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

Yan Zhang, Joel Desharnais, Thomas H. Marsilje, Chenglong Li, Michael P. Hedrick, Lata T. Gooljarsingh, Ali Tavassoli, Stephen Benkovic, Arthur J. Olson, Dale L. Boger, Ian A. Wilson

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Abstract

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-CF3CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (Ki = 15 nM), but is inactive (Ki > 100 μM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC50 = 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 Å 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.

Original languageEnglish (US)
Pages (from-to)6043-6056
Number of pages14
JournalBiochemistry
Volume42
Issue number20
DOIs
StatePublished - May 27 2003

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Zhang, Y., Desharnais, J., Marsilje, T. H., Li, C., Hedrick, M. P., Gooljarsingh, L. T., Tavassoli, A., Benkovic, S., Olson, A. J., Boger, D. L., & Wilson, I. A. (2003). 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, 42(20), 6043-6056. https://doi.org/10.1021/bi034219c