezolid to inhibit protein

2008), whereas in the case of H. marismortui, an IC50 of synthesis in eubacterial ribosomes (Skripkin et al., 2008), 4.96 μm was found for linezolid (E. M. Duffy, personal com- and shows an improved pattern of selectivity to bacterial munication). This also conveys a selectivity of linezolid for ribosomes (Zhou et al., 2008a). In contrast, rivaroxaban, an the eubacterial ribosome. To the best of our knowledge, no oral anticoagulant, does not bind to the ribosome, although corresponding value for D. radiodurans has been reported it is structurally related to linezolid and radezolid [ChEMBL in the literature. (Gaulton et al., 2011); access date: 11th May 2013]. Hence, The co-crystal structures of linezolid with D50S and rivaroxaban was used as a negative control in the course of H50S provided first insights into the structural basis this study. Overall, our analyses reveal an intricate interplay for the species selectivity of the oxazolidinone family of structural, energetic, and dynamic determinants of the (Figure 1B). As such, U2585 (Escherichia coli numbering) species selectivity of oxazolidinone antibiotics. forms a hydrogen bond with the morpholino ring of line zolid in the D50S (Wilson et al., 2008) but not in the H50S structure (Ippolito et al., 2008). In contrast, the phosphate group of G2505 forms a hydrogen bond with the acetamide Results NH group of linezolid in the H50S but not in the D50S structure. Otherwise, the overall position of linezolid is Overall stability of the oxazolidinone-50S similar in both species in terms of ring orientations and complexes interactions (Figure 1B). Obviously, the origin for the difference in the binding affinity of linezolid toward D50S or We studied the differences in oxazolidinone binding H50S cannot be deduced from structural data alone. to D50S and H50S by all-atom explicit solvent MD

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1531 simulations of the respective complexes of 50 ns length as revealed from the time series of these values (Figure each. This leads to a total of 300 ns of simulation time 2B). The average drifts in the effective binding energies, for systems of the size of ∼8.5 × 105 atoms. For investigat- as determined by the slopes of the linear regression lines, ing structural deviations along the trajectories, the overall are -0.19 kcal/(mol*ns) [0.50 kcal/(mol*ns)] for linezolid root mean-square deviation (RMSD) as well as the RMSD (radezolid) in D50S. While the magnitude of these drifts is of the ‘core’ residues were computed with respect to the comparable to those found for ligands binding to proteins starting structures. (The term ‘residue’ is used here for (Metz et al., 2012), the changes in the effective binding both amino acids and nucleotides.) The ‘core’ residues energies over the last 10 ns associated with it point at the were selected by regarding only those residues with the difficulty of obtaining converged estimates of effective

90% lowest root mean-square fluctuations (RMSF) of Cα binding energies for the investigated systems. and phosphate atoms. Both the overall RMSD (Figure S1) and the RMSD of the ‘core’ (Figure 2A) stabilized over the initial 30 ns of simulation time: the RMSD values of the Structural analysis of the oxazolidinone-50S ‘core’ residues reach a plateau of 2–3 Å (4–5 Å) for the complexes linezolid/radezolid-H50S (D50S) complexes (Figure 2A). These deviations are comparable to those found in related The crystal structures of linezolid-D50S and linezolid- simulations (Chen and Lin, 2010; Ge and Roux, 2010) and H50S complexes both show that linezolid binds to the PTC suggest that for the ‘core’ of the ribosome, stable trajec- with the acetamide end being located near the mouth of tories can be obtained already after a few tens of nano- the ribosomal exit tunnel, the oxazolidinone ring making seconds. The results of the RMSD analyses also point out stacking interactions with U2504, and the morpholino that the nucleotides surrounding the binding site in the ring approaching U2585 (Figure 1B) (Ippolito et al., 2008; oxazolidinone-50S complexes do not undergo major struc- Wilson et al., 2008). Despite their similar overall binding tural rearrangements. Consequently, the last 20 ns of the modes, the crystal structures also reveal differences MD trajectories were used for all subsequent structural (Figure 1B) (Auerbach et al., 2004; Yonath and Bashan, analyses. However, in the energetic analysis, the effective 2004). These include a rotation around the fluorophenyl binding energies showed considerably higher fluctuations ring relative to the oxazolidinone ring and different con- in the 30- to 40-ns range than in the 40- to 50-ns range formations of U2506 and U2585. (Figure S2). Hence, for the energetic analyses, only the In order to detect whether such differences in the last 10 ns were used. This ensured rather stable effective binding modes also lead to differences in specific inter- binding energies computed by the MM-PBSA approach actions, we analyzed the network of hydrogen bonds of

8 A Linezolid-H50S B 20 Radezolid-H50S 7 Linezolid-D50S 15 Radezolid-D50S 6 10

5 5

4 (kcal/mol) 0 e ectiv RMSD ( Å ) 3 -5 eff

2 ∆ G -10

1 -15

-20 0510 15 20 25 30 35 40 45 50 40 41 42 43 44 45 46 47 48 49 50 Time (ns) Time (ns)

Figure 2 Time course of root mean square deviations and effective binding energies of oxazolidinone-50S complexes.

