Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Modulation of the de novo purine nucleotide pathway as a therapeutic strategy in mitochondrial myopathy.

Mitochondrial myopathy (MM) is characterised by muscle weakness, exercise intolerance and various histopathological changes. Recently, a subset of MM has also been associated with aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle. This aberrant mTORC1 activation promotes increased de novo nucleotide synthesis, which contributes to abnormal expansion and imbalance of skeletal muscle deoxyribonucleoside triphosphates (dNTP) pools. However, the exact mechanism via which mTORC1-stimulated de novo nucleotide biosynthesis ultimately disturbs muscle dNTP pools remains unclear. In this article, it is proposed that mTORC1-stimulated de novo nucleotide synthesis in skeletal muscle cells with respiratory chain dysfunction promotes an asymmetric increase of purine nucleotides, probably due to NAD+ deficiency. This in turn could disrupt purine nucleotide-dependent allosteric feedback regulatory mechanisms, ultimately leading to dNTP pools aberration. Pharmacological down-modulation of aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) activity is also proposed as a potential therapeutic strategy in MM exhibiting mTORC1-driven abnormal metabolic reprogramming, including aberrant dNTPs pools.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization.

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

Discovery of 5-substituted pyrrolo[2,3-d]pyrimidine antifolates as dual-acting inhibitors of glycinamide ribonucleotide formyltransferase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis: implications of inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase to ampk activation and antitumor activity.

We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

The apo and ternary complex structures of a chemotherapeutic target: human glycinamide ribonucleotide transformylase.

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.

Discovery of AICAR Tfase inhibitors that disrupt requisite enzyme dimerization.

The discovery of a new class of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) inhibitors through screening peptidomimetic libraries (>40,000 compounds) that act by inhibiting requisite enzyme dimerization is disclosed. In addition to defining key structural features of the lead compounds responsible for the activity, kinetic analysis of the remarkably small inhibitors established that they act as noncompetitive, dissociative inhibitors of AICAR Tfase with the prototypical lead (A1B3, Cappsin 1) exhibiting a K(i) of 3.1 +/- 0.3 microM. Thus, the studies define a unique approach to selectively targeting AICAR Tfase over all other folate-dependent enzymes, and it represents only one of a few enzymes for which inhibition achieved by disrupting requisite enzyme dimerization has emerged from screening unbiased combinatorial libraries.

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, K(i) = 0.23 microM), 10-methanesulfonyl-5-DACTHF (2, K(i) = 0.58 microM), and 10-methylthio-DDACTHF (3, K(i) = 0.25 microM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50 = 100 nM) against the CCRF-CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50 = 1.0 microM and 2, IC50 = 2.0 microM).

Synthesis and biological evaluation of N-[4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis and evaluation of N-[4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid (2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The inhibitor 2 was prepared in a convergent synthesis involving C-alkylation of methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)benzoate with 1-chloro-3-iodopropane followed by construction of the pyrimidinone ring. Compound 2 was found to be an effective inhibitor of recombinant human GAR Tfase (K(i) = 0.50 microM), whereas it was inactive (K(i) > 100 microM) against E. coli GAR Tfase as well as recombinant human AICAR Tfase. Compound 2 exhibited modest, purine-sensitive growth inhibitory activity against the CCRF-CEM cell line (IC50 = 6.0 microM).

Synthesis and biological evaluation of alpha- and gamma-carboxamide derivatives of 10-CF3CO-DDACTHF.

Structurally-related, but non-polyglutamylatable, derivatives of 10-CF3CO-DDACTHF (1), which incorporate L-glutamine (2) and L-isoglutamine (3) in place of L-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR Tfase. While the L-glutamate alpha-carboxamide derivative 3 was much less effective as a rhGAR Tfase inhibitor (K(i) = 4.8 microM) and inactive in cellular functional assays, the gamma-carboxamide derivative 2 was found to be a potent and selective rhGAR Tfase inhibitor (K(i) = 0.056 microM) being only 4-fold less potent than 1 (K(i) = 0.015 microM). Moreover, 2 was effective in cellular functional assays exhibiting purine sensitive cytotoxic activity (IC50 = 300 nM, CCRF-CEM) only 20-fold less potent than 1 (IC50 = 16 nM), consistent with inhibition of de novo purine biosynthesis via selective inhibition of GAR Tfase. Like 1, 2 is transported into the cell by the reduced folate carrier. Unlike 1, the functional activity of 2 is not dependent upon FPGS polyglutamylation.

Virtual screening of human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI diversity set by use of AutoDock to identify novel nonfolate inhibitors.

AICAR transformylase (5-aminoimidazole-4-carboxamide ribonucleotide transformylase) is a folate-dependent activity of the bifunctional protein ATIC (AICAR transformylase and IMP cyclohydrolase) and is responsible for catalyzing the penultimate step of the de novo purine biosynthetic pathway. As such, AICAR transformylase has been proposed as a potential target for antineoplastic drug design. Virtual screening of the human AICAR transformylase active site by use of AutoDock against the NCI diversity set, a library of compounds with nonredundant pharmacophore profiles, has revealed 44 potential inhibitor candidates. In vitro inhibition assay of 16 soluble compounds from this list revealed that eight compounds with novel scaffolds, relative to the general folate template, had micromolar inhibition. Subsequent extension of docking trials on compounds with similar scaffolds from the entire NCI-3D database has unveiled 11 additional inhibitors that were confirmed by the in vitro inhibition assay. In particular, one compound, NSC30171, had nanomolar inhibition (K(i) = 154 nM, IC(50) = 600 nM) against AICAR transformylase. These 19 inhibitors serve as novel templates/scaffolds for development of more potent and specific non-folate-based AICAR transformylase inhibitors.

