Structure-based design of inhibitors specific for bacterial thymidylate synthase

Thymidylate synthase is an attractive target for antiproliferative drug des ign because of its key role in the synthesis of DNA. As such, the enzyme ha s been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In pract ice, TS is highly conserved across species, and it has proven to be difficu lt to develop inhibitors that are selective for microbial TS enzymes over t he human enzyme. Using the structure of TS from Lactobacillus casei in comp lex with the nonsubstrate analogue phenolphthalein, inhibitors were designe d to take advantage of features of the bacterial enzyme that differ from th ose of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and test ing showed that these second-round compounds inhibit the bacterial enzyme a t sub-micromolar concentrations, while the human enzyme was not inhibited a t detectable levels (selectivities of 100-1000-fold or greater). Although t hese inhibitors share chemical similarities, X-ray crystal structures revea l that the analogues bind to the enzyme in substantially different orientat ions. Site-directed mutagenesis experiments suggest that the individual inh ibitors may adopt multiple configurations in their complexes with TS.