The present application is to fully develop a new strategy (called """"""""tethering"""""""") to discover low molecular weight ligands (about 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 library of disulfide-containing molecules at concentrations typically used in drug screening. The cysteine-captured ligands, which are readily identified by mass spectroscopy, 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 and novel 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 about 1 mM) was improved 3000-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. In Phase I, we developed the overall strategy and applied it to bacterial thymidylate synthase. In Phase II, we will optimize the strategy and apply it to human thymidylate synthase, an important anti-cancer target.
As this technology is developed it will provide a new and general method for drug-lead discovery, applicable to many protein-ligand or protein-protein interactions. The method is more efficient than classic or combinatorial library screening, and will therefore allow us to find drug leads more rapidly and with less expense. The first protein studied, thymidylate synthase, is a validated target for both anti-cancer as well as anti-microbial agents, and will therefore lead to development of new drugs.
| Erlanson, Daniel A; Lam, Joni W; Wiesmann, Christian et al. (2003) In situ assembly of enzyme inhibitors using extended tethering. Nat Biotechnol 21:308-14 |
