A molecular docking strategy identifies Eosin B as a non-active site inhibitor of protozoal bifunctional thymidylate synthase-dihydrofolate reductase

J Biol Chem. 2003 Apr 18;278(16):14092-100. doi: 10.1074/jbc.M212690200. Epub 2003 Jan 29.


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

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antiprotozoal Agents / pharmacology*
  • Arginine / chemistry
  • Binding Sites
  • CHO Cells
  • Chromatography, High Pressure Liquid
  • Cricetinae
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Eosine I Bluish
  • Fluoresceins / chemistry*
  • Fluoresceins / metabolism
  • Fluoresceins / pharmacology
  • Glutamic Acid / chemistry
  • Hydrogen-Ion Concentration
  • Inhibitory Concentration 50
  • Kinetics
  • Leishmania major / metabolism
  • Models, Molecular
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Tetrahydrofolate Dehydrogenase / chemistry*
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Thymidylate Synthase / chemistry*
  • Thymidylate Synthase / metabolism
  • Time Factors
  • Toxoplasma / metabolism


  • Antiprotozoal Agents
  • Enzyme Inhibitors
  • Fluoresceins
  • Multienzyme Complexes
  • thymidylate synthase-dihydrofolate reductase
  • Glutamic Acid
  • Arginine
  • Tetrahydrofolate Dehydrogenase
  • Thymidylate Synthase
  • Eosine I Bluish