(A) Root mean-square deviations of Cα and phosphate atoms of the ‘core residues’ along the MD trajectories of oxazolidinone-50S complex structures determined with respect to the starting structure of the production run. The ‘core residues’ were defined to be those residues with the 90% lowest RMSF of the Cα and phosphate atoms. RMSD values for linezolid and radezolid in H50S are depicted in black and red, and for linezolid and radezolid in D50S in blue and green, respectively. (B) Time series of effective binding energies calculated for 500 snapshots extracted in 20-ps intervals from the last 10 ns of MD simulations of linezolid-D50S (blue) and radezolid-D50S complexes (green). The drifts in the effective binding energies, determined from the slopes of the linear regression lines, are -0.19 kcal/(mol*ns) and 0.50 kcal/(mol*ns), respectively.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1532 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics the antibiotics inside the binding pockets along the MD disrupted throughout the whole MD simulation in the lin- trajectories. In the linezolid-H50S crystal structure, the ezolid-H50S case (Figure 3A). Instead, in the latter case, acetamide NH of linezolid forms a hydrogen bond with the ligands’ acetamide NH group forms a strong hydro- the phosphate group of G2505 (Ippolito et al., 2008) gen bond with the sugar part of U2504 (occupancy: 75%) (Figure 1B). In contrast, the crystal structure of linezolid (Figure 3A). No hydrogen bond formation is observed for bound to D50S shows the involvement of the oxygen atom the morpholino and triazolyl-methylamino moieties, of the morpholino ring in a hydrogen bond interaction however, in both H50S simulations. with N3 of U2585 (Wilson et al., 2008) (Figure 1B). Figure 3 In the case of D50S, no hydrogen bond is found involv- depicts the shortest distances between these respective ing the acetamide NH of linezolid or radezolid, respectively, atoms (and those of the triazolyl-methylamino moiety of and the phosphate group of G2505 (Figure 3B, D). However, radezolid and the carbonyl oxygens’ of U2585 in the case in the linezolid-D50S case, the acetamide NH forms a weak of radezolid) along the MD simulations of the H50S and interaction with O6 of A2061 (occupancy: 20%) (Figure D50S complexes with linezolid and radezolid, respec- 3B). Furthermore, a stable interaction between linezolid’s tively. A distance < 3.2 Å between the acceptor and donor oxygen of the oxazolidinone core and the NH group of the atoms and an angle > 120° for the coordinate triple (accep- base of U2504 is observed (distance between O and N: tor atom, hydrogen, donor atom) are used as criteria for 3.08 ± 0.09 Å); this interaction does not qualify as a hydro- the existence of a hydrogen bond (Gohlke et al., 2003). gen bond according to our angle criterion, however (occu- In the case of radezolid-H50S, the hydrogen bond pancy: 0%). No hydrogen bond involving the morpholino between the acetamide NH of the ligand and the phos- moiety of linezolid is found. In contrast, in the case of rad- phate group of G2505 is present ∼90% of the time ezolid-D50S, hydrogen bond interactions of the triazolyl- (Figure 3C), whereas the respective hydrogen bond is methylamino moiety occur at ∼34 ns and around 37 ns.

H50S D50S 18 A G2505 B G2505 16 U2585 U2585 U2504 A2061 14 12 10 8 olid distance ( Å ) 6 Linez 4 2 30 35 40 45 50 30 35 40 45 50 18 16 C D 14 12 10

olid distance ( Å ) 8 6 Radez 4 2 30 35 40 45 50 30 35 40 45 50 Time (ns) Time (ns)

Figure 3 Distances monitoring hydrogen bond formation for (A) linezolid-H50S, (B) linezolid-D50S, (C) radezolid-H50S, and (D) radezolid- D50S complex simulations. Hydrogen bonds were defined by acceptor donor atom distances of < 3.2 Å and acceptor H-donor angles of > 120°. The distance between the ligands’ acetamide NH group and the oxygens’ of the phosphate group of G2505 is shown in black; the distance between the oxygen of the morpholino ring of linezolid and N3 of U2585 or any donor group of the triazolyl-methylamino moiety of radezolid and the carbonyl oxygens’ of U2585 are shown in red. Only the smallest distance found is plotted in all cases. In addition, the distance between linezolid’s acetamide NH group and O2′ of U2504 is shown for H50S (A, cyan) as is the distance between linezolid’s acetamide NH group and O6 of A2061 for D50S (B, blue).

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1533

Next, we analyzed interactions between the oxazolidi- Radezolid forms all contacts (Figure 4A, B, C, D, E, F). It none ring and the nucleobase of U2504 as well as between is obvious, however, that – when established – contacts the fluoro-phenyl ring and the nucleobases of A2451 and between the fluoro-phenyl ring and nucleobases of A2451 C2452, which form the so-called A-site cleft, by measuring or C2452 of linezolid and radezolid have a longer distance the distances between the centers of the respective rings in the D50S case (Figure 4D, F) than in the H50S case (Figure 4). These ring/nucleobase pairs have been identi- (Figure 4C, E). This suggests that weaker ring/nucleobase fied in the crystal structures of linezolid-D50S and line- interactions are formed in the former case. zolid-H50S as making important contacts. We used a ring As the A-site cleft is wider (Blaha et al., 2008) and center-to-ring center distance < 5.0 Å as a criterion to iden- more rigid (Fulle and Gohlke, 2009) in apo D50S than in tify such contacts, which allows identifying T-shaped or apo H50S, the dynamic behavior of the oxazolidinones parallel-displaced ring configurations in addition to per- inside the binding site could also differ between these fectly stacked ones (Arunan and Gutowsky, 1993; Ippolito eubacterial and archaeal ribosomes. We, thus, analyzed et al., 2008). The results show that linezolid engages in the RMSF of the bound oxazolidinones (Figure S3). The all such ring/nucleobase contacts (Figure 4A, B, D, E, F) RMSF values demonstrate that radezolid shows a higher except with A2451 when bound to H50S (Figure 4C). mobility in the acetamide regions than linezolid (in D50S

H50S D50S 10 A Linezolid B 9 Radezolid

5 U2504 distance ( Å ) 4

3 30 35 40 45 50 30 35 40 45 50 10 C D 9

5 A2451 distance ( Å ) 4

3 30 35 40 45 50 30 35 40 45 50 10 E F 9

5 C2452 distance ( Å ) 4

3 30 35 40 45 50 30 35 40 45 50 Time (ns) Time (ns)

Figure 4 Distances between the centers of mass of the oxazolidinone ring and the nucleobase of U2504 (A, B) as well as between the centers of mass of the fluoro-phenyl ring and the nucleobases of A2451 (C, D) and C2452 (E, F), respectively. (A), (C), (E) Distances for oxazolidinone-H50S complex simulations. (B), (D), (F) Distances for oxazolidinone-D50S complex simulations. Black curves are for linezolid, red curves are for radezolid.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1534 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics and H50S) and that radezolid’s mobility, itself, is higher (6.96 kcal/mol; Table S1), which is in line with experimen- when bound to D50S than when bound to H50S. The tal findings that rivaroxaban does not bind to the ribosome latter is also confirmed when analyzing structural varia- [ChEMBL (Gaulton et al., 2011); access date: 11th May 2013]. tions of radezolid over the course of the MD trajectories The analysis of the MD simulation of the rivaroxaban-D50S (Figure S4). The difference of RMSF values in the case of complex revealed that due to a displacement from its linezolid is less pronounced in the acetamide region. In initial position, rivaroxaban loses important interactions the region of the oxazolidinone core, the mobility is gener- inside the binding site that are present in the linezolid- ally low. In contrast, in the region of the morpholino and D50S complex crystal structure (data not shown). triazolyl-methylamino moieties, a generally high mobility The difference in the effective binding energy is observed. (ΔΔGeffective) for linezolid bound to D50S vs. H50S is Finally, as the negatively charged ribonucleic acids -6.93 ± 0.26 kcal/mol (Table 1), thus, demonstrating that need Mg2+ ions to maintain their structural stability (Ge binding of the antibiotic to D50S is favored. At T = 300 K, and Roux, 2009), a correct description of the properties this difference is equivalent to a 105-fold larger association and behavior of the Mg2+ ions in the simulations is essen- constant for linezolid-D50S than for linezolid-H50S. This tial. The RMSF of the Mg2+ ions over the course of the MD result agrees well with experiment according to which a simulations reveal that all Mg2+ ions remain fixed to their 103-fold higher concentration of the antibiotic is required starting positions (data not shown). Thus, it seemed rea- for a successful co-crystallization in H50S compared to sonable to consider these ions as part of the receptor in the D50S (Wilson, 2011). It is also in line with results from subsequent effective binding energy calculations. For the functional assays on S. aureus and H. marismortui ribo- D50S complexes, the simulations also showed that one somes, where a selectivity of linezolid toward the eubacte- of the Mg2+ ions remains in close proximity to the bound rical ribosome is found (Skripkin et al., 2008) (E. M. Duffy, oxazolidiones (Figure S5), as also observed in the crystal personal communication). The decomposition of ΔΔGeffective structure (Wilson et al., 2008).

