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Fig. (2). Schematic representation of interactions between ATP and GyrB24 [19,23]. Coordinate and H-bonds are presented by dashed lines. Hydrophobic interactions are shown as solid curves.

Figure 2 (2). Schematic representation of interactions between ATP and GyrB24 [19,23]. Coordinate and H-bonds are presented by dashed lines. Hydrophobic interactions are shown as solid curves.

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Lys337 from domain II, which forms an additional H-bond with the Y-phosphate group of ATP, also forms part of the ATP- binding site. Another important structural element is the crystal water molecule, which is part of the H-bond network formed by the adenine ring of ATP and residues Asp73, Gly77 and Thr165. This particular water molecule is also conserved in other crystal structures of the ATPase domain in its complex with other inhibitors [20,22,24,25,30]. The second monomer also contributes to the binding of the nucleotide, where the N- Fig. (1). (A) Crystal structure of the £. coli 43 kDa N-terminal fragment of GyrB (GyrB43) dimer in complex with ADPNP (black spheres) [19,23]. Domain I (GyrB24) and II are colored dark and light gray in the first monomer, whereas the second monomer is colored light gray. Also indicated are the beginning and the end of the N-terminal tail of domain I (Ser2—Gly15) and the glycine linker between the two domains (Gly220-Gly221). (B) Superposition of the crystal structures of ADPNP (black balls and sticks) and ATPase inhibitor novobiocin (dark gray balls and sticks) bound in the 24 kDa N-terminal fragment of GyrB (GyrB24) from E. coli (the secondary structural elements are colored light gray) [21,23]. The first crystal structure of any bacterial topo II enzyme to be solved was the 43kDa N-terminal fragment of GyrB from Escherichia coli (called GyrB43) in complex with ADPNP (the non-hydrolyzable analog of ATP) [19]. In the crystal structure, GyrB43 forms a dimer in the presence of ATP or ADPNP (Fig. 1A) [19,23], which was later corroborated by other biochemical evidence [29]. The nucleotide binding site is located in domain I (GyrB24, residues 2-220) comprising an eight-stranded B- sheet and five Q-helices. The tertiary structure of domain II (residues 221-392) consists of a four-stranded B-sheet and four a-helices. Interestingly, a large cavity with a diameter of 20 A is
Lys337 from domain II, which forms an additional H-bond with the Y-phosphate group of ATP, also forms part of the ATP- binding site. Another important structural element is the crystal water molecule, which is part of the H-bond network formed by the adenine ring of ATP and residues Asp73, Gly77 and Thr165. This particular water molecule is also conserved in other crystal structures of the ATPase domain in its complex with other inhibitors [20,22,24,25,30]. The second monomer also contributes to the binding of the nucleotide, where the N- Fig. (1). (A) Crystal structure of the £. coli 43 kDa N-terminal fragment of GyrB (GyrB43) dimer in complex with ADPNP (black spheres) [19,23]. Domain I (GyrB24) and II are colored dark and light gray in the first monomer, whereas the second monomer is colored light gray. Also indicated are the beginning and the end of the N-terminal tail of domain I (Ser2—Gly15) and the glycine linker between the two domains (Gly220-Gly221). (B) Superposition of the crystal structures of ADPNP (black balls and sticks) and ATPase inhibitor novobiocin (dark gray balls and sticks) bound in the 24 kDa N-terminal fragment of GyrB (GyrB24) from E. coli (the secondary structural elements are colored light gray) [21,23]. The first crystal structure of any bacterial topo II enzyme to be solved was the 43kDa N-terminal fragment of GyrB from Escherichia coli (called GyrB43) in complex with ADPNP (the non-hydrolyzable analog of ATP) [19]. In the crystal structure, GyrB43 forms a dimer in the presence of ATP or ADPNP (Fig. 1A) [19,23], which was later corroborated by other biochemical evidence [29]. The nucleotide binding site is located in domain I (GyrB24, residues 2-220) comprising an eight-stranded B- sheet and five Q-helices. The tertiary structure of domain II (residues 221-392) consists of a four-stranded B-sheet and four a-helices. Interestingly, a large cavity with a diameter of 20 A is
