Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

Abstract

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those 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 testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.Thymidylate synthase (TS) 1 is an attractive target for the design of drugs used against proliferative diseases because of its central role in the production of DNA. TS catalyzes the methylation of 2′-deoxyuridine 5′-monophosphate (dUMP) by N 5 ,N 10 -methylene tetrahydrofolate (CH 2 H 4 folate). This reaction is the terminal step in the only de novo synthetic pathway to thymidylate, which is essential for DNA production. Inhibition of TS stops the production of DNA, disrupting the progression through the cell cycle and eventually leading to "thymineless" cell death (1).Much effort in drug design against TS has focused on inhibitors that resemble the substrate, dUMP, or the cofactor, CH 2 H 4 folate. A mechanism-based inhibitor of TS (2), 5-fluorouridylate, which is administered as the premetabolite, 5-fluorouracil (5-FU), is used in chemotherapy. The TS inhibitor, 10-propargyl-5,8-dideazafolate (CB3717), is a mimic of the cofactor, CH 2 H 4 folate (3). Although CB3717 is a potent inhibitor of TS [K i of 40 nM (4)], it shows liver and kidney toxicity in a small number of patients (5). Recent structure-based drug design efforts against TS (6-11, 41, 42) have resulted in a series of potent compounds such as AG337 and Tomudex that bind in the folate binding site of the enzyme. These new compounds show promise as cancer chemotherapeutics.The amino acid sequence of TS is highly conserved across species, particularly among those residues that form the substrate and cofactor binding pockets (12). These residues also interact closely with inhi...

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

“…Human TS pharmacogenomics has been focused to 5' and 3' untranslated polymorphisms, tandem repeats, and also in mutations in the TS coding region (Barbour, Berger & Berger, 1990;Tong et al, 1998). Furthermore, TS stands as an important model to understand structure-function relationships and as a paradigm for structure based drug design against various bacterial and proliferative diseases (Stout et al, 1999).…”

Kinetic Template‐Guided Tethering of Fragments

“…In addition, TS has been used a number of times to validate other methods of drug discovery. [148][149][150][151] Significantly, E. coli TS was used to generate an initial proof-of-concept for disulfide tethering by Erlanson et al [51] and was therefore considered to provide an excellent platform for proof-of-concept KTGT studies. Although TS has 5 native cysteine residues, in agreement with previous studies, [152] Erlanson et al demonstrated that only the catalytic cysteine (C146) is solvent exposed.…”

A step further in the discovery of phthalein derivatives as Thymidylate Synthase inhibitors

“…TS and DHFR are two of the most known target in anticancer chemotherapy and only recently TS has been regarded as potential target for anti-infectious diseases. [2][3][4][5][6][7][8][9][10][11][12] In anticancer chemotherapy, TS is strongly inhibited by the nucleotide substrate (dUMP) analog, 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) and the folate substrate (mTHF, 5,10-methylenetetrahydrofolate) analogs, such as However severe resistance problems are encountered after prolonged administration of these drugs, owing to their high structural similarity to the folate cofactor. Many of the resistance mechanisms are related the fact that folate analogs drugs are metabolized through the same pathway that folate does.…”

“…Recently, new series of compounds, unrelated to mTHF, with TS inhibitory activity were discovered. 2,[7][8][9] In particular phenolphthalein (Pth, Figure 2), a well-known pH indicator, was identified in a pioneering work of structure-based drug design, in which an automated docking procedure (DOCK 3.0) was applied to the virtual screening of 55,313 compounds taken from the Fine Chemical Directory (FCD), against the three-dimensional structure of LcTS. 2 The compound shows an inhibition constant (K i ) of 4.4 µM against LcTS.…”

“…15 A superior homologous molecule, phenolnaphthalein, was also considered and many derivatives were designed and synthesized. 7,8 Phenolnaphthaleins, such as α156 (Figure 2), show an interesting specificity profile, owing to their attitude to preferably inhibit non-human TS enzymes (Table 2). 7,12 Concerning Pth, in alkaline medium it exists in different molecular states involved in a complex equilibrium including the open forms responsible of its properties as a pH indicator (see Results and Discussion).…”