Energetic analysis of the oxazolidinone-50S Table 1 Differences in the energy and entropy components for oxazolidinone binding to D50S and H50S.a complexes Contributionb Linezolid Radezolid In order to obtain insights into the energetic determinants (D50S-H50S) (D50S-H50S) of the selectivity of linezolid and radezolid with respect Meanc σd Meanc σd to D50S and H50S, effective binding energies (ΔGeffective), ΔΔ i.e., the sum of gas-phase energies and solvation free Helec -4.52 0.04 -8.61 0.10 ΔΔH 1.12 0.19 7.05 0.26 energies, were computed by the MM-PBSA method using vdW ΔΔH -3.39 0.21 -1.56 0.30 the single-trajectory approach (Table 1; Table S1; Eqs. 1, gas ΔΔG -3.64 0.20 21.82 0.25 2). Although the alternative three-trajectory MM-PBSA PB ΔΔGnonpolar 0.07 0.09 0.48 0.09 approach (Homeyer and Gohlke, 2012) has the advantage ΔΔGeffective -6.93 0.26 20.73 0.31 e e that changes in the receptor and ligand conformation upon TΔSR, T, V, ligand in complex 3.66 – 3.15 – TΔS -4.46 –e 7.29 –e complex formation are taken into account, the computa- V, receptor in complex TΔΔS -0.80 –e 10.44 –e tionally less demanding single-trajectory approach has tot ΔΔG -6.16 –e 10.29 –e proven to be a good approximation in several ligand-binding energy studies (Huo et al., 2002; Hou et al., 2011; Yang aGas phase and solvation energy contributions were determined by et al., 2011). Here, ΔG was computed by averaging the MM-PBSA approach, and entropy contributions were calculated effective by quasiharmonic analysis (QHA) considering 500 snapshots from the over 500 snapshots extracted at 20-ps intervals from the last 10 ns of MD simulations of oxazolidione-50S complexes; T = 300 K. last 10 ns of the MD simulations. The associated standard b Helec, electrostatic energy; HvdW, van der Waals energy; Hgas, gas error in the mean determined according to Gohlke et al. phase energy; GPB, polar solvation free energy; Gnonpolar, nonpolar solvation free energy; G , effective energy; S , (2003) is ∼0.25 kcal/mol (Table S1). The computed ΔGeffective effective R, T, V, ligand in complex of linezolid (-6.73 kcal/mol; Table S1) and radezolid (-0.05 translational, rotational, and vibrational entropy of the ligand in the complex; S , vibrational entropy of the receptor in the kcal/mol; Table S1) bound to D50S are (weakly) negative, V, receptor in complex complex. indicating that the sum of gas-phase and solvation free cAverage contributions in kcal/mol. energies favor binding of these antibiotics. In contrast, for dStandard error in the mean values in kcal/mol. e rivaroxaban, a considerably positive ΔGeffective was obtained No values available.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1535 with respect to the energy terms contributing to it reveals the effective binding energy in terms of contributions from that the electrostatic (ΔΔHelec = -4.52 kcal/mol) and polar structural subunits of D50S and H50S. Such structural solvation free energy (ΔΔGPB = -3.64 kcal/mol) contributions decompositions have been successfully applied before favor binding to D50S over H50S, whereas van der Waals in the context of protein-protein interactions (Gohlke interactions (ΔΔHvdW = 1.12 kcal/mol) disfavor it. et al., 2003; Wichmann et al., 2010; Metz et al., 2012) and

In contrast, for radezolid, ΔΔGeffective = 20.73 kcal/mol protein-peptide interactions (Fulle et al., 2013) by us. The was computed (Table 1), which would suggest that, based standard error in the mean ΔGeffective value for one residue only on the sum of gas phase and solvation free ener- is assumed to be of the same order of magnitude than the gies, binding to H50S is favored over D50S. This result is one for the overall effective binding energy (∼0.25 kcal/ at variance with the experiment: while to the best of our mol). Note that the mean ΔGeffective values do not include knowledge no specific experimental data of radezolid contributions due to changes in the configurational binding to D50S or H50S has been reported, it has been entropy of the receptor residues upon ligand binding. stated that radezolid is > 100-fold selective for bacterial Such contributions could be of significant influence, ribosomes over those of rabbit reticulocyte (Zhou et al., however, according to the TSV, receptor in complex computations

2008a). Regarding the decomposition of ΔΔGeffective with above. As expected, the structural decomposition reveals respect to the energy terms contributing to it, we find that that first shell nucleotides contribute most to the effec- the polar solvation free energy (ΔΔGPB = 21.82 kcal/mol) tive binding energy (between -13.06 and -10.03 kcal/mol) highly favors binding to H50S compared to D50S. The van (Figure 5; Tables S3, S4). Still, second shell nucleotides der Waals interactions also considerably contribute to the show contributions of up to 1.76 kcal/mol, i.e., 15%. As a more favorable effective binding energy found for H50S single-trajectory alternative is used for the computations

(ΔΔHvdW = 7.05 kcal/mol), whereas the electrostatic energy of effective binding energies, long-range electrostatic

