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Insight into the catalytic hydrolysis mechanism of New Delhi metallo-β-lactamase to aztreonam by molecular modeling
Journal of Molecular Liquids 282 (2019) 244–250
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Insight into the catalytic hydrolysis mechanism of New Delhi metallo-β-lactamase to aztreonam by molecular modeling Yiding Yu a, Xiyan Wang a, Yawen Gao a, Yanan Yang a, Guizhen Wang a, Lin Sun a, Yan Zhou b,⁎, Xiaodi Niu a,⁎ a b
College of Food Science and Engineering, Jilin University, China The First Hospital of Jilin University, Pediatric Cardiovascular Department, Jilin University, China
a r t i c l e
i n f o
Article history: Received 21 August 2018 Received in revised form 24 January 2019 Accepted 1 March 2019 Available online 03 March 2019 Keywords: NDM-1 VIM-1 Aztreonam Molecular dynamics simulation
a b s t r a c t The monobactam antibiotic, aztreonam, can be hydrolyzed by some β-lactamases (NDM-1), whereas other β-lactamases, including VIM-1, have no hydrolytic activity on aztreonam. NDM-1 and VIM-1 are both metallo-β-lactamases (MBLs), but they catalyze hydrolysis at different active site residues, which is reflected in their obvious activity difference with aztreonam. In this work, to explore the catalytic hydrolysis mechanism of MBLs at the atomic level, molecular dynamic simulations were performed for NDM-1aztreonam and VIM-1-aztreonam complexes based on the molecular docking analysis. Molecular modeling revealed the binding of aztreonam to the active regions of NDM-1 and VIM-1. Residues Met67, Phe70, Asp124, Thr190, and His250 play key roles in the binding of aztreonam with NDM-1. His116, Asp118, Cys198, His201, and His240 are the critical residues for the binding of aztreonam with VIM-1. Interestingly, Asp124 displayed the strongest binding energy (ΔEtotal = −11.28 kcal/mol), which was nearly 10 times higher than that of the other residues in the NDM-1-aztreonam complex. A similar result was found for the binding of aztreonam with VIM-1, with Asp118 displaying the strongest binding energy (ΔEtotal = −2.95 kcal/mol). These results implicated aspartic acid as the critical active site in the catalytic hydrolysis pocket of NDM-1 and VIM-1. However, in the VIM-1-aztreonam complex, because of the strong π-π interaction between the thiazol ring group of aztreonam and the imidazole ring groups of His201 and His240, the binding energy obtained from Asp118 become significantly weaker than that of aztreonam with Asp124 of NDM-1. Analysis of the simulation trajectory indicated that the thiazol ring plane of aztreonam is almost parallel to the imidazole ring planes of His201 and His240, implying that they can form a strong π-π interaction, which is consistent with the above results. On the basis of the computational biology results, it was confirmed that aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote substrate hydrolysis, while histidine on the other side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. © 2019 Published by Elsevier B.V.
1. Introduction The extensive use of antibiotics has driven the increasing development of antibiotic resistance in bacteria, which complicates the treatment of bacterial infections. Bacteria can adapt to their environment, and can become drug-resistant by genetic and structural alterations [1]. Drug resistance can involve the expression of detoxifying enzymes and efflux pumps, and alterations in the binding sites of antibiotics. βlactam antibiotics comprise approximately 60% of all antibacterial agents used in the clinical treatment of bacterial infection [2,3]. Bacteria have developed β-lactam resistance by several means, most effectively by the expression of β-lactamases [4,5]. β-lactamases act by catalyzing the hydrolysis of antibiotics containing a β-lactam ring.
⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Zhou), [email protected] (X. Niu).
https://doi.org/10.1016/j.molliq.2019.03.006 0167-7322/© 2019 Published by Elsevier B.V.
In 1995, the first structure of metallo-β-lactamases (MBL) from Bacillus cereus was identified [6]. Over 1000 β-lactamases have since been identified. These enzymes are divided into four classes (A, B, C, and D) [7]. Class A, C, and D are serine-based β-lactamases (SBLs), which can use the active site serine as the nucleophile [8]. Class B are MBLs, which apply Zn2+ ions to hydrolyze β-lactam antibiotics [9]. Based on the variation in the number of metal ions and the sequence differences of enzymes [10,11], MBLs also consists of three groups: B1, B2, and B3 [12,13]. MBLs can hydrolyze almost all β-lactam antibiotics used in the clinical treatment of bacterial infection, including the latest antibiotics, cephamycins and imipenem [14–16]. More seriously, the activity of MBLs cannot be effectively inhibited by SBL inhibitors [3,17,18]. Therefore, there is an urgent need for the development of new strategies for the treatment of newly emerging drug-resistant bacterial pathogens, especially for the bacteria that can evolve MBLs, including New Delhi metallo-β-lactamase-1 (NDM-1), Verona integrin-encoded (VIM-1), and imipenemase (IMP-1).
