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PDBsum entry 3dat
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Oxidoreductase
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PDB id
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3dat
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References listed in PDB file
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Key reference
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Title
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X-Ray structure of the ternary mtx.Nadph complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-Site inhibitor design.
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Authors
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B.C.Bennett,
Q.Wan,
M.F.Ahmad,
P.Langan,
C.G.Dealwis.
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Ref.
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J Struct Biol, 2009,
166,
162-171.
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PubMed id
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Abstract
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For reasons of bioterrorism and drug resistance, it is imperative to identify
and develop new molecular points of intervention against anthrax. Dihydrofolate
reductase (DHFR) is a highly conserved enzyme and an established target in a
number of species for a variety of chemotherapeutic programs. Recently, the
crystal structure of Bacillus anthracis DHFR (baDHFR) in complex with
methotrexate (MTX) was determined and, based on the structure, proposals were
made for drug design strategies directed against the substrate-binding site.
However, little is gleaned about the binding site for NADPH, the cofactor
responsible for hydride transfer in the catalytic mechanism. In the present
study, X-ray crystallography at 100 K was used to determine the structure of
baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly
identical to that seen in other DHFR ternary complex structures, the adenine
moiety adopts an off-plane tilt of nearly 90 degrees and this orientation is
stabilized by hydrogen bonds to functionally conserved Arg residues. A
comparison of the binding site, focusing on this region, between baDHFR and the
human enzyme is discussed, with an aim at designing species-selective
therapeutics. Indeed, the ternary model, refined to 2.3 A resolution, provides
an accurate template for testing the feasibility of identifying dual-site
inhibitors, compounds that target both the substrate and cofactor-binding site.
With the ternary model in hand, using in silico methods, several compounds were
identified which could potentially form key bonding contacts in the substrate
and cofactor-binding sites. Ultimately, two structurally distinct compounds were
verified that inhibit baDHFR at low microM concentrations. The apparent Kd for
one of these,
(2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone
oxime), was measured by fluorescence spectroscopy to be 5.3 microM.
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