Crystal structure of avian aminoimidazole-4-carboxamide ribonucleotide transformylase in complex with a novel non-folate inhibitor identified by virtual ligand screening.

Aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase), one of the two folate-dependent enzymes in the de novo purine biosynthesis pathway, is a promising target for anti-neoplastic chemotherapy. Although classic antifolates, such as methotrexate, have been developed as anticancer agents, their general toxicity and drug resistance are major issues associated with their clinical use and future development. Identification of inhibitors with novel scaffolds could be an attractive alternative. We present here the crystal structure of avian AICAR Tfase complexed with the first non-folate based inhibitor identified through virtual ligand screening of the National Cancer Institute Diversity Set. The inhibitor 326203-A (2-[5-hydroxy-3-methyl-1-(2-methyl-4-sulfophenyl)-1H-pyrazol-4-ylazo]-4-sulfo-benzoic acid) displayed competitive inhibition against the natural cofactor, 10-formyl-tetrahydrofolate, with a K(i) of 7.1 mum. The crystal structure of AICAR Tfase with 326203-A at 1.8 A resolution revealed a unique binding mode compared with antifolate inhibitors. The inhibitor also accessed an additional binding pocket that is not occupied by antifolates. The sulfonate group of 326203-A appears to form the dominant interaction of the inhibitor with the proposed oxyanion hole through interaction with a helix dipole and Lys(267). An aromatic interaction with Phe(316) also likely contributes to favorable binding. Based on these structural insights, several inhibitors with improved potency were subsequently identified in the National Cancer Institute Compound Library and the Available Chemical Directory by similarity search and molecular modeling methods. These results provide further support for our combined virtual ligand screening rational design approach for the discovery of novel, non-folate-based inhibitors of AICAR Tfase.

A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example.

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.

A defect in the p53 response pathway induced by de novo purine synthesis inhibition.

p53 is believed to sense cellular ribonucleotide depletion in the absence of DNA strand breaks and to respond by imposition of a p21-dependent G1 cell cycle arrest. We now report that the p53-dependent G1 checkpoint is blocked in human carcinoma cell lines after inhibition of de novo purine synthesis by folate analogs inhibitory to glycinamide ribonucleotide formyltransferase (GART). p53 accumulated in HCT116, MCF7, or A549 carcinoma cells upon GART inhibition, but, surprisingly, transcription of several p53 targets, including p21cip1/waf1, was impaired. The mechanism of this defect was examined. The p53 accumulating in these cells was nuclear but was not phosphorylated at serines 6, 15, and 20, nor was it acetylated at lysines 373 or 382. The DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo but did not activate histone acetylation over the p53 binding sites in the p21 promoter that is an integral part of the transcriptional response mediated by the DNA damage pathway. We concluded that the robust initial response of the p53 pathway to GART inhibitors is not transcriptionally propagated to target genes due to a defect in p53 post-translational modifications and a failure to open chromatin structure despite promoter binding of this unmodified p53.

Deaza analogs of folic acid as antitumor agents.

Derivatives of the vitamin folic acid function in the body for the synthesis of thymidylate, purines and amino acids and are necessary for normal metabolism and growth. Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR) is the outstanding example of an antitumor antifolate. MTX is clinically useful in the treatment of childhood leukemia, choriocarcinoma and psoriasis, where it corrects abnormal growth, and in rheumatoid arthritis and other autoimmune diseases where it corrects abnormal immune function. Since 1949, when the chemical synthesis of MTX was reported by workers at the Lederle Laboratories of the American Cyanamid Company, much has been learned about the basis of antifolate cytotoxicity and selectivity. This review will focus on deaza antifolates which are: 1). presently under clinical development and 2). less developed compounds which represent novel approaches. Compounds will be grouped according to their enzyme targets; DHFR, thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFT). In addition to inhibition of target enzymes, antifolate membrane transport into cells and conversion to poly-L-gamma-glutamate forms are important considerations in drug design along with the reverse processes, cellular hydrolysis of antifolate poly-L-gamma-glutamates to monoglutamates and the extrusion of the monoglutamates through the cell membrane. These processes can be modulated by competition with folates.

Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase.

A series of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues lacking the benzoylglutamate subunit were prepared and examined as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase).

10-(2-benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: a potential inhibitor of GAR transformylase and AICAR transformylase.

The design and synthesis of 10-(2-benzoxazolcarbonyl)-DDACTHF (1) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Ketone 1 and the corresponding alcohol 13 were evaluated for inhibition of GAR Tfase and AICAR Tfase and the former was found to be a potent inhibitor of recombinant human (rh) GAR Tfase (Ki=600 nM).

Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway.

The synthesis and evaluation of analogues and key derivatives of 10-CF3CO-DDACTHF as inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. Polyglutamate analogues of 1 were evaluated as inhibitors of Escherichia coli and recombinant human (rh) GAR Tfase, and AICAR Tfase. Although the pentaglutamate 6 was found to be the most active inhibitor of the series tested against rhGAR Tfase (Ki=0.004 microM), little distinction between the mono-pentaglutamate derivatives was observed (Ki=0.02-0.004 microM), suggesting that the principal role of the required polyglutamation of 1 is intracellular retention. In contrast, 1 and its defined polyglutamates 3-6 were much less inactive when tested against rhAICAR Tfase (Ki=65-0.120 microM) and very selective (> or =100-fold) for rh versus E. coli GAR Tfase. Additional key analogues of 1 were examined (7 and 8) and found to be much less active (1000-fold) highlighting the exceptional characteristics of 1.