Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A

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1 Indian Journal of Biochemistry & Biophysics Vol. 46, April 2009, pp Interaction of nalidixic acid and ciprofloxacin with wild type and mutated quinolone-resistance-determining region of DNA gyrase A Jitendra Vashist 1, Vishvanath 1, Renuka Kapoor 2, Arti Kapil 2, Ragothaman Yennamalli 3, N Subbarao 3 and Moganty R Rajeswari 1 * 1 Department of Biochemistry, 2 Department of Microbiology, All India Institute of Medical Sciences, New Delhi , India 3 School of Information Technology, Jawaharlal Nehru University, New Delhi , India Received 13 October 2008; revised 28 January 2009 The quinolones exert their anti-bacterial activity by binding to DNA gyrase A (GyrA), an essential enzyme in maintenance of DNA topology within bacterial cell. The mutations conferring resistance to quinolones arise within the quinolone-resistancedetermining region (QRDR) of GyrA. Therefore, quinolones interaction with wild and mutated GyrA can provide the molecular explanation for resistance. Resistant strains of Salmonella enterica of our hospital have shown mutations in the QRDR of GyrA of serine 83 (to phenylalanine or tyrosine) or aspartic acid 87 (to glycine or tyrosine). In order to understand the association between observed resistance and structural alterations of GyrA with respect to quinolone binding, we have studied the interaction of mutated QRDR of GyrA with nalidixic acid and ciprofloxacin by molecular modeling using GLIDE v4. Analysis of interaction parameters like G-score has revealed reduced interaction between nalidixic acid/ciprofloxacin with QRDR of GyrA in all four mutated cases of resistant strains. The mutation of Ser83 to Phe or Tyr shows least binding for nalidixic acid, while Asp87 to Gly or Tyr exhibits minimal binding for ciprofloxacin. The study also highlights the important role of arginines at 21, 91 and His at 45, which form strong hydrogen bonds (at < 3 Å) with quinolones. The hydrophilic OH group of Serine 83, which is in close proximity to the quinolone binding site is replaced by aromatic moieties of Tyr or Phe in mutated GyrA. This replacement leads to steric hindrance for quinolone binding. Therefore, quinolone resistance developed by Salmonella appears to be due to the decreased selectivity and affinity of nalidixic acid/ciprofloxacin to QRDR of GyrA. Keywords: Gyrase A, Ciprofloxacin, Nalidixic acid, GLIDE, Quinolone-resistance-determining region mutation Quinolones are a group of antibacterial agents used for various kinds of bacterial infections including Salmonella enterica and E. coli. These agents generally consist of a 1-substituted-1, 4-dihydro-4- oxopyridine-3-carboxylic acid moiety combined with an aromatic or heteroaromatic ring fused at the 5- and 6-positions. The first member of this group to be synthesized was nalidixic acid (Fig. 1A), which showed activity against several Gram-negative bacteria. Derivatives of nalidixic acid have shown antibacterial potencies 1000-times greater than that of parent compound and are active against both Gramnegative and Gram-positive organisms. Many of the new drugs have a fluorine at position 6 of the quinolone structure e.g. ciprofloxacin (Fig. 1B), which significantly enhances antibacterial potency 1. Ciprofloxacin has a broad-spectrum antimicrobial *Author for correspondence mrraji@hotmail.com Ph: Fax: Abbreviations: GyrA, gyrase A; GyrB, gyrase B; QRDR, quinolone-resistance-determining region Fig. 1 Chemical structure of (A) nalidixic acid (CID 4421) and (B) ciprofloxacin (CID 2764) activity and is the first-line drug used for the treatment of typhoid fever. However, its widespread use has resulted in an increasing incidence of ciprofloxacin resistance 2. There is increased evidence to prove that the nalidixic acid-resistant strains also exhibit decreased susceptibilities to the most recent fluoroquinolones (ciprofloxacin) 3. Recently, we have reported reduced susceptibility of S. enterica strains to ciprofloxacin (fluoroquinolones) 4. Similarly, quinolone resistance in E. coli is also reported 5,6. DNA gyrase consists of two proteins gyrase A (GyrA) and gyrase B (GyrB), which form an A 2 B 2 complex in the active enzyme. Gyrase introduces changes in the topology of closed circular DNA by