Y. Yu et al. / Journal of Molecular Liquids 282 (2019) 244–250
Advances in the knowledge of the structure and reactivity of the active site of MBLs by crystallographic [19–22], spectroscopic [23–25], and computational [26–28] methods have led to the development of MBL inhibitors [29–35]. Comparison and analysis of the crystal structures of NDM-1 and NDM-1 complexes have revealed that B1 and B3 MBLs use the Zn1 ion as a Lewis acid to generate a nucleophilic hydroxide in the active site, while B2 employs the hydrogen bond network for this purpose instead of the metal ion. The His120, His122, His189, Asp124, Cys208, and His 250 residues of NDM-1 are crucial in the binding with Zn (II) ions [36]. In 2017, molecular dynamic (MD) simulation and quantum mechanical/ molecular mechanical (QM/MM) methods along with density functional theory (DFT) approach were used to detail the catalytic hydrolysis mechanisms of NDM-1 for cephalexin and meropenem [37]. Lys211 was demonstrated to contribute to the proton transfer through a chain of two water molecules. The same year, Chen et al. reported that the change in the zinc ion coordination was of significance for the conformational changes of NDM-1 to meet the needs of hydrolysis and to develop potent inhibitors targeting NDM-1 by MD simulations and the umbrella sampling method [38]. Another promising finding was that zinc ions play a key role in the binding of the antibiotic ampicillin (AMP), based on statistical analyses of interaction contacts of AMP with residues of NDM-1. The combined use of avibactam (the chemical structure was shown in Fig. 1) and the monobactam antibiotic aztreonam has become the latest strategy for the treatment of drug-resistant bacterial infection in clinical trials [39]. However, some MBLs can effectively hydrolyze aztreonam, such as NDM-1, which challenges the widely held view that aztreonam cannot be hydrolyzed by MBLs [40]. By contrast, the VIM-1 β-lactamase displays no hydrolysis activity for aztreonam. NDM-1 and VIM-1 are both MBLs and yet have different active site residues of the catalytic hydrolysis, as reflected in their obviously different activities for aztreonam. We have exploited this difference in the present study to conduct an integrated molecular modeling and binding free energy calculation to explore the interaction mechanisms of aztreonam with NDM-1 and VIM-1 at the atom level. We anticipate that the results will promote the development of efficient inhibitors of MBLs. 2. Method and computation The crystal structures of NDM-1 (PDB code 3ZR9) [41] and VIM-1 (PDB code 5N5G) were used as the initial coordinates of NDM-1 and VIM-1 for the molecular docking by the Autodock 4.0 package [42]. The molecular structure of aztreonam was obtained through the optimization by Gaussian 09 software, which were used as the initial structure of molecular dynamics simulation.
Fig. 1. The chemical structure of aztreonam.
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The processes and methods are described in detail in the Supplementary materials. 3. Results and discussion 3.1. Identification of potential binding modes of aztreonam with NDM-1 and VIM-1 We confirmed the prior observation [40] of the direct interaction of aztreonam with NDM-1 and VIM-1. Molecular docking and molecular dynamics simulation were performed to explore the interaction mechanism between ligand and proteins. As shown in Fig. 2, in the simulation after 20 ns, the RMSD average values of complexes were 0.33 and 0.27 nm for NDM-1-aztreonam and VIM-1-aztreonam, indicating that the two complexes reached equilibrium at 20 ns. During the 100-ns simulation, the potential stable three-dimensional structures of aztreonam with NDM-1 and VIM-1 were obtained (Fig. 3). aztreonam bound to the active sites of NDM-1, and except for zinc ions, Ile35, Met67, Phe70, His122, Asp124, Thr190, and Cys208 residues appeared to strongly interact with aztreonam (Fig. 3A). In particular, Ile35, Phe70, and His250 were crucial in the immobilization of the thiazol ring group of aztreonam, and His122 and Asp124 anchored the sulfanolate moiety of aztreonam. Curiously, aztreonam also bound to the active sites of VIM-1, and interacted with residues in the active region, which included His116, Asp118, Cys198, His201, and His240. The thiazol ring group of aztreonam formed an interaction with Cys198, His201, and His240. His116 and Asp118 were critical in the anchorage of the sulfanolate moiety with protein (Fig. 3B). Root mean square fluctuations (RMSFs) were calculated to analysis the conformational changes of the residue side-chains around the binding sites before and after binding to aztreonam (Fig. 4). In the NDM-1-aztreonam complex, the flexibility of the residues bound to aztreonam differed from those of residues in the free protein. The flexibilities of the binding site residues (65–70, 120–125, 185–210, and 245–250) in NDM-1 bound to aztreonam were less than the flexibilities in the free protein, with RMSFs b0.2 nm, indicating that these residues were more rigid after binding to aztreonam (Fig. 4A). The same results were obtained with the VIM-1-aztreonam complex. Residues 110–120, 180–220, 185–210, and 240–245, which were involved in the binding of VIM-1 to aztreonam, were less flexible than those of the free protein (Fig. 4B). The results of the RMSF calculations were consistent with the three-dimensional structures of the NDM-1-aztreonam and VIM-1-aztreonam complexes based on the
Fig. 2. The RMSD displayed by the backbone atoms of protein during MD simulations of NDM-1-aztreonam (black line) and VIM-1-aztreonam (red line).
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Fig. 3. Potential binding modes of aztreonam with NDM-1 (A) and VIM-1 (B) based on the molecular dynamics simulation. In the NDM-1-aztreonam complex, the Ile35, Met67, Phe70, His122, Asp124, Thr190, Cys208, and His250 residues play key roles in the binding of aztreonam with NDM-1. In the VIM-1-aztreonam complex, His116, Asp118, Cys198, His201, and His240 residues contribute the major binding energy.
MD simulation, implying that the binding modes of aztreonam with NDM-1 and VIM-1 are reliable. 3.2. Confirmation of the aztreonam binding sites with NDM-1 and VIM-1 The Molecular Mechanics Generalized Born Surface Area (MMGBSA) method was used to calculate the detailed binding affinities
contributed by the residues in the binding of NDM-1 or VIM-1 to aztreonam. Asp124 displayed the strongest binding energy, with ΔEtotal values of −11.3 kcal/mol (Fig. 5A). The binding energy was almost 10 times higher than that of the other residues in the NDM-1-aztreonam complex, implying that Asp124 plays a key role in the binding of substrate with NDM-1 via interaction with the sulfanolate moiety of aztreonam.
Fig. 4. Root mean square fluctuations (RMSFs) of the all residues of NDM-1 (A) and VIM-1 (B) over the last 50 ns of simulations with respect to their initial position in the free protein (black line) and in the complexes (red line). The region of the protein backbone exhibits different fluctuations that are dependent on the local environment (bound with aztreonam), residues 65–70, 120–125, 185–210, and 245–250 of NDM-1, and residues 110–120, 180–220, 185–210, and 240–245 of VIM-1. The regions are highlighted with gray bars.
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Fig. 5. Decomposition of the binding energy on a per-residue basis in the NDM-1-aztreonam (A) and VIM-1-aztreonam (B) by the MM-GBSA method. The histogram chart shows the van der Waals (cyan), electrostatic (LT gray), solvation (LT magenta), and total (orange) contributions for the complexes.
Met67, Phe70, Thr190, and His250 also displayed strong binding energies, with ΔEtotal b −1.00 kcal/mol. Analysis of the complex structure based on the MD simulation, it was confirmed that Met67 could form a strong interaction with the central part of aztreonam, with the side chains of Phe70 and His250 having strong affinity for the thiazol ring group of aztreonam. In the VIM-1-aztreonam complex, Asp118 also displayed the strongest binding energy to the binding of VIM-1 to aztreonam, with ΔEtotal values of −2.95 kcal/mol (Fig. 5B). Except for Asp118,
aztreonam also formed interactions with His116, His179, Cys198, His201, and His240 residues, with ΔEtotal b −1.00 kcal/mol. The distances between all residues and aztreonam in the NDM-1aztreonam and VIM-1-aztreonam complexes were calculated during the last 50 ns of the simulation. Consistent with the results described above, the distances between residues (Met67, Phe70, Asp124, Thr190, and His250) with higher binding energies and aztreonam were lower (b0.3 nm) in the NDM-1-aztreonam complex system
Fig. 6. Distance between the whole residues of NDM-1 (A) and aztreonam, the distance between residues in the binding sites (B) and aztreonam; Distance between the whole residues of VIM-1 (C) and aztreonam, the distance between residues in the binding sites (D) and aztreonam. The black line ranges represent the binding regions of complexes.
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(Fig. 6A and B). For the VIM-1-aztreonam, His116, Asp118, His179, Cys198, His201, and His240 residues displayed the minimum distance with aztreonam (b0.3 nm; Fig. 6C and D). The results support the suggestions that Met67, Phe70, Asp124, Thr190, and His250 are crucial in the binding of NDM-1 to aztreonam, and that the His116, Asp118, His179, Cys198, His201, and His240 residues contribute the major binding affinity in the VIM-1-aztreonam complex. Based on the previous literatures [10,43], the active site key residues of NDM-1 were exactly H120, H189, C208, K211, N220, H250, and for VIM-1, the active key residues were Cys221, His224, Ser228 (the number of residues increased 23 than ours in this work). These results implied that aztreonam can exactly bind to the active sites of NDM-1 and VIM-1. Comparison of the binding energy contributions of the residues in the binding sites revealed that Asp118 displayed the strongest binding affinity with aztreonam, while the binding energy between Asp118 and the sulfanolate moiety of aztreonam in the VIM-1aztreonam complex was considerably lower than that in the NDM1-aztreonam complex. On the contrary, the binding energies between the side chains of His201 and His240 and the thiazol ring group of aztreonam in VIM-1-aztreonam complex were obviously higher than those contributed by Phe70 and His250 to the thiazol ring group of aztreonam in the NDM-1-aztreonam complex. It follows that the binding affinity of the thiazol ring group of aztreonam with the histidine in the active site has a direct impact on the binding strength between sulfanolate moiety of aztreonam and aspartic acid in the active site, leading to a change in the hydrolysis activity of the enzyme to aztreonam. 