Helicobacter pylori-selective Antibacterials Based on Inhibition of Pyrimidine Biosynthesis

Copeland, Robert A.; Marcinkeviciene, Jovita; Haque, Tasir S.; Kopcho, Lisa M.; Jiang, Wenjun; Wang, Kathy; Ecret, Lisa D.; Sizemore, Christine; Amsler, Karen; Foster, Lori; Tadesse, Seifu; Combs, Andrew P.; Stern, Andrew M.; Trainor, George L.; Slee, Andrew M.; Rogers, Michael J.; Hobbs, Frank W.

doi:10.1074/jbc.m004451200

Cited by 59 publications

(55 citation statements)

References 23 publications

(27 reference statements)

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“…H. pylori has no homologs of the pyrimidine salvage enzymes uracil PRTase (Upp) or uridine kinase (Udk), which convert uracil to UMP (37). Although our results could be explained by poor uptake of medium supplements, they are consistent with recent reports that the conserved gene HP1084 (pyrB), encoding aspartate carbamoyltransferase, is also essential (13) and that inhibitors of dihydroorotate dehydrogenase (PyrD) are lethal to H. pylori (17). Taken together these data appear to demonstrate that de novo pyrimidine biosynthesis is required for survival of H. pylori.…”

Section: Resultssupporting

confidence: 82%

“…Importantly, a further degree of selectivity may be conferred by the fact that the nearest neighbors of some of these genes are not required for viability in other bacteria. The potential of this type of approach has been graphically illustrated by the recent identification of pyrazole-based anti-H. pylori compounds which specifically inhibit the pyrimidine biosynthesis enzyme PyrD in H. pylori but are inactive against orthologous enzymes in other bacteria and humans (17). We therefore suggest that the products of these highly diverged essential genes merit further investigation as potential targets for novel, highly specific anti-H. pylori agents.…”

Section: Resultsmentioning

confidence: 99%

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Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

et al. 2001

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A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

et al. 2001

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“…The R265A mutation affected some compounds by up to 35-fold, whereas there was no effect on others. This site is also thought to bind CoQ, although direct structural evidence for the CoQ site is still lacking (12,14,31). In support of a common binding site for the malaria enzyme, the most potent benzamidine derivatives (compound 6) appeared to be a simple competitive inhibitor with respect to CoQ utilization.…”

Section: Discussionmentioning

confidence: 99%

“…1). Thirdly, species-selective pyrazole-based inhibitors of Hylicobacter pylori and of Escherichia coli DHODH have been reported that were identified by highthroughput screening of chemical libraries (13,14). Finally, we previously demonstrated that P. falciparum DHODH is poorly inhibited by the potent human DHODH inhibitors redoxal, dichloroallyl lawsone, and A77-1726 analogs (15).…”

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confidence: 99%

High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

Baldwin

Michnoff

Malmquist

et al. 2005

Journal of Biological Chemistry

184

210

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Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and ureabased compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC 50 values below 600 nM were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC 50 against malarial DHODH of 16 nM, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

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“…An inhibitor of human DHODH (hDHODH) (A77 1726 the active metabolite of leflunomide) is marketed for the treatment of rheumatoid arthritis, illustrating that DHODH is a druggable target (10,11). Finally, biochemical (12,13) and structural studies (14,15) suggested that the identification of species-selective inhibitors against this target was feasible.…”

mentioning

confidence: 99%

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

Deng

Gujjar

Mazouni

et al. 2009

Journal of Biological Chemistry

113

214

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Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into speciesselective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.The human malaria parasite is endemic in 87 countries putting 2.5 billion people in the poorest nations of the tropics at risk for the disease (1, 2). Despite intensive efforts to control malaria through combination drug therapy and insect control programs, malaria remains one of the largest global health problems. The most severe form of the disease is caused by Plasmodium falciparum, which kills 1-2 million people yearly, primarily children and pregnant woman. Effective vaccines have not been developed, and chemotherapy remains the mainstay of both treatment and prevention of the disease. Unfortunately widespread drug resistance to almost every known antimalarial agent has compromised the effectiveness of malaria control programs (3). The introduction of artemisinin combination chemotherapy has provided new treatment options to combat drug-resistant parasites (4). However, recent reports by the World Health Organization suggest that resistance to artemisinin is developing along the Thai-Cambodian border, underscoring the need for a continual pipeline of new drug development to combat this disease.The malaria parasite relies exclusively on de novo pyrimidine biosynthesis to supply precursors for DNA and RNA biosynthesis (5, 6). In contrast, the human host cells contain the enzymatic machinery for both de novo pyrimidine biosynthesis and for salvage of preformed pyrimidine bases and nucleosides. The lack of a redundant mechanism to acquire pyrimidines in malaria...

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Helicobacter pylori-selective Antibacterials Based on Inhibition of Pyrimidine Biosynthesis

Cited by 59 publications

References 23 publications

Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis

High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

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