10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway

The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.     

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.     

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

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.     

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.     

Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells

The folate compound 10-formyldihydrofolate (H2folate) has not been found as a component of intracellular folates in normal tissues but has been identified in the cytosol of methotrexate (MTX)-treated MCF-7 breast cancer cells and normal human myeloid precursor cells. Its identity was verified by coelution of this compound with a synthetic marker on high pressure liquid chromatography, its reduction to 10-formyltetrahydrofolate (H4folate) in the presence of dihydrofolate reductase, and its enzymatic deformylation to dihydrofolate in the presence of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase. Chemically synthesized monoglutamated or pentaglutamated 10-formyl-H2folate was examined for its interaction with three folate-dependent enzymes: AICAR transformylase, glucinamide ribotide (GAR) transformylase, and thymidylatesynthase. 10-Formyl-H2folate-Glu5 was a competitive inhibitor of thymidylate synthase (Ki = 0.16 microM with 5,10-methylene-H4folate-Glu1 as substrate and 1.6 microM with 5,10-methylene-H4folate-Glu5) and inhibited GAR transformylase (Ki = 2.0 microM). It acted as a substrate for AICAR transformylase (Km = 5.3 microM), and its efficiency was equal to that of the natural substrate 10-formyl-H4folate-Glu5. The inhibition of thymidylate synthase by 10-formyl-H2folate was highly dependent on the inhibitor's polyglutamation state, the -Glu5 derivative having a 52-85-fold greater affinity as compared to the affinity of -Glu1. Polyglutamation of 10-formyl-H2folate did not affect its inhibition of GAR transformylase. While the actual role of 10-formyl-H2folate contributing to the cytotoxicity of MTX has not been determined, this compound has the potential to enhance inhibition of GAR transformylase and thymidylate synthase, and at the same time provides additional substrate for AICAR transformylase. The MTX-induced intracellular accumulation of 10-formyl-H2folate and H2folate may play a role in the drug-related cytotoxicity through the contribution of these folates to the inhibition of thymidylate synthase and de novo purine synthesis.     

Human AICAR transformylase: role of the 4-carboxamide of AICAR in binding and catalysis

We have prepared 4-substituted analogues of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to investigate the specificity and mechanism of AICAR transformylase (AICAR Tfase). Of the nine analogues of AICAR studied, only one analogue, 5-aminoimidazole-4-thiocarboxamide ribonucleotide, was a substrate, and it was converted to 6-mercaptopurine ribonucleotide. The other analogues either did not bind or were competitive inhibitors, the most potent being 5-amino-4-nitroimidazole ribonucleotide with a K(i) of 0.7 +/- 0.5 microM. The results show that the 4-carboxamide of AICAR is essential for catalysis, and it is proposed to assist in mediating proton transfer, catalyzing the reaction by trapping of the addition compound. AICAR analogues where the nitrogen of the 4-carboxamide was derivatized with a methyl or an allylic group did not bind AICAR Tfase, as determined by pre-steady-state burst kinetics; however, these compounds were potent inhibitors of IMP cyclohydrolase (IMP CHase), a second activity of the bifunctional mammalian enzyme (K(i) = 0.05 +/- 0.02 microM for 4-N-allyl-AlCAR). It is proposed that the conformation of the carboxamide moiety required for binding to AICAR Tfase is different than the conformation required for binding to IMP CHase, which is supported by inhibition studies of purine ribonucleotides. It is shown that 5-formyl-AICAR (FAICAR) is a product inhibitor of AICAR Tfase with K(i) of 0.4 +/- 0.1 microM. We have determined the equilibrium constant of the transformylase reaction to be 0.024 +/- 0.001, showing that the reaction strongly favors AICAR and the 10-formyl-folate cofactor. The coupling of the AICAR Tfase and IMP CHase activities on a single polypeptide allows the overall conversion of AICAR to IMP to be favorable by coupling the unfavorable formation of FAICAR with the highly favorable cyclization reaction. The current kinetic studies have also indicated that the release of FAICAR is the rate-limiting step, under steady-state conditions, in the bifunctional enzyme and channeling is not observed between AICAR Tfase and IMP CHase.     

Design, synthesis, and biological evaluation of fluoronitrophenyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase

The examination results of a novel series of potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported. These agents incorporate an electrophilic fluoronitrophenyl group that can potentially react with an active site nucleophile or the substrate GAR/AICAR amine via nucleophilic aromatic substitution.     

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).     

Conformationally restricted analogues designed for selective inhibition of GAR Tfase versus thymidylate synthase or dihydrofolate reductase

The synthesis and evaluation of a series of conformationally restricted analogues of 10-formyl-tetrahydrofolate as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide transformylase (AICAR Tfase) are reported.     

Crystal structures of human GAR Tfase at low and high pH and with substrate beta-GAR

Glycinamide ribonucleotide transformylase (GAR Tfase) is a key folate-dependent enzyme in the de novo purine biosynthesis pathway and, as such, has been the target for antitumor drug design. Here, we describe the crystal structures of the human GAR Tfase (purN) component of the human trifunctional protein (purD-purM-purN) at various pH values and in complex with its substrate. Human GAR Tfase exhibits pH-dependent enzyme activity with its maximum around pH 7.5-8. Comparison of unliganded human GAR Tfase structures at pH 4.2 and pH 8.5 reveals conformational differences in the substrate binding loop, which at pH 4.2 occupies the binding cleft and prohibits substrate binding, while at pH 8.5 is permissive for substrate binding. The crystal structure of GAR Tfase with its natural substrate, beta-glycinamide ribonucleotide (beta-GAR), at pH 8.5 confirms this conformational isomerism. Surprisingly, several important structural differences are found between human GAR Tfase and previously reported E. coli GAR Tfase structures, which have been used as the primary template for drug design studies. While the E. coli structure gave valuable insights into the active site and formyl transfer mechanism, differences in structure and inhibition between the bacterial and mammalian enzymes suggest that the human GAR Tfase structure is now the appropriate template for the design of anti-cancer agents.     

Unexpected formation of an epoxide-derived multisubstrate adduct inhibitor on the active site of GAR transformylase.

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transformylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta-GAR substrate, typically exhibit K(i)'s in the picomolar range. However, these compounds have reduced bioavailability due to the incorporation of a negatively charged phosphate moiety that prevents effective cellular uptake. Thus, a folate analogue that is capable of adduct formation with the substrate on the enzyme active site could lead to a potent GAR Tfase inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10-trideazafolic acid, that was an intermediate designed to assemble with the substrate beta-GAR on the enzyme active site. We have now determined the crystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 A resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the existence of a covalent adduct between the substrate beta-GAR and the folate analogue (K(i) = 20 microM). However, the electron density map surprisingly indicated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but proceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epoxide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was strongest when the dibromide or epoxide is preincubated with both enzyme and substrate, indicating that inhibition occurs via the enzyme-dependent formation of the multisubstrate adduct. Thus, the crystal structure revealed the successful formation of an enzyme-assembled multisubstrate adduct and highlighted a potential application for epoxides, and perhaps aziridines, in the design of efficacious GAR Tfase inhibitors.     

A quantum chemical study on the mechanism of glycinamide ribonucleotide transformylase inhibitor: 10-Formyl-5,8,10-trideazafolic acid

A density functional theory (DFT) study is presented on the reaction mechanism of glycinamide ribonucleotide (GAR) with 10-formyl-5,8,10-trideazafolic acid (10f-TDAF), which is an inhibitor designed for GAR transformylase (GAR Tfase). There are three different paths for this system and the results indicate that inhibitor 10f-TDAF can form a very stable intermediate with the substrate GAR or generate an imine bond with GAR by elimination of water. The results have verified the presumption from available experiments and implied that 10f-TDAF would be an important target for anti-neoplastic intervention.     

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.     

Structural and mechanistic studies on the HeLa and chicken liver proteins that catalyze glycinamide ribonucleotide synthesis and formylation and aminoimidazole ribonucleotide synthesis

Glycinamide ribonucleotide (GAR) transformylase from HeLa cells has been purified 200-fold to apparent homogeneity with a procedure using two affinity resins. The activities glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase were found to copurify with GAR transformylase. Glycinamide ribonucleotide synthetase and GAR transformylase were separable only after exposure to chymotrypsin. Antibodies raised to pure L1210 cell GAR transformylase were able to precipitate the glycinamide ribonucleotide transformylase and GAR synthetase activities from HeLa and L1210 cells both in their native and in their proteolytically shortened forms. The compound N-10-(bromoacetyl)-5,8-dideazafolate was found to inhibit formylation but to leave the ATP-requiring synthetase activities intact.     

Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase

The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.     

Heterocyclic substituted cantharidin and norcantharidin analogues--synthesis, protein phosphatase (1 and 2A) inhibition, and anti-cancer activity

Norcantharidin (3) is a potent PP1 (IC(50)=9.0+/-1.4 microM) and PP2A (IC(50)=3.0+/-0.4 microM) inhibitor with 3-fold PP2A selectivity and induces growth inhibition (GI(50) approximately 45 microM) across a range of human cancer cell lines including those of colorectal (HT29, SW480), breast (MCF-7), ovarian (A2780), lung (H460), skin (A431), prostate (DU145), neuroblastoma (BE2-C), and glioblastoma (SJ-G2) origin. Until now limited modifications to the parent compound have been tolerated. Surprisingly, simple heterocyclic half-acid norcantharidin analogues are more active than the original lead compound, with the morphilino-substituted (9) being a more potent (IC(50)=2.8+/-0.10 microM) and selective (4.6-fold) PP2A inhibitor with greater in vitro cytotoxicity (GI(50) approximately 9.6 microM) relative to norcantharidin. The analogous thiomorpholine-substituted (10) displays increased PP1 inhibition (IC(50)=3.2+/-0 microM) and reduced PP2A inhibition (IC(50)=5.1+/-0.41 microM), to norcantharidin. Synthesis of the analogous cantharidin analogue (19) with incorporation of the amine nitrogen into the heterocycle further increases PP1 (IC(50)=5.9+/-2.2 microM) and PP2A (IC(50)=0.79+/-0.1 microM) inhibition and cell cytotoxicity (GI(50) approximately 3.3 microM). These analogues represent the most potent cantharidin analogues thus reported.     

Inhibition of poly(ADP-ribose) synthetase by unsaturated fatty acids, vitamins and vitamin-like substances

Various vitamins and vitamin-like substances inhibited the activity of poly(ADP-ribose) synthetase in vitro. The most potent were essential fatty acids, i.e. arachidonic acid, linoleic acid, and linolenic acid; their 50% inhibitory concentrations (IC50) were 44-110 microM, indicating a higher potency than nicotinamide, a well-known vitamin inhibitor (IC50 = 210 microM). Vitamins K3, K1, and retinal were the next strongest inhibitors, followed by alpha-lipoic acid, coenzyme Q0, and pyridoxal 5-phosphate. Nicotinamide and vitamin K3 exhibited mixed-type inhibition with respect to NAD+, while arachidonic acid exhibited dual inhibitions, competitive at 50 microM and mixed-type at 100 microM.     

New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid

The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, beta-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol. The presence of the hydrated aldehyde was confirmed in solution by (13)C-(1)H NMR spectroscopy of the ternary GAR Tfase-beta-GAR-10-formyl-TDAF complex using the (13)C-labeled 10-formyl-TDAF. This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents.