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PDBsum entry 1f4e
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Site-Directed ligand discovery.
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Authors
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D.A.Erlanson,
A.C.Braisted,
D.R.Raphael,
M.Randal,
R.M.Stroud,
E.M.Gordon,
J.A.Wells.
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Ref.
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Proc Natl Acad Sci U S A, 2000,
97,
9367-9372.
[DOI no: ]
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PubMed id
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Abstract
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We report a strategy (called "tethering") to discover low molecular
weight ligands ( approximately 250 Da) that bind weakly to targeted sites on
proteins through an intermediary disulfide tether. A native or engineered
cysteine in a protein is allowed to react reversibly with a small library of
disulfide-containing molecules ( approximately 1,200 compounds) at
concentrations typically used in drug screening (10 to 200 microM). The
cysteine-captured ligands, which are readily identified by MS, are among the
most stable complexes, even though in the absence of the covalent tether the
ligands may bind very weakly. This method was applied to generate a potent
inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism
with therapeutic applications in cancer and infectious diseases. The affinity of
the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by
synthesis of a small set of analogs with the aid of crystallographic structures
of the tethered complex. Such site-directed ligand discovery allows one to
nucleate drug design from a spatially targeted lead fragment.
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Figure 5.
Fig. 5. Grafting a glutamate residue onto
N-tosyl-D-proline improves the affinity 50-fold, and adding a
negatively charged appendage further increases the affinity by
an additional 70-fold.
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Figure 6.
Fig. 6. Overlay of three crystallographically determined
structures; only the inhibitor is shown for clarity. The
inhibition constant (K[i]) of each inhibitor is also shown.
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