Journal of Medicinal Chemistry
Article
X-ray Data Collection. Initialdata from a single crystal were collected
on a ADSC Quantum 210 CCDdetector using synchrotron radiation
(λ = 1 Å) at the IMCA-CATbeamline 17-ID of the Argonne Photon
Source. Data were collected usingφ rotations of 0.5°, and 180° of total
data were collected.
ACKNOWLEDGMENTS
■
We thank Karl Gunderson and Bing Wang for their analytical
support and the Natural Products Unit for their collaboration.
X-ray Data Processing, Structure Determination, and Refinement. Data
were processed using the HKL2000 Suite, version 0.95. Data-
processing statistics are shown in Table 7.Crystals were diffracted to
2.15 Å resolution in the space group C222(1) with a unit cell of a =
97.246Å, b = 100.376 Å, c =156.483 Å, α = β = γ = 90°. Data
collectionstatistics are shown in Table 7. The structureof the EF-Tu/
GDP/48 complex was determined by molecularreplacement as
implemented in PHASER (20), using E. coli EF-Tu protein as a
search model (1D8T). The resulting molecularreplacement solution
contained two EF-Tu/48 protein moleculesin the asymmetric unit.
Refinement was carried out with CNX (21) usinga single round of
rigid-body refinement, following several cyclesof simulated annealing
refinement, B-factor refinement,and model building with the COOT
software package. Water molecules,GDP, and the Mg2+ ion were added
prior to addition of theligand 48. Refinement produced a final model
with excellentgeometry (rmsd bond length of 0.011 Å, rmsd bond
angle of 1.4°)and Rwork and Rfree of 21.1% and 27.3%, respectively.
Crystallization and X-ray of Compound 58. To form the EF-
Tu/58 complex, 10 mg/mL protein (227μM) E. coli EF-Tu protein in
a buffer containing50 mM Tris, pH 8, and 50 mM NaCl were
incubated with 1 mM compound 1 for 1 h at 4 °C. The sample was
centrifuged at 20000g to remove any resulting precipitant.
Crystallization wascarried out using 300 nL of the protein sample
plus 300 nL of crystallizationsolution containing 0.1 M Hepes, pH 7.5,
22% PEG 3350, 0.2 M MgCl2, using a sitting drop format and
equilibrated against a reservoirof the crystallization solution. The
crystals were flash frozen withliquid nitrogen after being stabilized in a
cryobuffer containing0.1 M Hepes, pH 7.5, 22% PEG 3350, 0.2 M
MgCl2, 20% ethyleneglycol.
ABBREVIATIONS USED
■
MIC, minimum inhibitory concentration; EC50, 50% effective
dose; G+, Gram positive; MRSA, methicillin resistant Staph-
ylococcus aureus; VRE, vancomycin resistant enterococci; S.
aureus, Staphylococcus aureus; E. faecalis, Enterococcus faecalis; E.
faecium, Enterococcus faecium; S. pyogenes, Streptococcus pyogenes;
EF-Tu, elongation factor Tu
REFERENCES
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X-ray Data Collection. Initial datafrom a single crystal were collected
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data were collected.
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for the Treatment of Acne. Patent WO03105881, 2003.
(7) Chemical stability was measured by incubation of compound at
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Degradation was measured by LC/MS and quantified by AUC.
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X-ray Data Processing, Structure Determination, and Refinement. Data
were processed using the HKL2000 Suite, version 0.95. Data-
processing statistics are shown in Table 7.Crystals were diffracted to
2.45 Å resolution in the space group P2(1)2(1)2(1) with a unit cell of
a = 80.372Å, b = 82.216 Å, c = 129.9Å, α = β = γ = 90°. Data
collection statisticsare shown in Table 7. The structure of theEF-Tu/
GDP/58 complex was determined by molecular replacementas
implemented in PHASER, using E. coli EF-Tu proteinas a search
model (1D8T). The resulting molecular replacement solutioncon-
tained two EF-Tu/58 protein molecules in the asymmetricunit.
Refinement was carried out with CNX using a single round ofrigid-
body refinement, following several cycles of simulated annealingrefine-
ment, B-factor refinement, and model buildingwith the COOT
software package. Water molecules, GDP, and the Mg2+ ion were
added prior to addition of the ligand. Refinementproduced a final
model with excellent geometry (rmsd bond length of0.011 Å, rmsd
bond angle of 1.2°) and Rwork and Rfree of 22.4% and29.8%,
respectively.
ASSOCIATED CONTENT
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S
* Supporting Information
Detailed synthetic procedures and compound characterization
data. This material is available free of charge via the Internet at
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: (617) 871-7729. Fax: 617-871-4081. E-mail:
8108
dx.doi.org/10.1021/jm200938f|J. Med. Chem. 2011, 54, 8099−8109