Site-Directed Ligand Discovery

We report a strategy (called "tethering") to discover low molecular weight ligands (≈ 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 disulfi...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-08, Vol.97 (17), p.9367-9372
Main Authors: Erlanson, Daniel A., Braisted, Andrew C., Raphael, Darren R., Randal, Mike, Stroud, Robert M., Gordon, Eric M., Wells, James A.
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Binding Sites
Biochemistry
Chemical bonds
Chemistry
Crystal structure
Crystallography, X-Ray
Cysteine - genetics
Cysteine - metabolism
Disulfides
Disulfides - metabolism
Drug Design
Enzymes
Escherichia coli - enzymology
Esters
Glutamic Acid - metabolism
Lead
Libraries
Ligands
Models, Molecular
Molecular Weight
Molecules
Peptide Library
Pharmacology
Physical Sciences
Proline - analogs & derivatives
Proline - metabolism
Protein Conformation
Protein Engineering
Proteins
Thermodynamics
Thymidylate Synthase - antagonists & inhibitors
Thymidylate Synthase - chemistry
Thymidylate Synthase - genetics
Thymidylate Synthase - metabolism
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Summary:We report a strategy (called "tethering") to discover low molecular weight ligands (≈ 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 (≈ 1,200 compounds) at concentrations typically used in drug screening (10 to 200 μ M). 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 (Ki≈ 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.97.17.9367