(ΔΔHelec = -8.61 kcal/mol), as in the case of linezolid, shows influences are most likely responsible for the contribution the opposite trend, i.e., is more favorable in the D50S case. by the second shell nucleotides (Selzer et al., 2000). While the continuum solvation model provides Regarding linezolid, the most pronounced difference estimates of the free energy of solvation and includes is observed for A2451 in the first shell (Tables S3–S5): this entropic contributions of the solvent, changes in the nucleotide contributes strongly (-3.61 kcal/mol) to the entropy of the solute molecules upon binding have been binding of the antibiotic in D50S (Table S3) but shows neglected so far. However, such changes can signifi- an almost neutral influence (-0.12 kcal/mol) in the case cantly influence the binding affinity (Brady and Sharp, of H50S (Table S4). The difference in the contribution of 1997; Singh and Warshel, 2010; Hou et al., 2011). Hence, the highly favorable C2452-ligand interaction is not as we estimated differences in the changes in the con- pronounced (-4.43 kcal/mol in D50S vs. -5.54 kcal/mol in figurational entropy (TΔΔS at T = 300 K) upon binding H50S), but amounts up to ∼1 kcal/mol in favor of H50S. of linezolid (or radezolid) to D50S and H50S based on Notably, also small differences in the effective binding motions of the ligands and the nucleotides forming the energies of second shell nucleotides are observed: nucleo- first shell of the binding site by quasiharmonic analy- tides 2055 (2572) contribute favorably (neutrally) to binding sis (QHA) (Table 1; Tables S1 and S2; Eqs. S2a–d; Figure in the case of D50S [-0.40 kcal/mol (0.15 kcal/mol)], S6). The librational/vibrational entropy of the ligand whereas they disfavor binding to H50S [0.29 kcal/mol inside the binding site (TSR,T,V, ligand in complex) was found to be (0.80 kcal/mol)]. higher in the D50S complex than in the H50S complex by As to radezolid, again A2451 and C2452 show signifi- 3–4 kcal/mol for both ligands (Table 1). In contrast, ligand cant and opposing differences in their contributions to binding has converse effects on the vibrational entropy the effective binding energy (Table S5): both nucleotides of the first shell nucleotides (TSV, receptor in complex): linezolid contribute highly favorably to binding in D50S and H50S, (radezolid) binding leads to ∼4 kcal/mol lower (∼7 kcal/mol but A2451 shows a higher effective binding energy in D50S higher) values in the case of D50S than H50S. Taken (-4.16 kcal/mol) than in H50S (-3.21 kcal/mol), whereas together, the difference in the changes in the configu- C2452 exhibits a lower effective binding energy in D50S rational entropy is close to zero for linezolid binding to (-2.21 kcal/mol) than in H50S (-3.44 kcal/mol). For U2504, D50S vs. H50S; in contrast, the changes in the configura- which shows the second largest difference, the same trend tional entropy favor radezolid binding to D50S over H50S as for A2451 is observed: this nucleotide’s contribution by ∼10 kcal/mol (Table 1). is more favorable in the D50S case (-3.54 kcal/mol) than To gain insights into the origin of the selectivity of oxa- in the H50S case (-2.22 kcal/mol). Finally, G2505 zolidinone binding on an atomic level, we decomposed and U2506 contribute to the effective binding energy by

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1536 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics

Figure 5 Per-residue contributions to the effective binding energy as calculated by a structural decomposition via MM-PBSA using ensem- bles from MD simulations of (A) linezolid-H50S, (B) linezolid-D50S, (C) radezolid-H50S, and (D) radezolid-D50S complex structures. Each sphere represents the center of mass of the nucleobase of the respective nucleotide; nucleotides of the first shell are labeled with straight letters, those of the second shell in italics. Ligands are shown as sticks. The per-residue contributions are mapped using a color code with a linear scale (red: ≤ -3 kcal/mol; white: 0 kcal/mol; blue: ≥ +3 kcal/mol). Hydrogen bonds observed during the MD simulations between oxazolidinones and nucleotides (Figure 3) are indicated by dotted green lines; ring/nucleobase contacts observed during the MD simulations (Figure 4) are shown as dotted black lines.

-1.57 and -2.09 kcal/mol in H50S, whereas their contribu- contributions, and configurational entropies with respect tions remain almost neutral in the case of D50S. As to the to the binding of linezolid or radezolid to D50S and H50S. contributions from second shell nucleotides, again differences are found between D50S and H50S: nucleotides 2055 and 2572 both disfavor binding to H50S, whereas their contribution to binding in D50S is neutral. Discussion In summary, the energetic analyses of the oxazolidione-50S complexes reveal significant differences in the The highly conserved functional sites in the ribo- effective binding energies, also in terms of structural some make the task of developing selective antibiotics

