Site-directed ligand discovery

Abstract

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.

Figure 1

(A) Schematic illustration of the tethering approach: a cysteine-containing protein is equilibrated with a disulfide-containing library in the presence of a reducing agent such as 2-mercaptoethanol. Most of the library members will have little or no inherent affinity for the protein, and thus by mass action the equilibrium will lie toward the unmodified protein. However, if a library member does show inherent affinity for the protein, the equilibrium will shift toward the modified protein. (B) Schematic illustration of a generic disulfide library derived from carboxylic acids. Other functional groups have also been converted to disulfide libraries, as described in Materials and Methods. In the present case, 1,200 compounds were screened against TS in pools of 8 to 15 compounds.

Figure 2

(A) Two representative tethering experiments. The protein TS is present at a concentration of 15 μM, and each of the 10 disulfide library members in each pool is present at 200 μM. The buffer contains 25 mM potassium phosphate (pH 7.5) and 1 mM 2-mercaptoethanol, and the samples were allowed to equilibrate at ambient temperature for 1 h before analysis. (B) Three tethering experiments in which the concentrations of 2-mercaptoethanol were varied as stated. The pool of disulfides is the same as in A Right, and the conditions (other than 2-mercaptoethanol concentration) were the same as above. (C) Three tethering experiments in which the pool size and concentrations were varied as stated. All other conditions were the same as above.

Figure 3

Compounds either selected by covalent tethering (Left) or present in the disulfide library but not selected (Right). Except in the case of N-tosyl-d-proline, all compounds tested were racemic unless otherwise indicated. For N-tosyl-d-proline, both stereoisomers were screened separately in different pools, and both were identified as hits, although the d-isomer appeared to be selected slightly more strongly than the l-isomer.

Figure 4

Overlay of three crystallographically determined structures. The structure in green was determined after soaking N-tosyl-d-proline (free acid) into crystals of unmodified TS. The structure in red is TS covalently modified by N-tosyl-d-proline disulfide-bonded to C146 (the active-site cysteine). Finally, the structure in blue is mutant TS (C146S/L143C) covalently modified by N-tosyl-d-proline disulfide bonded to L143C.

Figure 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.

Figure 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.