2 148 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009 cleaving the helix in both strands and passing another segment of DNA through the break, and finally resealing the broken ends 5. The double-stranded breaks in DNA that are created by GyrA are stabilized by quinolones. The quinolones exert the antibacterial activity by giving unfavorable conditions for DNAligation and thereby blocking DNA replication. It is thought that blocking of DNA polymerase by the quinolone-topoisomerase complex triggers the release of broken DNA ends by a yet-undefined mechanism. The proposed model of quinolone binding is complex involving GyrA, GyrB and DNA 7-9. Therefore, any mutation either in GyrA/B can cause resistance. However, the mutations in GyrA lead to a 20-fold resistance, while in GyrB result only a 4-fold resistance. Further, in GyrB region where mutations are reported, is in fact distal (40 Å away) to the active site, while the quinolone resistance determining region (QRDR), where mutations are seen in GyrA is proximal to the active site 8. Therefore, any slight conformational change in the QRDR of GyrA results in a drastic change in the cellular function of gyrase. This suggests that mutations in QRDR GyrA play a crucial role as compared to GyrB in causing resistance. In view of its important role of GyrA in the survival of bacteria like E. coli and Salmonella, it is a good candidate to study the effect of mutations on quinolone resistance. However, the mode of interaction of quinolone with GyrA is still unclear and needs to be explored. In earlier study in S. enterica, we found that majority of resistant clinical isolates contained substitutions between positions 67 and 106 (inclusive) of GyrA, leading to the categorization of this section as the quinolone resistance determining region (Fig. 2A) 4. This region ( amino acids) is within the N-terminal domain (1-477 amino acids) of GyrA of E. coli, for which a high-resolution structure has been determined 10. The QRDR is situated close to the active site (shown in arrow in Fig. 2B). Spontaneous mutations in QRDR at Ser 83 and Asp 87 in GyrA subunit are reported in E. coli 7 and in S. enterica from North 4 and South India 11. Studies based on the site-directed mutagenesis at these positions to alanine in GyrA of E. coli have confirmed the role of 83 and 87 in quinolone resistance 7. This indicates that Ser 83 and Asp 87 are crucial for GyrA in quinolone binding but, molecular nature of these interactions is not yet known. Fig. 2 (A): Amino acid sequence of QRDR of gyrase A which is identical in Salmonella enterica and E. coli; the 83 and 87 are underlined; and (B): Structure of gyrase A highlighting the QRDR region in yellow color; image is made with the help of Insight II and using the PDB file 1ab4. Frequently found mutations at Ser 83 and Asp 87 are shown in green and red respectively. The active site location is shown by arrow Previously, we have reported GyrA gene mutations in Salmonella typhi and S. paratyphi A strains, isolated from patients presenting with enteric fever at All India Institute of Medical Sciences (A.I.I.M.S), New Delhi, India 4. The four point mutations found in QRDR of GyrA are Ser 83 to Phe, Ser 83 to Tyr, Asp 87 to Tyr and Asp 87 to Gly whose minimal inhibitory concentrations (MICs) for ciprofloxacin are in the range of µg/ml compared to that of wild type of 0.02 µg/ml. Although all the four groups of strains are highly resistant to nalidixic acid despite having the same kind of mutation, but show varying levels of MIC of ciprofloxacin in S. typhi and S. paratyphi A 4. Although we have not done the mutation analysis of GyrA in E. coli, frequent mutations at these same positions have been earlier reported 7. Therefore, in the present study, the effect of these mutations has been analyzed on the structure of QRDR of GyrA, which influence quinolone binding and finally causing resistance. Materials and Methods The interaction study on quinolones and DNA gyrase A (GyrA) was done using Glide v4.5 software from Schrodinger (Portland, Oreg.) ( com, 2006) 12. Glide v4.5 was used for ligand docking in flexible mode. This docking method is a fast and accurate and consists of mainly two steps: i) generation of receptor grids, and ii) ligand docking and scoring 13.

