Rational design, synthesis, evaluation, and crystal structure of a potent inhibitor of human GAR Tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid

Biochemistry. 2003 May 27;42(20):6043-56. doi: 10.1021/bi034219c.


Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

Publication types

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

MeSH terms

  • Binding Sites
  • Cell Line
  • Crystallography, X-Ray
  • Drug Design
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / enzymology
  • Humans
  • Hydroxymethyl and Formyl Transferases / antagonists & inhibitors*
  • Hydroxymethyl and Formyl Transferases / chemistry
  • In Vitro Techniques
  • Kinetics
  • Macromolecular Substances
  • Models, Molecular
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphoribosylglycinamide Formyltransferase
  • Protein Conformation
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Static Electricity
  • Tetrahydrofolates / chemical synthesis
  • Tetrahydrofolates / chemistry*
  • Tetrahydrofolates / pharmacology*


  • 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid
  • Enzyme Inhibitors
  • Macromolecular Substances
  • Recombinant Proteins
  • Tetrahydrofolates
  • lometrexol
  • Hydroxymethyl and Formyl Transferases
  • Phosphoribosylglycinamide Formyltransferase