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1537 challenging. In the present study, we aimed at identifying compared to apo H50S (Blaha et al., 2008) may contribute on an atomic level the determinants of selectivity of lin- to the observed higher mobility of radezolid particularly ezolid and radezolid, binding to the eubacterial D50S and in the acetamide region (Figures S3) and, consequently, to the archaeal H50S subunits, by means of MD simulations the higher vibrational/librational entropy (Table 1) when and free energy computations. Although structure-activ- bound to D50S compared to H50S. In turn, rigidity analy- ity relationships of oxazolidinones have been extensively ses on 50S subunits revealed that the glycosidic bond reported (Gregory et al., 1989; Gregory et al., 1990; Park of A2451 is flexible in H50S but rigid in D50S (Fulle and et al., 1992; Brickner et al., 1996; Barbachyn and Ford, Gohlke, 2009). Accordingly, binding of the antibiotics can 2003; Brickner et al., 2008; Zhou et al., 2008a,b; Locke rigidify this crevice only in the case of H50S, even more et al., 2010), none of these studies has considered aspects so if strong aromatic interactions are formed between the of structure, energetics, and dynamics to selectivity simul- fluoro-phenyl ring and the cleft’s nucleobases. The latter taneously. Furthermore, to the best of our knowledge, this is particularly observed for radezolid binding to H50S is the first study to investigate the selectivity of oxazolidi- (Table 1; Figure 5; Table S4). Thus, it does not come as none binding to 50S subunits by MD simulations and free a surprise that vibrational entropy contributions of first energy calculations. Preliminary results addressing this shell nucleotides of the binding sites favor radezolid question have been reported by Franceschi et al. as a con- binding to D50S over H50S (Table 1). ference contribution using MCPRO (MCPRO) for a residue- The structural analysis of the MD trajectories reveals by-residue analysis of interaction energies (Franceschi that linezolid does not form strong hydrogen bonds et al., 2010). between its acetamide group and the phosphate group From a global point of view, it is remarkable that the of G2505, neither when it is bound to D50S nor to H50S. MM-PBSA analyses including estimates of changes in Regarding the acetamide group, this observation agrees the configurational entropy agree well with results from with observations from the linezolid-D50S crystal struc- experiment on the selectivity of linezolid. These computa- ture (Wilson et al., 2008) but disagrees with what has tions furthermore reveal that linezolid’s selectivity toward been found for the linezolid-H50S structure (Ippolito D50S over H50S is favored by the effective energy, whereas et al., 2008). Rather, linezolid’s acetamide groups form there is only a vanishing contribution by the configura- hydrogen bonds with the sugar part of U2504 in H50S, and tional entropy (Table 1). A converse picture emerges for with O6 of A2061 in D50S. In the latter case, an additional the global selectivity determinants of radezolid: the effec- stable interaction between linezolid’s oxazolidinone core tive energy strongly favors binding to H50S over D50S and the base of U2504 was observed, too. now, but differences in the changes of the configurational Regarding the morpholino moiety, according to the entropy oppose this effect (Table 1). Still, in sum, binding structural analysis of the MD trajectories, linezolid neither of radezolid to H50S remains favorable. Note, however, forms hydrogen bonds when bound to D50S nor to H50S. that the contributions of the configurational entropy In the linezolid-D50S crystal structure, only a relatively only consider the effects of the first shell of binding site weak hydrogen bond has been found (Wilson et al., 2008), nucleotides due to the lack of convergence when includ- which stabilizes an otherwise highly flexible U2585. Thus, ing second (and, likely, higher) shell nucleotides (see the observed breaking of this hydrogen bond during Materials and methods, and Figure S6) such that entropy the MD simulations at 300 K (Figure 3A, B) is not unex- contributions from residues further away are neglected. pected. Interactions of linezolid’s fluoro-phenyl ring are Furthermore, when extending the computation of con- observed to C2452 in both the D50S and H50S simulations tributions of the configurational entropy to the last 20 ns (Figure 4E, F), in agreement with crystal structure analy- of the MD trajectories, the differences in the changes of ses (Ippolito et al., 2008; Wilson et al., 2008), with them the configurational entropy even more favor radezolid being tighter in the H50S case. In contrast, A2451 forms binding to D50S such that, in sum, binding of radezolid additional interactions with the fluoro-phenyl ring of lin- to D50S would become favorable (Table S2). The last ezolid only in D50S, which explains to a large extent why two points call for performing longer MD simulations in the effective binding energy of linezolid is more favorable related studies in the future. for D50S than for H50S (Table 1). These observations on the energetics on a global level Radezolid forms a strong hydrogen bond with its are consistent with structural and mobility analyses of acetamide group to G2505 in H50S only. Furthermore, oxazolidinone-50S complexes from the MD trajectories, although it forms tighter interactions with its oxazolidi- and structural analyses of apo and holo 50S crystal struc- none ring with U2504 in D50S, it does so too between its tures. As such, a wider A-site cleft in the case of apo D50S fluoro-phenyl ring and A2451 as well as C2452 in H50S.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1538 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics

Taken together, these differences in the interactions can when their accuracy is sufficiently high, as demonstrated explain to a large extent why the effective binding energy here for the species-selectivity of linezolid. However, such of radezolid is computed to be more favorable in the H50S analyses are computationally highly demanding. It may, case than in the D50S case (Table 1). thus, be worth to invest in the development of small RNA The results from the structural decomposition of the model systems of antibiotic binding sites, like those that effective binding energies confirm essentially these analy- have been extraordinarily useful to study the A-site of bac- ses of the influence of interactions of first shell nucleotides terial 16S rRNA in the 30S subunit (Hermann, 2006). of the binding sites on oxazolidinone selectivity. As an advantage, the structural decomposition provides a spa- tially resolved picture of the binding energetics (Figure 5), making the identification of selectivity ‘hot spots’ or ‘cold Materials and methods spots’ easier. This is particularly helpful for investigat- ing influences of the second (and higher) shell residues, Molecular dynamics simulations where often a direct link to interactions with the ligand is All MD simulations were performed using the AMBER 10 suite of not obvious. In the present case, the structural decomposi- programs (Case et al., 2005) together with the ff99SB force field (Cor- tion reveals the most pronounced influence of second shell nell et al., 1995; Hornak et al., 2006) for proteins and RNAs, and the residues on the selectivity of oxazolidinones for nucleotides general amber force field (GAFF) (Wang et al., 2004) for the ligands 2055 and 2572 (Figure 5; Table S5). This finding is in line with linezolid, radezolid, and rivaroxaban. Details on how the oxazolidi- previous reports according to which both nucleotides are one-50S systems were set up for these MD simulations are described involved in restraining the conformational space of U2504 in in the Supplemental Material as are details of the MD simulation pro- cedures for the D50S systems. The MD simulations of the H50S sys- eubacteria vs. archaea/eucaryotes and, hence, the selectiv- tems have been executed analogously. A detailed description of the ity of antibiotics binding to the A-site cleft (Davidovich et al., protocol used in these MD simulations will be published elsewhere 2008; Gürel et al., 2009): in general, in eubacteria, U2504 is (Saini, Fulle, Homeyer, Gohlke, unpublished results). Details of the sterically fostered by C2055 and A2572 to adopt a conforma- structural analysis of the MD trajectories are given in the Supplemen- tion that favors interactions with antibiotics; in contrast, in tal Material, too. archaea/eucaryotes, U2504 adopts a conformation unsuita- ble for interactions with antibiotics due to A2055 and U2572. Oxazolidinone binding to the A-site cleft is now peculiar in Calculation of effective binding energies that U2504 in H50S adopts a eubacterial conformation. As We applied the MM-PBSA method (Homeyer and Gohlke, 2012) as a consequence, the induced fit should incur a cost in free implemented in the mm_pbsa.pl module of the AMBER 10 (Case energy. In line with this, the structural decomposition iden- et al., 2005) suite of programs. MM-PBSA estimates the free energy of x tifies U2504 to disfavor oxazolidinone binding to H50S over a molecule x as the sum of its gas phase energy (Hgas), solvation free x x D50S. Notably, the conformational strain is also relayed to energy (Gsolv), and configurational entropy (TS ) (Eq. 1). nucleotides 2055 and 2572 as these disfavor oxazolidinone Gixx()=+Hi() Gixx()-(TS i) (1) binding to H50S over D50S, too (Figure 5; Table S5). This gassolv finding provides direct evidence for the energetic involve- The effective binding energy (Gohlke and Case, 2004) was com- ment of second shell residues in oxazolidinone selectivity. puted as the mean difference of the effective energies (Geffective = Hgas+Gsolv) In summary, our analyses reveal an intricate inter- of the complex and the receptor and ligand (Eq. 2). < · > denotes an play of structural, energetic, and dynamic determinants average over snapshots i taken from MD trajectories. of the species selectivity of oxazolidinone antibiotics. complexreceptorligand ∆=GG<>()iG-(iG)- ()i (2) Even for the structurally rather similar members linezolid e ective e ective e ective e ective and radezolid investigated here, significant differences in Details of the calculations of effective binding energies are the (opposing) contributions from interaction energies, described in the Supplemental Material. solvation, and entropic factors have been identified, as have been influences of first and second shell nucleotides detected. These findings add another layer of complexity to the current knowledge on structure-activity relation- Structural decomposition of the effective ships of oxazolidinones and suggest that selectivity analy- binding energies ses solely based on static crystal structures and qualitative The contributions to the effective binding energies calculated accord- arguments on interactions may not reach far enough. Com- ing to Eqs. (1 and 2) (but without considering the configurational putational analyses may aid in filling this gap, particularly entropy) were decomposed at the per-residue level. Residue-wise gas