3 VASHIST et al.: INTERACTION OF QUINOLONES WITH GYRASE A 149 preparation The initial structures of two ligands ciprofloxacin and nalidixic acid were taken from the data bank pubchem ( whose identification numbers were 2764 and 4421, respectively. Their three-dimensional structure were generated by using CORNIA v and then minimized by using the premin option in Glide with OPLS_2005 force field. Nalidixic acid and ciprofloxacin were prepared by addition of hydrogen, followed by assigning appropriate ionization state of each ligand by using the ionizer option. Since ciprofloxacin and nalidixic acid have carboxylic acid moiety, they are expected to be ionised at physiological ph. Thirty-two stereoisomers and tautomers were generated for each ligand. At least one low energy ring conformation was allowed to generate per ligand. These structures were again minimized by using the premin option with the OPLS_2005 force field. After these steps the ligand was used for docking. Protein preparation The X-ray crystal structure of the protein GyrA of E. coli was retrieved from protein data bank (pdb code 1ab4). N-terminal sequence of S. enterica was retrieved from NCBI ( database. The sequence homology of GyrA of S. enterica (residue 29 to 522) and E. coli was about 82%, implying that both the sequences were nearly identical. The nearest mutation in the sequence of GyrA of E. coli at position 133 (changed from glutamic acid to glycine) in Salmonella. However, this was quite well separated (>10 Å) from the center of QRDR and, therefore, the N-terminal sequence of E. coli could be safely used as the protein template for docking studies. This protein template was referred in the study as wild type (Fig. 2A). Amino acid substitution corresponding to the mutation of either Ser 83 to Phe/Tyr or Asp 87 to Tyr/Gly was introduced in the wild type protein sequence. Wild type and mutated GyrA structures were subjected to protein preparation. The GyrA protein was prepared in Maestro ver8.0 by removing water molecules and metal ions. Hydrogen were added to the protein, so as to satisfy the valences and bond order were assigning during optimized steps. The modified structures were energy minimized to RMSD 0.30, after assigning the charge, so as to remove steric clashes between the and the resulting optimized structure was used in the study. Residue 83 and 87, which were involved in binding site were specified as the centroid for generating a grid cube of 20 Å length that covered the entire binding site. The grid was used for the next step of docking. Docking of quinolones to QRDR The minimized structures of ligands and GyrA were subsequently used in docking simulation. The conformation flexibility of ligands were considered by exhaustive conformational search within Glide augmented by heuristic screen that removed conformations which were not suitable for GyrA binding or had long range hydrogen bonds, whereas GyrA conformation remained fixed. An exhaustive systemic search of the conformational space and a series of hierarchical filters to locate the possible position of ligand in the QRDR during docking simulations were done. The shape and properties were represented on grid by different fields, which provided more accurate position and orientation of ligand (termed pose ) in the GyrA. A flexible Monte- Carlo sampling and minimization were used to identify the best ligand poses for scoring. Five poses were calculated per ligand molecule, which were docked to QRDR of GyrA. The resulting docked conformations were analyzed using Glide pose viewer tool. The conformation/poses that made the maximum number of interaction were considered to further analysis. Glide score (G-score) Schrodinger proprietary scoring function Glide score (G-score) was used in docking experiments. G-score was based on ChemScore, but include a steric clash term and adds buried polar terms devised by Schrodinger to penalize electrostatic mismatches. G-score takes into account a number of parameters hydrogen bonds (H bond), hydrophobic (Lipo), Vander-waals (vdw), columbic (Coul), polar interactions in the binding site (site), metal-binding term (metal) and penalty for buried polar group (BuryP) and freezing rotable bonds (RotB). (G-Score = H bond + Lipo + Metal + Site Coul vdw - BuryP RotB)... (1) Results and Discussion Quinolone resistance determining regions (QRDR) of GyrA in S. enterica and E. coli were identical and therefore, present study and conclusions drawn thereafter applied equally well in both organisms. The

