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L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
C-terminal 6ÂHis tagged N-terminal 43 kDa ATP-binding domain. Complexes
References and notes
with inhibitors were crystallized by sitting drop vapor diffusion, from 10 to
20 mg/mL protein solutions (in 20 mM Tris, pH 8.0, 100 mM NaCl) using the
following mother liquor solutions: E. faecalis GyrB, P212121 form; 20 °C, 25% (w/
v) PEG 1500, 3% (v/v) t-butanol, 20% (v/v) glycerol, 20 mM citrate pH 5.6. No
cryoprotection required. E. coli GyrB, P21 form: 4 °C, 30% (w/v) PEG 4000,
200 mM MgCl2, 100 mM Tris pH 8.0. Crystals cryoprotected with mother liquor
supplemented with 25% (v/v) ethylene glycol.E. coli ParE, P21 form: 20 °C, 5.6%
(w/v) PEG 4000, 30% glycerol, 70 mM acetate pH 4.6. no cryoprotection
required. F. tularensis ParE, P21 form: 4 °C, 10% (w/v) PEG 4000, 10% (v/v)
isopropanol, 100 mM citrate pH 5.4. Crystals cryoprotected with mother liquor
supplemented with 25% glycerol.E. faecalis ParE, P31 form: 20 °C, 20% PEG 3350,
200 mM ammonium phosphate. Crystals cryoprotected with mother liquor
supplemented with 25% ethylene glycol. With the exception of the E. coli
GyrB complex with N-(7-(1H-imidazol-1-yl)-2-(pyridin-3-yl)thiazolo[5,4-
d]pyrimidin-5-yl)cyclopropanecarboxamide (2.6 Å), and the E. faecalis and F.
tularensis ParE complexes with 10a (2.7 Å), all other crystals studied diffracted
to 2.2 Å or better resolution on a rotating anode or synchrotron radiation
source. Structures were solved by molecular replacement. Coordinates for all
crystal structures described in the text have been deposited in the Protein Data
with 7-(1H-imidazol-1-yl)-2-(pyridin-3-yl)thiazolo[5,4-d]pyrimidin-5-amine
(ligand not shown in text), 4HZ0. E. coli GyrB with N-(7-(1H-imidazol-1-yl)-
2-(pyridin-3-yl)thiazolo[5,4-d]pyrimidin-5-yl)cyclopropanecarboxamide
(ligand not shown in text), 4HYP. F. tularensis ParE with 10a, 51 and 54, 4HXZ,
4HY1 and 4HYM, respectively. E. faecalis ParE with 10a, 4HZ5. E. faecalis GyrB
with 19, 54, 56, and 62, 4HXW, 4GGL, 4GFN, and 4GEE, respectively.
14. Bellon, S.; Parsons, J. D.; Wei, Y.; Hayakawa, K.; Swenson, L. L.; Charifson, P. S.;
Lippke, J. A.; Aldape, R.; Gross, C. H. Antimicrob. Agents Chemother. 1856, 2004,
48.
1. (a) Walsh, C. Antibiotics: Actions, Origins, Resistance; American Society for
Microbiolgy (ASM) Press: Washington, DC, 2003; (b) Silver, L. L. Nat. Rev. Drug
Disc. 2007, 6, 41.
2. Oblak, M.; Kotnik, M.; Solmajer, T. Curr. Med. Chem. 2007, 14, 2033.
3. Sissi, C.; Palumbo, M. Cell. Mol. Life Sci. 2001, 2010, 67.
4. Collin, F. E.; Karkare, S.; Maxwell, A. Appl. Microbiol. Biotechnol. 2011, 92, 479.
5. Emmerson, A. M.; Jones, A. M. J. Antimicrob. Chemother. 2003, 51, 13.
6. Laponogov, I.; Sohi, M. K.; Veselkov, D. A.; McAuley, K. E.; Fisher, L. M.;
Sanderson, M. R. Nat. Struct. Mol. Biol. 2010, 16, 667.
7. Heide, L. Nat. Prod. Rep. 2009, 26, 1241.
8. (a) Hooper, D. C. Emerg. Infect. Dis. 2001, 7, 337; (b) Wolfson, J. S. Eur. J. Clin.
Microbiol. Infect. Dis. 1989, 8, 1080.
9. (a) Lambert, H. P.; O’Grady, F. W. In Coumarins; Lambert, H. P., O’Grady, F. W.,
Eds.; Churchill Livingstone: Edinburgh, 1992; pp 140–141; (b) Maxwell, A. Mol.
Microbiol. 1993, 9, 101.
10. Tari, L. W.; Bensen, D.; Trzoss, M.; Lam, T. et al. In: 51st Annual Meeting of the
Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago,
IL, September 16–20, 2011, F2–1872.
11. Creighton, C.; Tari, L.; Chen, Z.; Hilgers, M.; Lam, T.; Li, X.; Trzoss, M.; Zhang, J.;
Finn, J.; Bensen, D. Int. Patent Appl. Pub. WO/2011/032050, 2010.
12. GyrB and ParE activity was evaluated using the coupled spectrophotometric
Enzchek™ assay in which the enzyme-dependent release of inorganic
phosphate from ATP hydrolysis was measured. The assay comprises between
20 and 100 nM GyrB or ParE (active site concentrations) in 50
buffer (pH 7.6), 2 M MgCl2, 125 M NaCl, 0.2 M 7-methyl-6-thioguanosine,
1 U/ml purine nucleoside phosphorylase. The reaction is initiated by addition
of 3 M ATP and monitored at 360 nm for 30 min at 27 °C. Inhibitor potency is
determined by incubating the target enzyme in the presence of various
concentrations of inhibitor ranging between 1.5 nM and 50 M for 10 min
lM Tris–HCl
l
l
l
l
15. Ligand strain energy due to angular deformation of the linker atom angle was
calculated by taking the difference between the energy resulting from a partial
optimization of the bound crystal ligand geometry, holding only the linker
l
prior to addition of ATP substrate. The final concentration of DMSO is kept
constant at 2.5% (v/v). Enzyme activity in the presence of inhibitor is expressed
relative to the no-inhibitor control and Ki values determined using Morrison
tight-binding equation to account for ligand depletion. All analysis is carried
out using GraphPad Prism 4.0.
angle constant (at its bound value), and the energy from
a subsequent
optimization allowing the linker angle of the ligand to relax. DFT optimizations
using the B3LYP functional and 6–311+G⁄ basis set were used to calculate the
energies. Frequency calculations verified the energy minima of full
13. Crystallization of E. faecalis GyrB, E. faecalis ParE, E. coli GyrB, and E. coli ParE
was conducted using C-terminal 6xHis tagged versions of the N-terminal
27 kDa ATP-binding domains of each protein, expressed in E. coli BL21 Star
(DE3). F. tularensis ParE was expressed in the same manner, using the
optimizations. Calculations were performed using GAUSSIAN03
Wallingford, CT).
(GAUSSIAN, Inc,