3.3. Analysis of the hydrolysis mechanism of NDM-1 and VIM-1 to aztreonam To verify the forementioned hypothesis, the dynamics analysis of the simulation trajectory was performed. Fig. 7A shows that the plane of the thiazol ring group could not be parallel to the benzene ring and imidazole ring planes of Phe70 and His250. Instead, in the VIM-1-aztreonam complex, the imidazole ring planes of His201 and His240 were very parallel to the plane of the thiazol ring group of aztreonam, implying a strong π-π interaction between aztreonam and His201 and His240. As is shown in Fig. 8A and B, in the NDM-1aztreonam complex, the dihedral angles of the imidazole ring plane of His250 and the benzene ring plane of Phe70 with the thiazol ring's plane of aztreonam fluctuated from 120° to −120° during the last 50 ns of the simulation, while the dihedral angles of the imidazole ring plane of His240 and the imidazole ring plane of His201 with the thiazol ring plane of aztreonam always fluctuated around 0° during the last 50 ns of the simulation in the VIM-1-aztreonam complex. The distances between the conjugated groups of residues and the thiazol ring moiety of aztreonam were analyzed based on the simulation trajectory, as illustrated in Fig. 8C and D. Fig. 8C shows that the average distances between the imidazole ring group's plane of His250, the benzene ring of Phe70, and the thiazol ring of aztreonam exceeded 0.6 nm, while the average distances between the imidazole ring groups of His240, His201, and the thiazol ring of aztreonam were lower than those of the NDM-1-aztreonam complex, with an average distance of approximately 0.4 nm (Fig. 8D). These results indicated that in the VIM-1-aztreonam complex system, the ligand can form strong π-π interactions with His201 and His240, while Phe70 and His250 of NDM-1 weakly interact with aztreonam. Due to the strong interaction between the thiazol ring of aztreonam and the side chains of His201 and His240, the binding affinity of the sulfanolate moiety of aztreonam with Asp118 was decreased, leading to the hydrolytic activity loss of VIM-1 binding to aztreonam (Fig. 9). Thus, Aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote the substrate hydrolysis, while histidine on the other
Fig. 7. Significant differences in the dynamics of the interactions of aztreonam with Phe70 and His250 of NDM-1 (A), and aztreonam with His201 and His240 of VIM-1 (B) during the last 50-ns simulations. The conformations of aztreonam, Phe70, His250, His201, and His240 from the molecular dynamics simulations are presented. The 20 snapshots obtained from the simulations were superimposed using Pymol software.
side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. 4. Conclusion The interaction mechanisms of aztreonam with NDM-1 and VIM-1 were explored using MD simulations and calculation of binding free energies. Molecular modeling revealed that aztreonam can bind to the active sites of NDM-1 and VIM-1. In these two complex systems, aspartic acid (Asp124 of NDM-1, Asp118 of VIM-1) contributes to the major affinity in the binding with aztreonam, implying that the aspartic acid in the binding site plays a key role in the hydrolysis of the aztreonam. However, the binding energy between Asp118 and aztreonam was weaker than that between Asp124 and aztreonam based on the binding free energy calculation. On the basis of the analysis of the simulation trajectory, it was confirmed that due to the π-π interaction between the side chains of His201, His240, and the thiazol ring of aztreonam, the binding energy of the sulfanolate moiety of aztreonam with Asp118 decreased, leading to the hydrolysis activity loss of VIM1 to aztreonam. Therefore, we believe that Asp124 of NDM-1 and Asp118 of VIM-1 can effectively promote substrate hydrolysis, while His201 and His240
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Fig. 8. Comparison of the dihedrals and distances in the different systems. (A) The dihedral angles of the imidazole ring plane of His250 (black line) and the benzene ring plane of Phe70 (red line) with the thiazol ring plane of aztreonam as a function of time. (B) The dihedral angles of the imidazole ring plane of His240 (black line) and the imidazole ring plane of His201 (red line) with the thiazol ring plane of aztreonam as a function of time. (C) The distance between the imidazole ring plane of His250 (black line), the benzene ring of Phe70 (red line), and the thiazol ring of aztreonam as a function of time. (D) The distance between the imidazole ring groups of His240 (black line), His201 (red line), and the thiazol ring of aztreonam as a function of time.
of VIM-1 on the other side of the active pocket can block the binding of the substrate with Aspartic acid. These results provide foundational data that will help in the development of new and more effective anti-drug resistance inhibitors.
Acknowledgments The authors acknowledge the financial support from the National Natural Science Foundation of China [Grant no. 31572566 to X. D. N].
