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

Boger, Dale L.; Marsilje, Thomas H.; Castro, René A.; Hedrick, Michael; Jin, Qiong‐Hua; Baker, Stephen J.; Shim, Jae Hoon; Benkovic, Stephen J.

doi:10.1016/s0960-894x(00)00271-7

Cited by 11 publications

(5 citation statements)

References 23 publications

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“…For example, some inactivation mechanisms use nucleophilic aromatic substitution,25 and others use nucleophilic attack on a dihydropyridinium metabolite 26. There are also similarities in catalytic mechanisms.…”

Section: Resultsmentioning

confidence: 99%

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

et al. 2011

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In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time-and concentration-dependent inactivation of DDAH and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK a ca. 8.8) and 1 does not inactivate papain (Cys pK a ca. ≤ 4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore represent a first-in-class example of a novel type of quiescent affinity label.

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Section: Resultsmentioning

confidence: 99%

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

et al. 2011

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“…The E. coli enzyme's lack of structural complexity, but close mechanistic similarity to the human trifunctional protein, made it an attractive candidate for previous studies of the biological role of this enzyme in purine biosynthesis and for the design of novel inhibitors for antineoplastic intervention. Extensive kinetic and structural studies have since then used E. coli GAR Tfase as the template for mechanistic studies ( 3 , 22−24 ) and for drug design ( − ), although some limited design on the human enzyme has also been reported ().…”

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confidence: 99%

Crystal Structures of Human GAR Tfase at Low and High pH and with Substrate β-GAR^,

et al. 2002

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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.

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“…These two enzymes carry out similar chemistry in catalyzing the transfer of a formyl group from 10-formyltetrahydrofolate to the amino group of the substrates GAR and AICAR to form fGAR and fAICAR. Investigators have invested their efforts in understanding the mechanisms of the two transformylases with the express purpose of rationally designing inhibitors that specifically target them, and in so doing disrupt purine biosynthesis (4)(5)(6)(7)(8). These and other purine enzymes may function in concert as a multienzyme complex, thus providing another target for chemotherapeutic intervention.…”

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Localization of GAR transformylase in Escherichia coli and mammalian cells

Gooljarsingh¹,

Ramcharan²,

Gilroy³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway. One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein-protein interactions. To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Genes encoding human as well as E. coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway. However, discrete clusters of the pathway may still exist throughout the cytoplasm.

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

Cited by 11 publications

References 23 publications

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Crystal Structures of Human GAR Tfase at Low and High pH and with Substrate β-GAR^,

Localization of GAR transformylase in Escherichia coli and mammalian cells

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

Cited by 11 publications

References 23 publications

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Discovery of Halopyridines as Quiescent Affinity Labels: Inactivation of Dimethylarginine Dimethylaminohydrolase

Crystal Structures of Human GAR Tfase at Low and High pH and with Substrate β-GAR,

Localization of GAR transformylase in Escherichia coli and mammalian cells

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Crystal Structures of Human GAR Tfase at Low and High pH and with Substrate β-GAR^,