4 150 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009 results of nalidixic acid or ciprofloxacin with respect to wild type (SAB4) and four mutated GyrA are discussed below in terms of G-score, hydrogen bonding and hydrophobic interactions (Tables 1-3 and Fig. 3). It must be mentioned here that in general a high G-score (on negative scale) reflects a strong interaction between the ligand and protein. On visual inspection, the displacement of molecules docked was clearly shown in all the mutated forms of GyrA, as compared to that of wild type. Nalidixic acid binding with QRDR of gyrase A The docking of nalidixic acid with QRDR of GyrA (wild type) in sensitive strain showed a G-score of kcal/mol, involving three hydrogen bonds via His 45, Ser 172 and Arg 91. The best G-score of kcal/mol suggested a strong binding between nalidixic acid and QRDR of GyrA in the wild type (Table 1). After introducing mutation by site-directed mutagenesis at Ser 83 to Phe or Tyr, G-score decreased reasonably ( -4.8 kcal/mol) and one hydrogen bond was observed between nalidixic acid and Arg 121 of GyrA. Mutation of Asp 87 to Tyr or Gly also involved only one hydrogen bond, but amino acids involved in the interaction with nalidixic acid were different i.e., Gln 94 or Ala 117, respectively. Although the G-score of GyrA with mutation at 87 (Tyr and Gly) as compared to wild type showed a decreasing trend after binding of nalidixic acid ( -5.2 kcal/mol), however, the change was not as significant as in the case of 83 mutated GyrA (Table 1). It may be noticed that mutation of Ser 83 or Asp 87 in GyrA resulted a relatively low G-score. Loss of hydrogen bonds involving His 45, Ser 172 and Arg 91 (seen in wild type) in mutated GyrA suggested the critical importance of these in binding of nalidixic acid with GyrA. It is important to mention here that these low G-scores were not only because of loss of hydrogen bonds, but also due to the hydrophobic interactions that took place between newly introduced aromatic namely Phe or Tyr in case of Ser 83 mutation. Predicted docking of Fig. 3 Predicted poses for nalidixic acid in wild type (A) and mutated S83F (B) and D87N (C) QRDR of Gyr A. Similarly, (D) represents QRDR complex with ciprofloxacin-wild type, (E) mutated (S83F) and (F) mutated (D87G). Where residue 83 is represented by green, residue 87 by red, nalidixic acid/ciprofloxacin in cyan and surface by yellow. Images have been made using Pymol ( Arrow shows the nalidixic acid/ciprofloxacin binding site with respect to wild type gyrase A Table 1 Interaction energies obtained with nalidixic acid and ciprofloxacin binding with QRDR of gyrase A in wild type and mutated GyrA using docking program Glide v4.5 and Getneares Amino acid changes Nalidixic acid Ciprofloxacin (Point mutation) G-score (Kcal/mol) No. of hydrogen bonds (Residues involved) G-score (Kcal/mol) No. of hydrogen bonds (Residues involved ) Wild type (His 45, Ser 172, Arg 91) (Asp 87, Arg 121, Met 120) Ser 83 to Phe (Arg 121) (Asp 87, Arg 121) Ser 83 to Tyr (Arg 121) (Arg 121) Asp 87 to Tyr (Gln 94) (Ser 172, His 45, Lys 42) Asp 87 to Gly (Ala 117) (Arg 91, Gln 94, Ala 117)

5 VASHIST et al.: INTERACTION OF QUINOLONES WITH GYRASE A 151 Table 2 Interacting, of GyrA and nalidixic acid involved in GyrA-nalidixic acid interaction using Getneares program [Interaction within a distance 4 Å are shown] Wild type *HIS 45 NE2 O Ser 83 Tyr contd *SER 172 N O ASP 82 O C *ARG 91 NE O TYR 83 CD2 C LYS 42 NZ C ALA 117 O C THR 88 OG1 C SER 171 C O ASP 87 Tyr *GLN94 NE2 N SER111 O C Ser 83 Phe *ARG 121 NH1 O TYR87 CE2 C MET 120 SD C ARG91 CD O ALA 119 CB O ASP115 O C ASP 87 PHE 83 OD1 CD2 C8 C VAL90 CG1 C ASP 82 OD2 C Asp 87 Gly *ALA117 N O TYR 86 CD2 N GLY 87 CA O ALA 117 O C GLN94 NE2 C ASP115 O C Ser 83 Tyr *ARG 121 NH1 O SER116 CA O MET 120 SD C ARG91 CB C ASP 87 OD1 C PHE96 CE2 C TYR 86 CB C SER 83 O O ALA 119 CB O * Represents hydrogen bond forming Table 3 Interacting, of GyrA and ciprofloxacin involved in GyrA-ciprofloxacin interaction using Getneares program [Interaction within a distance 4 Å are shown] Wild Type *ASP87 OD1 N Ser 83 Tyr contd *MET120 N O ALA117 O C *ARG121 NH1 O MET120 CG C ALA119 CB O TYR86 CD2 F Asp 87 Tyr *HIS45 NE2 O SER83 CB C *LYS42 NZ O *SER172 N O Ser 83 Phe *ARG121 NH1 O GLN267 OE1 C *ASP87 OD1 N ARG91 CD C ASP82 OD2 C SER171 CA O TYR86 ALA117 CD2 O C16 C TYR87 CB F PHE83 CD2 C Asp 87 Gly *ARG91 NH1 O MET120 CG C *GLN94 NE2 O *ALA117 N F ALA119 CB C GLY87 O C Ser 83 Tyr *ARG121 NH1 O SER97 OG O ASP87 OD1 C SER116 CA F ASP82 OD2 C TYR86 CD2 C TYR86 CD2 C VAL90 CG1 C TYR83 CD2 C PHE96 CE2 O ALA119 CB C *Represents hydrogen bond forming

