title: 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.
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-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.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Joel
authors.initials: J
authors.lastname: Desharnais
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Marsilje
authors.forename: Chenglong
authors.initials: C
authors.lastname: Li
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hedrick
authors.forename: Lata T
authors.initials: LT
authors.lastname: Gooljarsingh
authors.forename: Ali
authors.initials: A
authors.lastname: Tavassoli
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Benkovic
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Olson
authors.forename: Dale L
authors.initials: DL
authors.lastname: Boger
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D051026:Phosphoribosylglycinamide Formyltransferase; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D055672:Static Electricity; D013763:Tetrahydrofolates / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: C475104:10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; C045894:lometrexol; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase