Benzo[f]quinazoline Inhibitors of Thymidylate Synthase: Methyleneamino-Linked Aroylglutamate Derivatives

Background Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.Methodology/Principal FindingsAfter cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k cat values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Conclusions/SignificanceThus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis.

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“…aFrom Dev et al [46].bFrom Dolnick and Cheng [25].cFrom Davisson et al [26].dFrom Pendergast et al [44].…”

Section: Resultsmentioning

confidence: 99%

“…mTHF solutions were stored under argon at −20°C. 1843U89 was synthesized as previously reported [44].…”

Section: Methodsmentioning

confidence: 99%

Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

et al. 2008

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“…TS methylates the 5-carbon of 5’-deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (TMP) using 5,10-methylene THF as a cofactor. TS is the target for many anticancer agents including pemetrexed, ZD1694, 1843U89, AG331, and AG337 [11, 13–18]. …”

Section: Introductionmentioning

confidence: 99%

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase

Pang

Hunter

Gujjar

et al. 2009

Molecular and Biochemical Parasitology

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The plant-like, bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from malaria parasites has been a good target for drug development. Dihydrofolate reductase (DHFR) is inhibited by clinically established antimalarials, pyrimethamine and cycloguanil. Thymidylate synthase (TS) is the target of potent experimental antimalarials such as 5-fluoroorotate and 1843U89. Another enzyme in folate recycling, serine hydroxymethyltransferase (SHMT), produces 5,10-methylenetetrahydrofolate which, in many cells, is required for the de novo, biosynthesis of thymidine and methionine. Thus, the biochemical characterization of malarial SHMT was of interest. The principle, active P. falciparum SHMT (PfSHMT) was expressed in E. coli and purified using an N-terminal histidine tag. Unlike the plant enzyme, but like the host enzyme, PfSHMT requires the cofactor pyridoxal 5’-phosphate for enzymatic activity. The substrate specificities for serine, tetrahydrofolate, and pyridoxal 5’-phosphate were comparable to those for SHMT from other organisms. Antifolates developed for DHFR and TS inhibited SHMT in the mid-micromolar range, offering insights into the binding preferences of SHMT but clearly leaving room for improved new inhibitors. As previously seen with P. falciparum DHFR-TS, PfSHMT bound its cognate mRNA but not control RNA for actin. RNA-binding was not reversed with enzyme substrates. Unlike DHFR-TS, the SHMT RNA-protein interaction was not tight enough to inhibit translation. Another gene PF14_0534, previously proposed to code for an alternate mitochondrial SHMT, was also expressed in E. coli but found to be inactive. This protein, nor DHFR-TS, enhanced the catalytic activity of PfSHMT. The present results set the stage for developing specific, potent inhibitors of SHMT from P. falciparum.

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“…effects. 33–39 For example, Pendergast et al 40 observed that a 2′-fluoro substitution of a benzo[ f ]quinazoline antifolate increased antitumor activity, which was attributed to the conformational restriction of the side-chain L-glutamate via a fluorine−hydrogen bond. 41 …”

Section: Introductionmentioning

confidence: 99%

Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

et al. 2018

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Novel fluorinated 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine analogues 7–12 were synthesized and tested for selective cellular uptake by folate receptors (FRs) α and β or the proton-coupled folate transporter (PCFT) and for antitumor efficacy. Compounds 8, 9, 11, and 12 showed increased in vitro antiproliferative activities (~11-fold) over the nonfluorinated analogues 2, 3, 5, and 6 toward engineered Chinese hamster ovary and HeLa cells expressing FRs or PCFT. Compounds 8, 9, 11, and 12 also inhibited proliferation of IGROV1 and A2780 epithelial ovarian cancer cells; in IGROV1 cells with knockdown of FRα, 9, 11, and 12 showed sustained inhibition associated with uptake by PCFT. All compounds inhibited glycinamide ribonucleotide formyltransferase, a key enzyme in the de novo purine biosynthesis pathway. Molecular modeling studies validated in vitro cell-based results. NMR evidence supports the presence of an intramolecular fluorine−hydrogen bond. Potent in vivo efficacy of 11 was established with IGROV1 xenografts in severe compromised immunodeficient mice.

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Benzo[f]quinazoline Inhibitors of Thymidylate Synthase: Methyleneamino-Linked Aroylglutamate Derivatives

Cited by 30 publications

References 10 publications

Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

Kinetics and Ligand-Binding Preferences of Mycobacterium tuberculosis Thymidylate Synthases, ThyA and ThyX

Catalytic and ligand-binding characteristics of Plasmodium falciparum serine hydroxymethyltransferase

Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

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