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1539 phase energies were determined using the decomposition scheme due to ligand binding considering only the ligand and ribosomal implemented in the sander and mm_pbsa.pl modules of AMBER 10 residues of the ligand-binding site by QHA. We showed recently that (Case et al., 2005). Per-residue contributions of the polar part of the changes of the vibrational entropy due to ligand binding originate solvation free energy were computed on an atomic level as described mostly from changes in the degrees of freedom of residues to which for the total polar solvation contribution in the Supplemental Mate- the ligand forms direct interactions in the complex (Kopitz et al., rial; then, the energy contributions of all atoms belonging to a resi- 2012). Details on how individual entropy contributions were calcu- due were summed up. Per-residue contributions of the nonpolar part lated are described in the Supplemental Material. of the solvation free energy were obtained with the ICOSA method (Case et al., 2005). Finally, the effective binding energy per residue was calculated from the gas phase and solvation free energy contri- Acknowledgements: We gratefully acknowledge sup- butions using the mm_pbsa_statistics.pl script of AMBER 10 (Case port (and training) from the International NRW Research et al., 2005). School BioStruct, granted by the Ministry of Innovation, Science and Research of the State North Rhine-West- phalia, the Heinrich-Heine-University of Düsseldorf, and Approximation of entropy components the Entrepreneur Foundation at the Heinrich-Heine-Uni- versity of Düsseldorf. We are grateful to the Jülich Super- The configurational entropy of a molecule consists of translational, computing Center (NIC project 5921) and to the ‘Zentrum rotational, and vibrational contributions. Obtaining accurate estimates of vibrational entropy contributions is difficult and compu- für Informations- und Medientechnologie’ (ZIM) at the tationally demanding (Gohlke and Case, 2004). Determining such HHU for providing computational support. contributions for a system of the size of the ribosome did not seem feasible to us, neither by normal mode analysis (NMA) (Case, 1994) nor by QHA (Teeter and Case, 1990; Gohlke and Case, 2004; Baron Received May 20, 2013; accepted September 1, 2013; previously pub- et al., 2009). Thus, we decided to estimate changes in the entropy lished online September 3, 2013

References

Aleksandrov, A. and Simonson, T. (2008). Molecular dynamics Brickner, S.J., Hutchinson, D.K., Barbachyn, M.R., Manninen, P.R., simulations of the 30S ribosomal subunit reveal a preferred Ulanowicz, D.A., Garmon, S.A., Grega, K.C., Hendges, S.K., tetracycline binding site. J. Am. Chem. Soc. 130, 1114–1115. Toops, D.S., Ford, C.W., et al. (1996). Synthesis and Arunan, E. and Gutowsky, H.S. (1993). The rotational spectrum, antibacterial activity of U-100592 and U-100766, two structure and dynamics of a benzene dimer. J. Chem. Phys. 98, oxazolidinone antibacterial agents for the potential treatment 4294–4296. of multidrug-resistant gram-positive bacterial infections. Auerbach, T., Bashan, A., and Yonath, A. (2004). Ribosomal J. Med. Chem. 39, 673–679. antibiotics: structural basis for resistance, synergism and Brickner, S.J., Barbachyn, M.R., Hutchinson, D.K., and selectivity. Trends Biotechnol. 22, 570–576. Manninen, P.R. (2008). Linezolid (ZYVOX), the first member Barbachyn, M.R. and Ford, C.W. (2003). Oxazolidinone structure- of a completely new class of antibacterial agents for treatment activity relationships leading to linezolid. Angew. Chem. Int. of serious gram-positive infections. J. Med. Chem. 51, Ed. 42, 2010–2023. 1981–1990. Baron, R., Hünenberger, P.H., and McCammon, J.A. (2009). Brodersen, D.E., Clemons, W.M. Jr., Carter, A.P., Morgan- Absolute single-molecule entropies from quasi-harmonic Warren, R.J., Wimberly, B.T., and Ramakrishnan, V. (2000). The analysis of microsecond molecular dynamics: correction terms structural basis for the action of the antibiotics tetracycline, and convergence properties. J. Chem. Theory Comput. 5, pactamycin, and hygromycin B on the 30S ribosomal subunit. 3150–3160. Cell 103, 1143–1154. Belousoff, M.J., Shapira, T., Bashan, A., Zimmerman, E., Carter, A.P., Clemons, W.M., Brodersen, D.E., Morgan-Warren, R.J., Rozenberg, H., Arakawa, K., Kinashi, H., and Yonath, A. (2011). Wimberly, B.T., and Ramakrishnan, V. (2000). Functional Crystal structure of the synergistic antibiotic pair, lankamycin insights from the structure of the 30S ribosomal subunit and and lankacidin, in complex with the large ribosomal subunit. its interactions with antibiotics. Nature 407, 340–348. Proc. Natl. Acad. Sci. USA 108, 2717–2722. Case, D.A. (1994). Normal mode analysis of protein dynamics. Curr. Blaha, G., Gürel, G., Schroeder, S.J., Moore, P.B., and Steitz, T.A. Opin. Struct. Biol. 4, 285–290. (2008). Mutations outside the anisomycin-binding site can Case, D.A., Cheatham, T.E. 3rd, Darden, T., Gohlke, H., Luo, R., make ribosomes drug-resistant. J. Mol. Biol. 379, 505–519. Merz, K.M. Jr., Onufriev, A., Simmerling, C., Wang, B., and Bottger, E.C., Springer, B., Prammananan, T., Kidan, Y., and Woods, R.J. (2005). The Amber biomolecular simulation Sander, P. (2001). Structural basis for selectivity and toxicity of programs. J. Comput. Chem. 26, 1668–1688. ribosomal antibiotics. EMBO Rep 2, 318–323. Chen, S.Y. and Lin, T.H. (2010). A molecular dynamics study on Brady, G.P. and Sharp, K.A. (1997). Entropy in protein folding and binding recognition between several 4,5 and 4,6-linked in protein-protein interactions. Curr. Opin. Struct. Biol. 7, aminoglycosides with A-site RNA. J. Mol. Recognit. 23, 215–221. 423–434.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 1540 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics

Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, K.M., Hansen, J.L., Moore, P.B., and Steitz, T.A. (2003). Structures of five Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., and antibiotics bound at the peptidyl transferase center of the Kollman, P.A. (1995). A second generation force field for the large ribosomal subunit. J. Mol. Biol. 330, 1061–1075. simulation of proteins, nucleic acids, and organic molecules. Hermann, T. (2006). A-site model RNAs. Biochimie 88, 1021–1026. J. Am. Chem. Soc. 117, 5179–5197. Homeyer, N. and Gohlke, H. (2012). Free energy calculations by David-Eden, H., Mankin, A.S., and Mandel-Gutfreund, Y. (2010). the molecular mechanics Poisson–Boltzmann surface area Structural signatures of antibiotic binding sites on the method. Mol. Inf. 31, 114–122. ribosome. Nucleic Acids Res. 38, 5982–5994. Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., and Davidovich, C., Bashan, A., and Yonath, A. (2008). Structural basis Simmerling, C. (2006). Comparison of multiple Amber force for cross-resistance to ribosomal PTC antibiotics. Proc. Natl. fields and development of improved protein backbone Acad. Sci. USA 105, 20665–20670. parameters. Proteins Struct. Funct. Bioinf. 65, 712–725. Franceschi, F., DeVito, J., Dalton, J., Remy, J., Skripkin, E., Huo, S., Wang, J., Cieplak, P., Kollman, P.A., and Kuntz, I.D. (2002). Ippolito, J.A., and Duffy, E. (2010). Radezolid Overcomes Molecular dynamics and free energy analyses of cathepsin CFR-Mediated Linezolid-Resistance by Efficiently Inhibiting D-inhibitor interactions: insight into structure-based ligand Protein Synthesis of CFR-Methylated Ribosomes. Paper design. J. Med. Chem. 45, 1412–1419. presented at: Interscience Conference on Antimicrobial Agents Hou, T., Wang, J., Li, Y., and Wang, W. (2011). Assessing the and Chemotherapy (Boston, Massachusetts, USA). performance of the MM/PBSA and MM/GBSA methods. 1. Fulle, S. and Gohlke, H. (2009). Statics of the ribosomal exit tunnel: The accuracy of binding free energy calculations based on implications for cotranslational peptide folding, elongation molecular dynamics simulations. J. Chem. Inf. Model 51, regulation, and antibiotics binding. J. Mol. Biol. 387, 502–517. 69–82. Fulle, S., Withers-Martinez, C., Blackman, M.J., Morris, G.M., and Ippolito, J.A., Kanyo, Z.F., Wang, D., Franceschi, F.J., Moore, P.B., Finn, P.W. (2013). Molecular determinants of binding to the Steitz, T.A., and Duffy, E.M. (2008). Crystal structure of the plasmodium subtilisin-like protease 1. J. Chem. Inf. Model 53, oxazolidinone antibiotic linezolid bound to the 50S ribosomal 573–583. subunit. J. Med. Chem. 51, 3353–3356. Gaulton, A., Bellis, L.J., Bento, A.P., Chambers, J., Davies, Kopitz, H., Cashman, D.A., Pfeiffer-Marek, S., and Gohlke, H. (2012). M., Hersey, A., Light, Y., McGlinchey, S., Michalovich, Influence of the solvent representation on vibrational entropy D., Al-Lazikani, B., et al. (2011). ChEMBL: a large-scale calculations: generalized born versus distance-dependent bioactivity database for drug discovery. Nucleic Acids Res. dielectric model. J. Comput. Chem. 33, 1004–1013. 40, D1100–D1107. Leach, K.L., Brickner, S.J., Noe, M.C., and Miller, P.F. (2011). Ge, X. and Roux, B. (2009). Calculation of the standard binding free Linezolid, the first oxazolidinone antibacterial agent. Ann. NY energy of sparsomycin to the ribosomal peptidyl-transferase Acad. Sci. 1222, 49–54. P-site using molecular dynamics simulations with restraining Locke, J.B., Finn, J., Hilgers, M., Morales, G., Rahawi, S., potentials. J. Mol. Recognit. 23, 128–141. Kedar, G.C., Picazo, J.J., Im, W., Shaw, K.J., and Stein, J.L. Ge, X. and Roux, B. (2010). Absolute binding free energy (2010). Structure-activity relationships of diverse oxazoli- calculations of sparsomycin analogs to the bacterial ribosome. dinones for linezolid-resistant Staphylococcus aureus J. Phys. Chem. B 114, 9525–9539. strains possessing the CFR methyltransferase gene or Gohlke, H. and Case, D.A. (2004). Converging free energy estimates: ribosomal mutations. Antimicrob. Agents Chemother. 54, MM-PB(GB)SA studies on the protein-protein complex Ras-Raf. 5337–5343. J. Comput. Chem. 25, 238–250. Ma, C., Baker, N.A., Joseph, S., and McCammon, J.A. (2002). Binding Gohlke, H., Kiel, C., and Case, D.A. (2003). Insights into protein- of aminoglycoside antibiotics to the small ribosomal subunit: a protein binding by binding free energy calculation and continuum electrostatics investigation. J. Am. Chem. Soc. 124, free energy decomposition for the Ras-Raf and Ras-RalGDS 1438–1442. complexes. J. Mol. Biol. 330, 891–913. Mankin, A.S. and Garrett, R.A. (1991). Chloramphenicol resistance Gregory, W.A., Brittelli, D.R., Wang, C.L., Wuonola, M.A., mutations in the single 23S rRNA gene of the archaeon McRipley, R.J., Eustice, D.C., Eberly, V.S., Bartholomew, P.T., Halobacterium halobium. J. Bacteriol. 173, 3559–3563. Slee, A.M., and Forbes, M. (1989). Antibacterials. Synthesis MCPRO (2009), Schrödinger, LLC, New York, NY, USA. and structure-activity studies of 3-aryl-2-oxooxazolidines. Metz, A., Pfleger, C., Kopitz, H., Pfeiffer-Marek, S., Baringhaus, K.H., 1. The “B” group. J. Med. Chem. 32, 1673–1681. and Gohlke, H. (2012). Hot spots and transient pockets: Gregory, W.A., Brittelli, D.R., Wang, C.L., Kezar, H.S. 3rd, predicting the determinants of small-molecule binding to a Carlson, R.K., Park, C.H., Corless, P.F., Miller, S.J., protein-protein interface. J. Chem. Inf. Model 52, 120–133. Rajagopalan, P., Wuonola, M.A., et al. (1990). Antibacterials. Moellering, R.C. (2003). Linezolid: the first oxazolidinone antimi- Synthesis and structure-activity studies of 3-aryl-2-oxooxazo- crobial. Ann. Intern. Med. 138, 135–142. lidines. 2. The “A” group. J. Med. Chem. 33, 2569–2578. Park, C.H., Brittelli, D.R., Wang, C.L., Marsh, F.D., Gregory, W.A., Gürel, G., Blaha, G., Moore, P.B., and Steitz, T.A. (2009). U2504 Wuonola, M.A., McRipley, R.J., Eberly, V.S., Slee, A.M., determines the species specificity of the A-site cleft and Forbes, M. (1992). Antibacterials. synthesis and antibiotics: the structures of tiamulin, homoharringtonine, and structure-activity studies of 3-aryl-2-oxooxazolidines. bruceantin bound to the ribosome. J. Mol. Biol. 389, 146–156. 4. Multiply-substituted aryl derivatives. J. Med. Chem. 35, Hansen, J.L., Ippolito, J.A., Ban, N., Nissen, P., Moore, P.B., and 1156–1165. Steitz, T.A. (2002). The structures of four macrolide antibiotics Romanowska, J., McCammon, J.A., and Trylska, J. (2011). bound to the large ribosomal subunit. Mol. Cell 10, 117–128. Understanding the origins of bacterial resistance to aminogly-

