Abstract
Protozoal parasites are unusual in that their thymidylate synthase (TS) and dihydrofolate reductase (DHFR) enzymes exist on a single polypeptide. In an effort to probe the possibility of substrate channeling between the TS and DHFR active sites and to identify inhibitors specific for bifunctional TS-DHFR, we used molecular docking to screen for inhibitors targeting the shallow groove connecting the two active sites. Eosin B is a 100 microm non-active site inhibitor of Leishmania major TS-DHFR identified by molecular docking. Eosin B slows both the TS and DHFR reaction rates. When Arg-283, a key residue to which eosin B is predicted to bind, is mutated to glutamate, however, eosin B only minimally inhibits the TS-DHFR reaction. Additionally, eosin B was found to be a 180 microm inhibitor of Toxoplasma gondii in both biochemical and cell culture assays.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antiprotozoal Agents / pharmacology*
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Arginine / chemistry
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Binding Sites
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CHO Cells
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Chromatography, High Pressure Liquid
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Cricetinae
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Dose-Response Relationship, Drug
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Enzyme Inhibitors / pharmacology
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Eosine I Bluish
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Fluoresceins / chemistry*
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Fluoresceins / metabolism
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Fluoresceins / pharmacology
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Glutamic Acid / chemistry
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Hydrogen-Ion Concentration
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Inhibitory Concentration 50
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Kinetics
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Leishmania major / metabolism
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Models, Molecular
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Multienzyme Complexes / chemistry*
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Multienzyme Complexes / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Tetrahydrofolate Dehydrogenase / chemistry*
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Tetrahydrofolate Dehydrogenase / metabolism
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Thymidylate Synthase / chemistry*
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Thymidylate Synthase / metabolism
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Time Factors
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Toxoplasma / metabolism
Substances
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Antiprotozoal Agents
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Enzyme Inhibitors
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Fluoresceins
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Multienzyme Complexes
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thymidylate synthase-dihydrofolate reductase
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Glutamic Acid
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Arginine
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Tetrahydrofolate Dehydrogenase
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Thymidylate Synthase
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Eosine I Bluish