6 152 INDIAN J. BIOCHEM. BIOPHYS., VOL. 46, APRIL 2009 nalidixic acid-qrdr of GyrA clearly showed the phenyl ring making π-π interaction in GyrA Ser 83 Phe (Fig. 3B) and nalidixic acid binding was away from its usual binding site of wild type. It may be mentioned that the hydrophobic due to their π-π interaction increased G-score (Eq. 1). Based on their G scores and nature of molecular forces involved, nalidixic acid showed less affinity towards GyrA in 83, 87 mutated forms of GyrA. Wild type > (Asp 87 to Tyr/Gly) > (Ser 83 to Phe/Tyr). This decreasing order of binding suggested that mutation at position of Ser 83 induced more resistance than Asp 87. Ciprofloxacin binding with QRDR of gyrase A Ciprofloxacin bound to GyrA (SAB4) of sensitive strain (Fig. 3D) through three hydrogen bonds involving Asp 87, Arg 121 and Met 120. However, ciprofloxacin also made hydrophobic interactions with several nearby with a high G-score of kcal/mol (see Table 3 and Fig. 3D). Fluorine of ciprofloxacin made close contacts with Tyr 86. The mutation of Ser 83 to Tyr in GyrA allowed only one hydrogen bond with ciprofloxacin involving Arg 121. While Ser 83 to Phe allowed two hydrogen bonds involving amino acids Arg 121 and Asp 87. G-score remained relatively similar, but number of hydrogen bonds were reduced which lead to decrease in specificity of binding. Bulky aromatic group, Phe at 83 position brought conformation changes near binding site, in which ciprofloxacin fitted well and increased its hydrophobic interactions with GyrA. Since Asp 87 directly makes hydrogen bond with N3 of ciprofloxacin (in wild type), the mutation at this position was expected to bring drastic changes in molecular interactions involving ciprofloxacin. The hydrogen bonds between Asp 87, Met 120, Arg 121 in GyrA with ciprofloxacin in wild type were ruptured on introducing the mutation at 87, however, the mutated GyrA-ciprofloxacin complex was stabilized by two new sets of hydrogen bonds from (Ser 172, His 45, Lys 42)/(Arg 91, Gln 94, Ala 117) in 87 Tyr and 87 Gly, respectively. These changes made ciprofloxacin binding to new position, which was away from the natural binding site of quinolone in GyrA (Fig. 3D-F). The low G-score of approximately -3.9 kcal/mol in 87 mutated GyrA explained weak binding of ciprofloxacin to GyrA. The MIC data (earlier reported by us 4 ) showed good agreement with the docking results, as the mutated GyrA with high MIC showed maximum change in the G-score. As seen in all the mutations, in this study, we found there was a reduction of directionality and specificity between GyrA and ciprofloxacin. (Wild type) > (Ser 83 to Tyr/Phe) > (Asp 87 to Tyr/Gly). It appeared that position Asp 87 was more important for ciprofloxacin binding to QRDR, because it was directly involved in ciprofloxacin binding. Concurrently, Ser 83 was also important because it induced the proper conformation for nalidixic acid binding in wild type. Binding site of nalidixic acid/ciprofloxacin was shifted in mutated GyrA structures. However, the shift in the binding site in the mutated conformation of GyrA was prominent for nalidixic acid, as compared to ciprofloxacin (Tables 2 and 3). A very high IC 50 value for ciprofloxacin (13.1 µg/ml) of Asp 87 mutated GyrA of E. coli as compared to that of wild type (4.6 µg/ml) and Ser 83 mutated GyrA (8.62 µg/ml) indicated that mutation at Asp 87 provided more resistance for ciprofloxacin than at 83 position in GyrA 6. Therefore, the experimental data shown above correlated well the conclusions arrived from the present in silico study. In conclusion, both mutations Ser 83 and/or Asp 87 are responsible for the reduction in the specific interaction between quinolones and QRDR of gyrase A which finally leads to the resistance. The computational docking studies and the experimentally obtained IC 50 and MIC show good correlation and provide a possible explanation for the observed quinolone-resistance in S. enterica. Acknowledgement Jitendra Vashist and Vishvanath thank the Council of Scientific and Industrial Research, India for providing fellowship. References 1 Maxwell A (1992) J Antimicrob Chemother 30, Weigel L M, Steward C D & Tenover F C (1998) Antimicrob Agents Chemother 42, Hirose K, Hashimoto A, Tamura K, Kawamura Y, Ezaki T, Sagara H & Watanabe H (2002) Antimicrob Agents Chemother 46, Renuka, Kapil A, Kabra S K, Wig N, Das B K, Prasad V V S P, Chaudhry R & Seth P (2004) Microbial Drug Resist 10, Yoshida H, Bogaki M, Nakamura M & Nakamura S (1990) Antimicrob Agents Chemother 34,

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