Site-directed ligand discovery

We report a strategy (catled "tethering") to discover low molecular weight ligands (approximate to 50 Da) that bind weakly to targeted sites on protei ns through an intermediary disulfide tether. A native or engineered cystein e in a protein is allowed to react reversibly with a small library of disul fide-containing molecules (approximate to 1,200 compounds) at concentration s typically used in drug screening (10 to 200 mu M). The cysteine-captured ligands, which are readily identified by MS, are among the most stable comp lexes, even though in the absence of the covalent tether the ligands may bi nd very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with the rapeutic applications in cancer and infectious diseases. The affinity of th e untethered ligand (K(i)approximate to 1 mM) was improved 3,000-fold by sy nthesis of a small set of analogs with the aid of crystallographic strucutr es of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.