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.