Site-Directed Ligand Discovery

• 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
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.17.9367

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.