Table 1. A List of Solved Crystal Structures of DNA Gyrase (GyrB) and Topoisomerase IV (ParE) Fragments in Complex with ATPase Inhibitors, which are Available in the PDB Databank [28]
Table 1. A List of Solved Crystal Structures of DNA Gyrase (GyrB) and Topoisomerase IV (ParE) Fragments in Complex with ATPase Inhibitors, which are Available in the PDB Databank [28]
Fig. (3). Natural coumarin antibiotics isolated from Streptomyces microorganisms. Extensive site-directed mutagenesis studies were undertaken to identify residues essential for ATP binding and hydrolysis [33-36]. Mutation of residues His38, Glu42, Asn46, Glu50, Asp73, Arg76, Gly77, Ile78 and Thr165 to alanine and residue Lys103 to isoleucine resulted in significantly reduced or even zero ATPase activity of the mutant proteins [33-36]. In addition, mutation of Lys337 to glutamine had no major effect on the binding of the nucleotide, but, on the other hand, terminal tail of domain I’ (residues 2-15) stabilizes the conformation of a loop (residues 100-120) that forms a lid over the ATP-binding site. The side chain of Tyr5’ forms two H- bonds with the 2’-hydroxyl group of ribose and a nitrogen atom of the adenine ring at position 3, while the side chain of Ile10’ stabilizes the dimer through hydrophobic interactions [23,31]. Tyr5’ is conserved by all the bacterial gyrases and it is not found in any eukaryotic and/or viral topo II [32].
Fig. (3). Natural coumarin antibiotics isolated from Streptomyces microorganisms. Extensive site-directed mutagenesis studies were undertaken to identify residues essential for ATP binding and hydrolysis [33-36]. Mutation of residues His38, Glu42, Asn46, Glu50, Asp73, Arg76, Gly77, Ile78 and Thr165 to alanine and residue Lys103 to isoleucine resulted in significantly reduced or even zero ATPase activity of the mutant proteins [33-36]. In addition, mutation of Lys337 to glutamine had no major effect on the binding of the nucleotide, but, on the other hand, terminal tail of domain I’ (residues 2-15) stabilizes the conformation of a loop (residues 100-120) that forms a lid over the ATP-binding site. The side chain of Tyr5’ forms two H- bonds with the 2’-hydroxyl group of ribose and a nitrogen atom of the adenine ring at position 3, while the side chain of Ile10’ stabilizes the dimer through hydrophobic interactions [23,31]. Tyr5’ is conserved by all the bacterial gyrases and it is not found in any eukaryotic and/or viral topo II [32].
“AG= AH-TAS. “Average value from Refs. [21,22,37]. Data from Ref. [25]. Table 2. Thermodynamic Parameters for Binding of Coumarins to GyrB24"
“AG= AH-TAS. “Average value from Refs. [21,22,37]. Data from Ref. [25]. Table 2. Thermodynamic Parameters for Binding of Coumarins to GyrB24"
Fig. (4). Schematic diagram of interactions between novobiocin and GyrB24 [19,23]. H-bonds are presented by dashed lines. Hydrophobic interactions are shown as solid curves.
Fig. (4). Schematic diagram of interactions between novobiocin and GyrB24 [19,23]. H-bonds are presented by dashed lines. Hydrophobic interactions are shown as solid curves.
Fig. (5). Synthetic coumarin inhibitors of GyrB.
Fig. (5). Synthetic coumarin inhibitors of GyrB.
Fig. (6). Cyclothialidine (top) and seco-cyclothialidine (bottom) inhibitors of DNA gyrase. Screening of natural products for inhibition of DNA gyrase supercoiling activity yielded a cyclothialidine (Ro 09-1437), which was isolated from the fermentation broth of Streptomyces filipinesis NR0484 [58,59]. Cyclothialidine displays a two fold higher antigyrase activity compared to novobiocin and is highly selective against homologous eukaryotic topoisomerases [59], but due to poor penetration has only weak antibacterial activity [58].
Fig. (6). Cyclothialidine (top) and seco-cyclothialidine (bottom) inhibitors of DNA gyrase. Screening of natural products for inhibition of DNA gyrase supercoiling activity yielded a cyclothialidine (Ro 09-1437), which was isolated from the fermentation broth of Streptomyces filipinesis NR0484 [58,59]. Cyclothialidine displays a two fold higher antigyrase activity compared to novobiocin and is highly selective against homologous eukaryotic topoisomerases [59], but due to poor penetration has only weak antibacterial activity [58].
‘Data from ref. [58]. Data from Ref. [61]. “Data from Ref. [63]. {Data from Ref. [57]. “MNEC values (maximum non-efficient concentration), which are around 2—S- fold lower than ICs in this assay [61], were measured. fno data. ible 3. Enzymatic and Antibacterial Activity of Cyclothialidines and seco-Cyclothialidines
‘Data from ref. [58]. Data from Ref. [61]. “Data from Ref. [63]. {Data from Ref. [57]. “MNEC values (maximum non-efficient concentration), which are around 2—S- fold lower than ICs in this assay [61], were measured. fno data. ible 3. Enzymatic and Antibacterial Activity of Cyclothialidines and seco-Cyclothialidines
Fig. (7). Triazine inhibitors of DNA gyrase B. Seco-cyclothialidines are highly active and selective DNA gyrase inhibitors (Fig. 6 bottom, Table 3), having “an opened” lactone ring in the chemical structure [63,64]. Suitable substitution at position 4 of seco-cyclothialidine Ro 61-6653 from Hoffman La Roche, which was later followed by a similar compound from Bayer (BAY 50-7952), immensely improved permeation through the bacterial cell wall. Seco- cyclothialidines display very good antibacterial activity
Fig. (7). Triazine inhibitors of DNA gyrase B. Seco-cyclothialidines are highly active and selective DNA gyrase inhibitors (Fig. 6 bottom, Table 3), having “an opened” lactone ring in the chemical structure [63,64]. Suitable substitution at position 4 of seco-cyclothialidine Ro 61-6653 from Hoffman La Roche, which was later followed by a similar compound from Bayer (BAY 50-7952), immensely improved permeation through the bacterial cell wall. Seco- cyclothialidines display very good antibacterial activity
A patent application by AstraZeneca described 1,3,5-triazine derivatives (Fig. 7) as antibacterial agents that inhibit the DNA gyrase activity in Gram-positive bacteria. [65]. Compound 14
A patent application by AstraZeneca described 1,3,5-triazine derivatives (Fig. 7) as antibacterial agents that inhibit the DNA gyrase activity in Gram-positive bacteria. [65]. Compound 14
shows very good activity in DNA gyrase supercoiling assay (ICs59= 0.55 pg/ml), but no data on its antibacterial activity are given. In addition, compound 15 shows very good antibacterial activity (MIC= 2 g/ml) against MRSA and MSSA, as well as novobiocin and quinolone resistant S. aureus. Fig. (8). Schematic representation of interactions between triazine derivative 17 and the ATP-binding site of GyrB24. Hydrophobic interactions are shown as solid curves and H-bonds as dotted lines.
shows very good activity in DNA gyrase supercoiling assay (ICs59= 0.55 pg/ml), but no data on its antibacterial activity are given. In addition, compound 15 shows very good antibacterial activity (MIC= 2 g/ml) against MRSA and MSSA, as well as novobiocin and quinolone resistant S. aureus. Fig. (8). Schematic representation of interactions between triazine derivative 17 and the ATP-binding site of GyrB24. Hydrophobic interactions are shown as solid curves and H-bonds as dotted lines.
Fig. (9). DNA gyrase inhibitors- derivatives of 4-amino-pyrazolo[3,4-d]pyrimidine (A) and 4-amino-pyrazolo[1,5-a] [1,3,5]triazine (B). Compound 17 retains the majority of interactions with the protein (Fig. 8), which were previously described for ADPNP, coumarins and cyclothalidines. The carboxylate of Asp73 forms Replacement of the hydroxyl group of the coumarin ring with an amide (compound 19) or urea (compound 20) weakens
Fig. (9). DNA gyrase inhibitors- derivatives of 4-amino-pyrazolo[3,4-d]pyrimidine (A) and 4-amino-pyrazolo[1,5-a] [1,3,5]triazine (B). Compound 17 retains the majority of interactions with the protein (Fig. 8), which were previously described for ADPNP, coumarins and cyclothalidines. The carboxylate of Asp73 forms Replacement of the hydroxyl group of the coumarin ring with an amide (compound 19) or urea (compound 20) weakens
Table 4. Inhibitory DNA gyrase Activity of Indazole Derivatives
Table 4. Inhibitory DNA gyrase Activity of Indazole Derivatives
Fig. (10). Different structural classes of ATPase inhibitors of GyrB obtained by fragment-based screening.
Fig. (10). Different structural classes of ATPase inhibitors of GyrB obtained by fragment-based screening.
Fig. (12). Azole inhibitors of GyrB. A patent application by Vertex described substituted bicyclic pyrrazole derivatives with the structural formula 29, where ring A is thiazole, oxazole, imidazole or pyrazole (Fig. 12) [69,70]. The compounds were tested for inhibition of ATPase activity of DNA gyrase and the inhibitory activities for 37 compounds were listed in three classes: class A (K;< 500 Only recently, pyrrole derivatives with antibacterial activity were revealed in a patent application by AstraZeneca [75]. Compound 35, which is one of more than 300 disclosed examples with IC59< 20 g/ml in the GyrB ATPase activity assay, possesses good antibacterial activity against S. pneumoniae (MIC= 2 pg/ml). No data about the mechanism is given, but it can be speculated that the methylpyrrole ring binds to the same hydrophobic pocket as in the case of clorobiocin.
Fig. (12). Azole inhibitors of GyrB. A patent application by Vertex described substituted bicyclic pyrrazole derivatives with the structural formula 29, where ring A is thiazole, oxazole, imidazole or pyrazole (Fig. 12) [69,70]. The compounds were tested for inhibition of ATPase activity of DNA gyrase and the inhibitory activities for 37 compounds were listed in three classes: class A (K;< 500 Only recently, pyrrole derivatives with antibacterial activity were revealed in a patent application by AstraZeneca [75]. Compound 35, which is one of more than 300 disclosed examples with IC59< 20 g/ml in the GyrB ATPase activity assay, possesses good antibacterial activity against S. pneumoniae (MIC= 2 pg/ml). No data about the mechanism is given, but it can be speculated that the methylpyrrole ring binds to the same hydrophobic pocket as in the case of clorobiocin.
Fig. (11). Indazole inhibitors of GyrB.
Fig. (11). Indazole inhibitors of GyrB.
Table 5. Inhibitory Activities of Aminobenzimidazoles Against Bacterial Gyrase and Topo IV activity still needs to be examined. Compound 40, another phenol derivative with moderate antibacterial activity, is one of 51 screening hits from the cell blue assay that inhibit DNA gyrase and/or topo IV in the micromolar range (ICs59= 50 pg/ml). Both compounds 36 and 37 show equal or better activity against gyrase compared to novobiocin, and on the other hand, much better activity against topo IV. They have excellent broad spectrum antibacterial activity against a variety of pathogens responsible for nocosomial and community infections. The mode of action in bacteria was characterized using an S. aureus strain containing a GyrB T1731 mutant, which has been associated with novobiocin resistance.
Table 5. Inhibitory Activities of Aminobenzimidazoles Against Bacterial Gyrase and Topo IV activity still needs to be examined. Compound 40, another phenol derivative with moderate antibacterial activity, is one of 51 screening hits from the cell blue assay that inhibit DNA gyrase and/or topo IV in the micromolar range (ICs59= 50 pg/ml). Both compounds 36 and 37 show equal or better activity against gyrase compared to novobiocin, and on the other hand, much better activity against topo IV. They have excellent broad spectrum antibacterial activity against a variety of pathogens responsible for nocosomial and community infections. The mode of action in bacteria was characterized using an S. aureus strain containing a GyrB T1731 mutant, which has been associated with novobiocin resistance.
