Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase

Background: The substrate sites of enzymes are attractive targets for struc ture-based inhibitor design. Two difficulties hinder efforts to discover an d elaborate new (nonsubstrate-like) inhibitors for these sites. First, nove l inhibitors often bind at nonsubstrate sites. Second, a novel scaffold int roduces chemistry that is frequently unfamiliar, making synthetic elaborati on challenging, Results: In an effort to discover and elaborate a novel scaffold for a subs trate site, we combined structure-based screening with in-parallel syntheti c elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied, The available chemicals directory was screened, using a molecular -docking computer program, for molecules that complemented the three-dimens ional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (K-i 65 mu M). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dis similar to the substrate but bind competitively with it. The most active an alog had a K-i of 1.3 mu M. The tighter binding inhibitors were also the mo st specific for LcTS versus related enzymes, Conclusions: TS can recognize inhibitors that are dissimilar to, but that b ind competitively with, the folate substrate. Combining structure-based dis covery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can b e envisaged.