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18 J.S. Saini et al.: Species selectivity of oxazolidinone antibiotics 1541

cosides through molecular dynamics mutational study of the Wichmann, C., Becker, Y., Chen-Wichmann, L., Vogel, V., ribosomal A-site. PLoS Comput. Biol. 7, 1–6. Vojtkova, A., Herglotz, J., Moore, S., Koch, J., Lausen, J., Sanz, J.L., Marín, I., Ureña, D., and Amils, R. (1993). Functional Mantele, W., et al. (2010). Dimer-tetramer transition controls analysis of seven ribosomal systems from extremely halophilic RUNX1/ETO leukemogenic activity. Blood 116, 603–613. archaea. Can. J. Microbiol. 39, 311–317. Wilson, D.N. (2011). On the specificity of antibiotics targeting the Schlunzen, F., Zarivach, R., Harms, J., Bashan, A., Tocilj, A., large ribosomal subunit. Ann. NY Acad. Sci. 1241, 1–16. Albrecht, R., Yonath, A., and Franceschi, F. (2001). Structural Wilson, D.N., Schluenzen, F., Harms, J.M., Starosta, A.L., basis for the interaction of antibiotics with the peptidyl Connell, S.R., and Fucini, P. (2008). The oxazolidinone transferase centre in eubacteria. Nature 413, 814–821. antibiotics perturb the ribosomal peptidyl-transferase center Selzer, T., Albeck, S., and Schreiber, G. (2000). Rational design of and effect tRNA positioning. Proc. Natl. Acad. Sci. USA 105, faster associating and tighter binding protein complexes. Nat. 13339–13344. Struct. Biol. 7, 537–541. Yang, T., Wu, J.C., Yan, C., Wang, Y., Luo, R., Gonzales, M.B., Shaw, K.J. and Barbachyn, M.R. (2011). The oxazolidinones: past, Dalby, K.N., and Ren, P. (2011). Virtual screening using present, and future. Ann. NY Acad. Sci. 1241, 48–70. molecular simulations. Proteins Struct. Funct. Bioinf. 79, Singh, N. and Warshel, A. (2010). A comprehensive examination 1940–1951. of the contributions to the binding entropy of protein-ligand Yonath, A. (2005a). Antibiotics targeting ribosomes: resistance, complexes. Proteins Struct. Funct. Bioinf. 78, 1724–1735. selectivity, synergism and cellular regulation. Annu. Rev. Skripkin, E., McConnell, T.S., DeVito, J., Lawrence, L., Ippolito, J.A., Biochem. 74, 649–679. Duffy, E.M., Sutcliffe, J., and Franceschi, F. (2008). Rχ-01, a Yonath, A. (2005b). Ribosomal crystallography: peptide bond new family of oxazolidinones that overcome ribosome-based formation, chaperone assistance and antibiotics activity. Mol. linezolid resistance. Antimicrob. Agents Chemother. 52, Cells 20, 1–16. 3550–3557. Yonath, A. and Bashan, A. (2004). Ribosomal crystallography: Teeter, M.M. and Case, D.A. (1990). Harmonic and quasiharmonic initiation, peptide bond formation, and amino acid polymer- descriptions of crambin. J. Phys. Chem. 94, 8091–8097. ization are hampered by antibiotics. Annu. Rev. Microbiol. 58, Trylska, J., McCammon, J.A., and Brooks III, C.L. (2005). 233–251. Exploring assembly energetics of the 30S ribosomal subunit Zhou, J., Bhattacharjee, A., Chen, S., Chen, Y., Duffy, E., Farmer, J., using an implicit solvent approach. J. Am. Chem. Soc. 127, Goldberg, J., Hanselmann, R., Ippolito, J.A., Lou, R., et al. 11125–11133. (2008a). Design at the atomic level: design of biaryloxazoli- Vaiana, A.C., Westhof, E., and Auffinger, P. (2006). A molecular dinones as potent orally active antibiotics. Bioorg. Med. Chem. dynamics simulation study of an aminoglycoside/A-site RNA Lett. 18, 6175–6178. complex: conformational and hydration patterns. Biochimie Zhou, J., Bhattacharjee, A., Chen, S., Chen, Y., Duffy, E., Farmer, J., 88, 1061–1073. Goldberg, J., Hanselmann, R., Ippolito, J.A., Lou, R., et al. Wang, J., Wolf, R.M., Caldwell, J.W., Kollman, P.A., and Case, D.A. (2008b). Design at the atomic level: generation of novel hybrid (2004). Development and testing of a general amber force biaryloxazolidinones as promising new antibiotics. Bioorg. field. J. Comput. Chem. 25, 1157–1174. Med. Chem. Lett. 18, 6179–6183.

Bereitgestellt von | Heinrich Heine Universität Düsseldorf Angemeldet | 134.99.237.121 Heruntergeladen am | 14.10.13 10:18