Fig. (14). Phenolic inhibitors of DNA gyrase. Recent experimental work on ester derivatives of quercetin has been shown to exhibit useful increase in systemic aglycone concentrations, thus improving the bioavailability [86]. The phenol derivative 38, which was identified by fragment- based de novo design described in one of the previous sections,
Fig. (14). Phenolic inhibitors of DNA gyrase. Recent experimental work on ester derivatives of quercetin has been shown to exhibit useful increase in systemic aglycone concentrations, thus improving the bioavailability [86]. The phenol derivative 38, which was identified by fragment- based de novo design described in one of the previous sections,
Fig. (15). (Aza-) flavone and indolinone inhibitors of DNA gyrase.
Fig. (15). (Aza-) flavone and indolinone inhibitors of DNA gyrase.
Fig. (16). Schematic diagram of H-bond interaction pattern between the adenine ring of ADPNP, a conserved water molecule and residues Asp73, Gly77 and Thr165, as observed in the crystal structure. Also presented are central fragments of other identified GyrB/ParE inhibitors with the H-bonding motif indicated. A combination of virtual screening and NMR spectroscopy was used for fragment-based discovery of the indolinone derivative 44, which is a micromolar inhibitor of DNA gyrase supercoiling activity (MNEC= 25 uM) and binds to the isolated GyrB24 fragment with a Kp value of 10 uM [77,88]. The binding of compound 44 to the ATPase domain was corroborated by mass spectrometry and NMR spectroscopy [88]. The binding affinity of indolinone derivative 44 is 500-fold lower compared to novobiocin. However, according to ligand efficiency [89], which can be calculated as the pKp/MW ratio [90], compound Bacterial DNA gyrase and topoisomerase IV are well established targets for antibacterial agents as evidenced by the fluoroquinolones, which are inhibitors of the GyrA/ParC subunits commonly used for clinical treatment of bacterial infections. Although there is currently no GyrB/ParE inhibitor in clinical use, according to reports indazole derivatives QX5- 3073 and QX5-3075 from Quorex (now Pfizer), and aminobenzimidazoles VX-692 and VX-883 from Vertex have already reached different phases of clinical trials. In addition, Prolysis plans clinical studies in 2008 for the derivatives of compound GYR-767 (undisclosed structure), which is an ATPase inhibitor of GyrB [91]. Furthermore, the need to discover new inhibitors of GyrB/ParE has motivated scientists from academia and industry to join forces in an EC-funded consortium [92].
Fig. (16). Schematic diagram of H-bond interaction pattern between the adenine ring of ADPNP, a conserved water molecule and residues Asp73, Gly77 and Thr165, as observed in the crystal structure. Also presented are central fragments of other identified GyrB/ParE inhibitors with the H-bonding motif indicated. A combination of virtual screening and NMR spectroscopy was used for fragment-based discovery of the indolinone derivative 44, which is a micromolar inhibitor of DNA gyrase supercoiling activity (MNEC= 25 uM) and binds to the isolated GyrB24 fragment with a Kp value of 10 uM [77,88]. The binding of compound 44 to the ATPase domain was corroborated by mass spectrometry and NMR spectroscopy [88]. The binding affinity of indolinone derivative 44 is 500-fold lower compared to novobiocin. However, according to ligand efficiency [89], which can be calculated as the pKp/MW ratio [90], compound Bacterial DNA gyrase and topoisomerase IV are well established targets for antibacterial agents as evidenced by the fluoroquinolones, which are inhibitors of the GyrA/ParC subunits commonly used for clinical treatment of bacterial infections. Although there is currently no GyrB/ParE inhibitor in clinical use, according to reports indazole derivatives QX5- 3073 and QX5-3075 from Quorex (now Pfizer), and aminobenzimidazoles VX-692 and VX-883 from Vertex have already reached different phases of clinical trials. In addition, Prolysis plans clinical studies in 2008 for the derivatives of compound GYR-767 (undisclosed structure), which is an ATPase inhibitor of GyrB [91]. Furthermore, the need to discover new inhibitors of GyrB/ParE has motivated scientists from academia and industry to join forces in an EC-funded consortium [92].
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