Abstract
Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme in the de novo purine biosynthesis pathway, which has long been considered a potential target for development of anti-neoplastic therapeutics. Here we report the biological and X-ray crystallographic evaluations of both independent C10 diastereomers, 10S- and 10R-methylthio-DDACTHF, bound to human GAR Tfase, including the highest-resolution apo GAR Tfase structure to date (1.52 Å). Both diastereomers are potent inhibitors (Ki = 210 nM for 10R, and Ki = 180 nM for 10S) of GAR Tfase and exhibit effective inhibition of human leukemia cell growth (IC₅₀ = 80 and 50 nM, respectively). Their inhibitory activity was surprisingly high, and these lipophilic C10-substituted analogues show distinct advantages over their hydrophilic counterparts, most strikingly in retaining potency in mutant human leukemia cell lines that lack reduced folate carrier protein activity (IC₅₀ = 70 and 60 nM, respectively). Structural characterization reveals a new binding mode for these diastereoisomers, in which the lipophilic thiomethyl groups penetrate deeper into a hydrophobic pocket within the folate-binding site. In silico docking simulations of three other sulfur-containing folate analogues also indicate that this hydrophobic cleft represents a favorable region for binding lipophilic substituents. Overall, these results suggest sulfur and its substitutions play an important role in not only the binding of anti-folates to GAR Tfase but also the selectivity and cellular activity (growth inhibition), thereby presenting new possibilities for the future design of potent and selective anti-folate drugs that target GAR Tfase.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Antimetabolites, Antineoplastic / chemistry*
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Antimetabolites, Antineoplastic / metabolism
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Antimetabolites, Antineoplastic / pharmacology
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Apoproteins / antagonists & inhibitors
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Apoproteins / chemistry
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Apoproteins / metabolism
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Binding Sites
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Carbon-Nitrogen Ligases / antagonists & inhibitors
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Carbon-Nitrogen Ligases / chemistry*
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Carbon-Nitrogen Ligases / genetics
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Carbon-Nitrogen Ligases / metabolism
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Enzyme Inhibitors / chemistry*
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Enzyme Inhibitors / metabolism
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Enzyme Inhibitors / pharmacology
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Humans
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Inhibitory Concentration 50
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Leukemia / drug therapy
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Leukemia / enzymology
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Models, Molecular*
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Molecular Conformation
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Molecular Docking Simulation
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Neoplasm Proteins / antagonists & inhibitors
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Neoplasm Proteins / chemistry
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Neoplasm Proteins / genetics
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Neoplasm Proteins / metabolism
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Peptide Fragments / antagonists & inhibitors
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Peptide Fragments / chemistry
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Peptide Fragments / metabolism
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Phosphoribosylaminoimidazolecarboxamide Formyltransferase / antagonists & inhibitors
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Phosphoribosylaminoimidazolecarboxamide Formyltransferase / chemistry
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Phosphoribosylaminoimidazolecarboxamide Formyltransferase / genetics
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Phosphoribosylaminoimidazolecarboxamide Formyltransferase / metabolism
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Phosphoribosylglycinamide Formyltransferase / antagonists & inhibitors
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Phosphoribosylglycinamide Formyltransferase / chemistry*
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Phosphoribosylglycinamide Formyltransferase / genetics
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Phosphoribosylglycinamide Formyltransferase / metabolism
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Stereoisomerism
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Structure-Activity Relationship
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Tetrahydrofolates / chemistry*
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Tetrahydrofolates / metabolism
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Tetrahydrofolates / pharmacology
Substances
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10-methylthio-DDACTHF
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Antimetabolites, Antineoplastic
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Apoproteins
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Enzyme Inhibitors
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Neoplasm Proteins
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Peptide Fragments
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Recombinant Proteins
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Tetrahydrofolates
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Phosphoribosylglycinamide Formyltransferase
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Phosphoribosylaminoimidazolecarboxamide Formyltransferase
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Carbon-Nitrogen Ligases
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GART protein, human
Associated data
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PDB/4EW1
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PDB/4EW2
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PDB/4EW3