Mark S. Warren

10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF): A potent inhibitor of glycinamide ribonucleotide transformylase

The synthesis of 10-formyl-5,8,10-trideazafolic acid (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) is reported. The target compound was prepared by a convergent synthesis utilizing the alkylation of hydrazone 5 with benzylic bromide 6 to construct the core heterocycle 7. The aldehyde 3 and related agents were evaluated as inhibitors of purN GAR Tfase and avian AICAR Tfase. Compound 3 exhibited potent inhibition of GAR Tfase with a Ki of 0.26 +/- 0.05 microM. In contrast, 3 exhibited more moderate inhibition of aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase), with Ki of 7.6 +/- 1.5 microM.

The transformylase enzymes of de novo purine biosynthesis

Formyl transfer reactions play a key role in the construction of the purine heterocycle during de now purine biosynthesis. Formylation is catalyzed early in the pathway by the purN glycinamide ribonucleotide transformylase (GAR Transformylase, EC 2.1.2.2) in a tetrahydrofolate-dependent manner and also by the purT GAR transformylase in a tetrahydrofolate-independent manner in bacteria. Late in the pathway, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Transformylase, EC 2.1.2.3) catalyzes the second and final formylation involved in purine nucleotide biosynthesis. This article summarizes the salient properties and mechanistic knowledge on the transformylases with special emphasis on the mechanism of the purN GAR transformylase as explored by mutagenesis studies. Introduction The de novo purine biosynthetic pathway produces purine nucleotides that are essential for many processes in the cell. Purines serve as building blocks in DNA and RNA synthesis, are utilized as an energy source for chemical reactions (ATP), are used in cellular redox reactions (NADH, NADPH, FAD, etc.), and also play key roles in regulatory functions (CAMP, ZTP, etc.). Virtually all organisms studied to date utilize this pathway to synthesize purines, with the exception of parasitic protozoa (1) which must scavenge purines from their environment. The overall de novo purine biosynthetic pathway consists of ten enzymatic reactions which serve to convert 5-phosphoribosyl-1 -pyrophosphate to inosine monophosphate, which can then be converted to adenosine monophosphate and guanine monophosphate. These reactions are invariant in all organisms synthesizing purines, although the organization and regulation may differ (2). Generally, prokaryotes tend to use smaller single function enzymes, while higher eukaryotic organisms place increased reliance on larger multifunctional enzymes in this pathway (2). Because cancer cells grow rapidly and require large amounts of purines to maintain such growth, the de novo purine biosynthetic pathway has attracted considerable attention as a target for cancer chemotherapy (3). Some of the most successful antiproliferative drugs developed to date have been folate antimetabolites. Two of the enzymes in this pathway require a reduced folate, and are thus natural targets for screening novel antifolates. These enzymes, glycinamide ribonucleotide transformylase (GAR Transformylase, EC 2.1.2.2) and 5-aminoimidazole-4-carboxamide ribonucleotide transformy lase (AICAR Transformy lase, EC 2.1.2.3) catalyze the third and ninth reactions of this pathway. Both of these enzymes are involved in formyl transfer reactions, and both use 10-formyl tetrahydrofolate as a cofactor. These two enzymes are products of the purN and purH genes in Escherichia coli. Recently, a second glycinamide ribonucleotide transformylase enzyme was isolated and characterized from E. coli (4, 5). This enzyme is the product of the purT gene and does not utilize a folate cofactor. The purN and purT enzymes both catalyze the formylation of P-glycinamide ribonucleotide (GAR) to produce formyl p-glycinamide ribonucleotide (fGAR), however, they do so using different cofactors and different mechanisms. The reactions catalyzed by the three transformylases of de novo purine biosynthesis are shown in Fig. 1. The E. coli purN GAR transformylase is a monomeric protein of 212 amino acids with a molecular weight of 23,200. The homologous enzyme in humans is a much larger trifunctional polypeptide encoding GAR synthase (EC 6.3.4.13) and aminoimidazole ribonucleotide synthetase (EC 6.3.3.1) activities in addition to a GAR transformylase activity (6). Sequence homology and mutagenesis of catalytic residues suggests that there is a substantial degree of mechanistic similarity between the human and E. coli enzymes (6-9). High resolution x-ray crystal structures of the E. coli purN GAR Transformylase have been reported in the absence of ligands (lo), in a ternary complex with substrate GAR and a folate inhibitor (1 l), and in a binary complex with a multisubstrate adduct inhibitor bound (12). These results have shown that the enzyme structure is composed of two domains. The amino terminal domain (residues 1 to 101) contains a

Functionalized analogues of 5,8,10-trideazafolate as potential inhibitors of GAR Tfase or AICAR Tfase

Abstract A series of TDAF-based analogues of 10-formyl-tetrahydrofolic acid are examined in efforts to explore the formyl transfer region of GAR Tfase and AICAR Tfase.

Functionalized analogues of 5,8,10-trideazafolate: development of an enzyme-assembled tight binding inhibitor of GAR Tfase and a potential irreversible inhibitor of AICAR Tfase.

A set of inhibitors 3 and 4 of GAR and AICAR Tfase based on the TDAF core which contain an sp2 C-10 carbon atom replacing N-10 of the natural cofactor are detailed. Both possess electrophilic olefins and the potential of trapping the reacting amine of the substrates GAR and AICAR by a Michael addition at the enzyme active site to provide an enzyme-assembled tight binding inhibitor. While these agents did not display such characteristics and served as simple competitive inhibitors of GAR Tfase and AICAR Tfase, inhibitor 15 prepared in the conversion of 3 to 4 may provide an enzyme-assembled tight binding inhibitor of GAR Tfase upon reaction with the substrate GAR and may inactivate AICAR Tfase by virtue of alkylation of an active site residue.

DESIGN, SYNTHESIS, AND EVALUATION OF POTENTIAL GAR AND AICAR TRANSFORMYLASE INHIBITORS

Abstract The synthesis and evaluation of 1 – 4 as potential inhibitors of GAR Tfase and AICAR Tfase are detailed.

Abenzyl 10-formyl-trideazafolic acid (abenzyl 10-formyl-TDAF): An effective inhibitor of glycinamide ribonucleotide transformylase

The synthesis of N-[7-(2-amino-3,4-dihydro-4-oxo-quinazolin-6-yl) -6-formyl-1-oxo-heptyl]-L-glutamic acid (2, abenzyl 10-formyl-5,8,10-trideazafolic acid) as a potential enzyme-assembled tight binding inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) or aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. The inhibitor was prepared by a convergent synthesis utilizing the sequential alkylations of acetaldehyde dimethylhydrazone with 6 and 8. The agent exhibited effective inhibition of GAR Tfase (Ki = 4.5 +/- 0.3 microM) and more modest inhibition of AICAR Tfase (Ki = 42 +/- 11 microM).

Multisubstrate analogue based on 5,8,10-trideazafolate.

Abstract The preparation and evaluation of the multisubstrate analogue 3 based on the 5,8,10-trideazafolate core for GAR and AICAR Tfase inhibition is detailed.