Fig. 9. Schematic representation of the interaction between residues of the binding sites and aztreonam. The π-π interaction of His201 and His240 with the thiazol ring of aztreonam decrease the binding affinity of Asp118 with the sulfanolate moiety of aztreonam. (Table 1)
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Table 1 Calculated energy components, binding free energy (kcal/mol) of NDM-1 and VIM-1 binding with ATM based on MM-GBSA. Energy components (kcal/mol)
NDM-1
VIM-1
ΔEele ΔEvdw ΔEMM ΔGele,sol ΔGnonpolar,sol ΔGsol ΔGele,sol + ΔEele ΔGnonpolar + ΔEvdw ΔGbind
−88.45 ± 2.44 −6.60 ± 0.65 −95.05 ± 6.08 −3.98 ± 0.08 66.81 ± 4.02 62.83 ± 3.93 −92.43 ± 1.92 60.21 ± 2.37 −32.22 ± 4.58
−74.05 ± 2.48 −20.59 ± 0.57 −94.64 ± 2.40 −4.89 ± 0.03 75.31 ± 2.78 70.42 ± 2.09 −78.94 ± 3.16 54.72 ± 1.82 −24.22 ± 0.90
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Insight into the catalytic hydrolysis mechanism of New Delhi metallo-β-lactamase to aztreonam by molecular modeling Yiding Yu a, Xiyan Wang a, Yawen Gao a, Yanan Yang a, Guizhen Wang a, Lin Sun a, Yan Zhou b,⁎, Xiaodi Niu a,⁎ a b
College of Food Science and Engineering, Jilin University, China The First Hospital of Jilin University, Pediatric Cardiovascular Department, Jilin University, China
a r t i c l e
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Article history: Received 21 August 2018 Received in revised form 24 January 2019 Accepted 1 March 2019 Available online 03 March 2019 Keywords: NDM-1 VIM-1 Aztreonam Molecular dynamics simulation
a b s t r a c t The monobactam antibiotic, aztreonam, can be hydrolyzed by some β-lactamases (NDM-1), whereas other β-lactamases, including VIM-1, have no hydrolytic activity on aztreonam. NDM-1 and VIM-1 are both metallo-β-lactamases (MBLs), but they catalyze hydrolysis at different active site residues, which is reflected in their obvious activity difference with aztreonam. In this work, to explore the catalytic hydrolysis mechanism of MBLs at the atomic level, molecular dynamic simulations were performed for NDM-1aztreonam and VIM-1-aztreonam complexes based on the molecular docking analysis. Molecular modeling revealed the binding of aztreonam to the active regions of NDM-1 and VIM-1. Residues Met67, Phe70, Asp124, Thr190, and His250 play key roles in the binding of aztreonam with NDM-1. His116, Asp118, Cys198, His201, and His240 are the critical residues for the binding of aztreonam with VIM-1. Interestingly, Asp124 displayed the strongest binding energy (ΔEtotal = −11.28 kcal/mol), which was nearly 10 times higher than that of the other residues in the NDM-1-aztreonam complex. A similar result was found for the binding of aztreonam with VIM-1, with Asp118 displaying the strongest binding energy (ΔEtotal = −2.95 kcal/mol). These results implicated aspartic acid as the critical active site in the catalytic hydrolysis pocket of NDM-1 and VIM-1. However, in the VIM-1-aztreonam complex, because of the strong π-π interaction between the thiazol ring group of aztreonam and the imidazole ring groups of His201 and His240, the binding energy obtained from Asp118 become significantly weaker than that of aztreonam with Asp124 of NDM-1. Analysis of the simulation trajectory indicated that the thiazol ring plane of aztreonam is almost parallel to the imidazole ring planes of His201 and His240, implying that they can form a strong π-π interaction, which is consistent with the above results. On the basis of the computational biology results, it was confirmed that aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote substrate hydrolysis, while histidine on the other side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. © 2019 Published by Elsevier B.V.
1. Introduction The extensive use of antibiotics has driven the increasing development of antibiotic resistance in bacteria, which complicates the treatment of bacterial infections. Bacteria can adapt to their environment, and can become drug-resistant by genetic and structural alterations [1]. Drug resistance can involve the expression of detoxifying enzymes and efflux pumps, and alterations in the binding sites of antibiotics. βlactam antibiotics comprise approximately 60% of all antibacterial agents used in the clinical treatment of bacterial infection [2,3]. Bacteria have developed β-lactam resistance by several means, most effectively by the expression of β-lactamases [4,5]. β-lactamases act by catalyzing the hydrolysis of antibiotics containing a β-lactam ring.
⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Zhou), [email protected] (X. Niu).
https://doi.org/10.1016/j.molliq.2019.03.006 0167-7322/© 2019 Published by Elsevier B.V.
In 1995, the first structure of metallo-β-lactamases (MBL) from Bacillus cereus was identified [6]. Over 1000 β-lactamases have since been identified. These enzymes are divided into four classes (A, B, C, and D) [7]. Class A, C, and D are serine-based β-lactamases (SBLs), which can use the active site serine as the nucleophile [8]. Class B are MBLs, which apply Zn2+ ions to hydrolyze β-lactam antibiotics [9]. Based on the variation in the number of metal ions and the sequence differences of enzymes [10,11], MBLs also consists of three groups: B1, B2, and B3 [12,13]. MBLs can hydrolyze almost all β-lactam antibiotics used in the clinical treatment of bacterial infection, including the latest antibiotics, cephamycins and imipenem [14–16]. More seriously, the activity of MBLs cannot be effectively inhibited by SBL inhibitors [3,17,18]. Therefore, there is an urgent need for the development of new strategies for the treatment of newly emerging drug-resistant bacterial pathogens, especially for the bacteria that can evolve MBLs, including New Delhi metallo-β-lactamase-1 (NDM-1), Verona integrin-encoded (VIM-1), and imipenemase (IMP-1).
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Advances in the knowledge of the structure and reactivity of the active site of MBLs by crystallographic [19–22], spectroscopic [23–25], and computational [26–28] methods have led to the development of MBL inhibitors [29–35]. Comparison and analysis of the crystal structures of NDM-1 and NDM-1 complexes have revealed that B1 and B3 MBLs use the Zn1 ion as a Lewis acid to generate a nucleophilic hydroxide in the active site, while B2 employs the hydrogen bond network for this purpose instead of the metal ion. The His120, His122, His189, Asp124, Cys208, and His 250 residues of NDM-1 are crucial in the binding with Zn (II) ions [36]. In 2017, molecular dynamic (MD) simulation and quantum mechanical/ molecular mechanical (QM/MM) methods along with density functional theory (DFT) approach were used to detail the catalytic hydrolysis mechanisms of NDM-1 for cephalexin and meropenem [37]. Lys211 was demonstrated to contribute to the proton transfer through a chain of two water molecules. The same year, Chen et al. reported that the change in the zinc ion coordination was of significance for the conformational changes of NDM-1 to meet the needs of hydrolysis and to develop potent inhibitors targeting NDM-1 by MD simulations and the umbrella sampling method [38]. Another promising finding was that zinc ions play a key role in the binding of the antibiotic ampicillin (AMP), based on statistical analyses of interaction contacts of AMP with residues of NDM-1. The combined use of avibactam (the chemical structure was shown in Fig. 1) and the monobactam antibiotic aztreonam has become the latest strategy for the treatment of drug-resistant bacterial infection in clinical trials [39]. However, some MBLs can effectively hydrolyze aztreonam, such as NDM-1, which challenges the widely held view that aztreonam cannot be hydrolyzed by MBLs [40]. By contrast, the VIM-1 β-lactamase displays no hydrolysis activity for aztreonam. NDM-1 and VIM-1 are both MBLs and yet have different active site residues of the catalytic hydrolysis, as reflected in their obviously different activities for aztreonam. We have exploited this difference in the present study to conduct an integrated molecular modeling and binding free energy calculation to explore the interaction mechanisms of aztreonam with NDM-1 and VIM-1 at the atom level. We anticipate that the results will promote the development of efficient inhibitors of MBLs. 2. Method and computation The crystal structures of NDM-1 (PDB code 3ZR9) [41] and VIM-1 (PDB code 5N5G) were used as the initial coordinates of NDM-1 and VIM-1 for the molecular docking by the Autodock 4.0 package [42]. The molecular structure of aztreonam was obtained through the optimization by Gaussian 09 software, which were used as the initial structure of molecular dynamics simulation.
Fig. 1. The chemical structure of aztreonam.
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The processes and methods are described in detail in the Supplementary materials. 3. Results and discussion 3.1. Identification of potential binding modes of aztreonam with NDM-1 and VIM-1 We confirmed the prior observation [40] of the direct interaction of aztreonam with NDM-1 and VIM-1. Molecular docking and molecular dynamics simulation were performed to explore the interaction mechanism between ligand and proteins. As shown in Fig. 2, in the simulation after 20 ns, the RMSD average values of complexes were 0.33 and 0.27 nm for NDM-1-aztreonam and VIM-1-aztreonam, indicating that the two complexes reached equilibrium at 20 ns. During the 100-ns simulation, the potential stable three-dimensional structures of aztreonam with NDM-1 and VIM-1 were obtained (Fig. 3). aztreonam bound to the active sites of NDM-1, and except for zinc ions, Ile35, Met67, Phe70, His122, Asp124, Thr190, and Cys208 residues appeared to strongly interact with aztreonam (Fig. 3A). In particular, Ile35, Phe70, and His250 were crucial in the immobilization of the thiazol ring group of aztreonam, and His122 and Asp124 anchored the sulfanolate moiety of aztreonam. Curiously, aztreonam also bound to the active sites of VIM-1, and interacted with residues in the active region, which included His116, Asp118, Cys198, His201, and His240. The thiazol ring group of aztreonam formed an interaction with Cys198, His201, and His240. His116 and Asp118 were critical in the anchorage of the sulfanolate moiety with protein (Fig. 3B). Root mean square fluctuations (RMSFs) were calculated to analysis the conformational changes of the residue side-chains around the binding sites before and after binding to aztreonam (Fig. 4). In the NDM-1-aztreonam complex, the flexibility of the residues bound to aztreonam differed from those of residues in the free protein. The flexibilities of the binding site residues (65–70, 120–125, 185–210, and 245–250) in NDM-1 bound to aztreonam were less than the flexibilities in the free protein, with RMSFs b0.2 nm, indicating that these residues were more rigid after binding to aztreonam (Fig. 4A). The same results were obtained with the VIM-1-aztreonam complex. Residues 110–120, 180–220, 185–210, and 240–245, which were involved in the binding of VIM-1 to aztreonam, were less flexible than those of the free protein (Fig. 4B). The results of the RMSF calculations were consistent with the three-dimensional structures of the NDM-1-aztreonam and VIM-1-aztreonam complexes based on the
Fig. 2. The RMSD displayed by the backbone atoms of protein during MD simulations of NDM-1-aztreonam (black line) and VIM-1-aztreonam (red line).
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Fig. 3. Potential binding modes of aztreonam with NDM-1 (A) and VIM-1 (B) based on the molecular dynamics simulation. In the NDM-1-aztreonam complex, the Ile35, Met67, Phe70, His122, Asp124, Thr190, Cys208, and His250 residues play key roles in the binding of aztreonam with NDM-1. In the VIM-1-aztreonam complex, His116, Asp118, Cys198, His201, and His240 residues contribute the major binding energy.
MD simulation, implying that the binding modes of aztreonam with NDM-1 and VIM-1 are reliable. 3.2. Confirmation of the aztreonam binding sites with NDM-1 and VIM-1 The Molecular Mechanics Generalized Born Surface Area (MMGBSA) method was used to calculate the detailed binding affinities
contributed by the residues in the binding of NDM-1 or VIM-1 to aztreonam. Asp124 displayed the strongest binding energy, with ΔEtotal values of −11.3 kcal/mol (Fig. 5A). The binding energy was almost 10 times higher than that of the other residues in the NDM-1-aztreonam complex, implying that Asp124 plays a key role in the binding of substrate with NDM-1 via interaction with the sulfanolate moiety of aztreonam.
Fig. 4. Root mean square fluctuations (RMSFs) of the all residues of NDM-1 (A) and VIM-1 (B) over the last 50 ns of simulations with respect to their initial position in the free protein (black line) and in the complexes (red line). The region of the protein backbone exhibits different fluctuations that are dependent on the local environment (bound with aztreonam), residues 65–70, 120–125, 185–210, and 245–250 of NDM-1, and residues 110–120, 180–220, 185–210, and 240–245 of VIM-1. The regions are highlighted with gray bars.
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Fig. 5. Decomposition of the binding energy on a per-residue basis in the NDM-1-aztreonam (A) and VIM-1-aztreonam (B) by the MM-GBSA method. The histogram chart shows the van der Waals (cyan), electrostatic (LT gray), solvation (LT magenta), and total (orange) contributions for the complexes.
Met67, Phe70, Thr190, and His250 also displayed strong binding energies, with ΔEtotal b −1.00 kcal/mol. Analysis of the complex structure based on the MD simulation, it was confirmed that Met67 could form a strong interaction with the central part of aztreonam, with the side chains of Phe70 and His250 having strong affinity for the thiazol ring group of aztreonam. In the VIM-1-aztreonam complex, Asp118 also displayed the strongest binding energy to the binding of VIM-1 to aztreonam, with ΔEtotal values of −2.95 kcal/mol (Fig. 5B). Except for Asp118,
aztreonam also formed interactions with His116, His179, Cys198, His201, and His240 residues, with ΔEtotal b −1.00 kcal/mol. The distances between all residues and aztreonam in the NDM-1aztreonam and VIM-1-aztreonam complexes were calculated during the last 50 ns of the simulation. Consistent with the results described above, the distances between residues (Met67, Phe70, Asp124, Thr190, and His250) with higher binding energies and aztreonam were lower (b0.3 nm) in the NDM-1-aztreonam complex system
Fig. 6. Distance between the whole residues of NDM-1 (A) and aztreonam, the distance between residues in the binding sites (B) and aztreonam; Distance between the whole residues of VIM-1 (C) and aztreonam, the distance between residues in the binding sites (D) and aztreonam. The black line ranges represent the binding regions of complexes.
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(Fig. 6A and B). For the VIM-1-aztreonam, His116, Asp118, His179, Cys198, His201, and His240 residues displayed the minimum distance with aztreonam (b0.3 nm; Fig. 6C and D). The results support the suggestions that Met67, Phe70, Asp124, Thr190, and His250 are crucial in the binding of NDM-1 to aztreonam, and that the His116, Asp118, His179, Cys198, His201, and His240 residues contribute the major binding affinity in the VIM-1-aztreonam complex. Based on the previous literatures [10,43], the active site key residues of NDM-1 were exactly H120, H189, C208, K211, N220, H250, and for VIM-1, the active key residues were Cys221, His224, Ser228 (the number of residues increased 23 than ours in this work). These results implied that aztreonam can exactly bind to the active sites of NDM-1 and VIM-1. Comparison of the binding energy contributions of the residues in the binding sites revealed that Asp118 displayed the strongest binding affinity with aztreonam, while the binding energy between Asp118 and the sulfanolate moiety of aztreonam in the VIM-1aztreonam complex was considerably lower than that in the NDM1-aztreonam complex. On the contrary, the binding energies between the side chains of His201 and His240 and the thiazol ring group of aztreonam in VIM-1-aztreonam complex were obviously higher than those contributed by Phe70 and His250 to the thiazol ring group of aztreonam in the NDM-1-aztreonam complex. It follows that the binding affinity of the thiazol ring group of aztreonam with the histidine in the active site has a direct impact on the binding strength between sulfanolate moiety of aztreonam and aspartic acid in the active site, leading to a change in the hydrolysis activity of the enzyme to aztreonam. 3.3. Analysis of the hydrolysis mechanism of NDM-1 and VIM-1 to aztreonam To verify the forementioned hypothesis, the dynamics analysis of the simulation trajectory was performed. Fig. 7A shows that the plane of the thiazol ring group could not be parallel to the benzene ring and imidazole ring planes of Phe70 and His250. Instead, in the VIM-1-aztreonam complex, the imidazole ring planes of His201 and His240 were very parallel to the plane of the thiazol ring group of aztreonam, implying a strong π-π interaction between aztreonam and His201 and His240. As is shown in Fig. 8A and B, in the NDM-1aztreonam complex, the dihedral angles of the imidazole ring plane of His250 and the benzene ring plane of Phe70 with the thiazol ring's plane of aztreonam fluctuated from 120° to −120° during the last 50 ns of the simulation, while the dihedral angles of the imidazole ring plane of His240 and the imidazole ring plane of His201 with the thiazol ring plane of aztreonam always fluctuated around 0° during the last 50 ns of the simulation in the VIM-1-aztreonam complex. The distances between the conjugated groups of residues and the thiazol ring moiety of aztreonam were analyzed based on the simulation trajectory, as illustrated in Fig. 8C and D. Fig. 8C shows that the average distances between the imidazole ring group's plane of His250, the benzene ring of Phe70, and the thiazol ring of aztreonam exceeded 0.6 nm, while the average distances between the imidazole ring groups of His240, His201, and the thiazol ring of aztreonam were lower than those of the NDM-1-aztreonam complex, with an average distance of approximately 0.4 nm (Fig. 8D). These results indicated that in the VIM-1-aztreonam complex system, the ligand can form strong π-π interactions with His201 and His240, while Phe70 and His250 of NDM-1 weakly interact with aztreonam. Due to the strong interaction between the thiazol ring of aztreonam and the side chains of His201 and His240, the binding affinity of the sulfanolate moiety of aztreonam with Asp118 was decreased, leading to the hydrolytic activity loss of VIM-1 binding to aztreonam (Fig. 9). Thus, Aspartic acid in the active pocket of NDM-1 and VIM-1 can effectively promote the substrate hydrolysis, while histidine on the other
Fig. 7. Significant differences in the dynamics of the interactions of aztreonam with Phe70 and His250 of NDM-1 (A), and aztreonam with His201 and His240 of VIM-1 (B) during the last 50-ns simulations. The conformations of aztreonam, Phe70, His250, His201, and His240 from the molecular dynamics simulations are presented. The 20 snapshots obtained from the simulations were superimposed using Pymol software.
side of the active pocket can block the binding of the substrate with aspartic acid, leading to the loss of hydrolysis. 4. Conclusion The interaction mechanisms of aztreonam with NDM-1 and VIM-1 were explored using MD simulations and calculation of binding free energies. Molecular modeling revealed that aztreonam can bind to the active sites of NDM-1 and VIM-1. In these two complex systems, aspartic acid (Asp124 of NDM-1, Asp118 of VIM-1) contributes to the major affinity in the binding with aztreonam, implying that the aspartic acid in the binding site plays a key role in the hydrolysis of the aztreonam. However, the binding energy between Asp118 and aztreonam was weaker than that between Asp124 and aztreonam based on the binding free energy calculation. On the basis of the analysis of the simulation trajectory, it was confirmed that due to the π-π interaction between the side chains of His201, His240, and the thiazol ring of aztreonam, the binding energy of the sulfanolate moiety of aztreonam with Asp118 decreased, leading to the hydrolysis activity loss of VIM1 to aztreonam. Therefore, we believe that Asp124 of NDM-1 and Asp118 of VIM-1 can effectively promote substrate hydrolysis, while His201 and His240
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Fig. 8. Comparison of the dihedrals and distances in the different systems. (A) The dihedral angles of the imidazole ring plane of His250 (black line) and the benzene ring plane of Phe70 (red line) with the thiazol ring plane of aztreonam as a function of time. (B) The dihedral angles of the imidazole ring plane of His240 (black line) and the imidazole ring plane of His201 (red line) with the thiazol ring plane of aztreonam as a function of time. (C) The distance between the imidazole ring plane of His250 (black line), the benzene ring of Phe70 (red line), and the thiazol ring of aztreonam as a function of time. (D) The distance between the imidazole ring groups of His240 (black line), His201 (red line), and the thiazol ring of aztreonam as a function of time.
of VIM-1 on the other side of the active pocket can block the binding of the substrate with Aspartic acid. These results provide foundational data that will help in the development of new and more effective anti-drug resistance inhibitors.
Acknowledgments The authors acknowledge the financial support from the National Natural Science Foundation of China [Grant no. 31572566 to X. D. N].
Fig. 9. Schematic representation of the interaction between residues of the binding sites and aztreonam. The π-π interaction of His201 and His240 with the thiazol ring of aztreonam decrease the binding affinity of Asp118 with the sulfanolate moiety of aztreonam. (Table 1)
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Table 1 Calculated energy components, binding free energy (kcal/mol) of NDM-1 and VIM-1 binding with ATM based on MM-GBSA. Energy components (kcal/mol)
NDM-1
VIM-1
ΔEele ΔEvdw ΔEMM ΔGele,sol ΔGnonpolar,sol ΔGsol ΔGele,sol + ΔEele ΔGnonpolar + ΔEvdw ΔGbind
−88.45 ± 2.44 −6.60 ± 0.65 −95.05 ± 6.08 −3.98 ± 0.08 66.81 ± 4.02 62.83 ± 3.93 −92.43 ± 1.92 60.21 ± 2.37 −32.22 ± 4.58
−74.05 ± 2.48 −20.59 ± 0.57 −94.64 ± 2.40 −4.89 ± 0.03 75.31 ± 2.78 70.42 ± 2.09 −78.94 ± 3.16 54.72 ± 1.82 −24.22 ± 0.90
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