Selective inhibition of bacterial dihydroorotate dehydrogenases by thiadiazolidinediones

Self Cite

We report the discovery of a class of pyrazole-based compounds that are potent inhibitors of the dihydroorotate dehydrogenase of Helicobacter pylori but that do not inhibit the cognate enzymes from Gram-positive bacteria or humans. In culture these compounds inhibit the growth of H. pylori selectively, showing no effect on other Gram-negative or Gram-positive bacteria or human cell lines. These compounds represent the first examples of H. pylori-specific antibacterial agents. Cellular activity within this structural class appears to be due to dihydroorotate dehydrogenase inhibition. Minor structural changes that abrogate in vitro inhibition of the enzyme likewise eliminate cellular activity. Furthermore, the minimum inhibitory concentrations of these compounds increase upon addition of orotate to the culture medium in a concentration-dependent manner, consistent with dihydroorotate dehydrogenase inhibition as the mechanism of cellular inhibition. The data presented here suggest that targeted inhibition of de novo pyrimidine biosynthesis may be a valuable mechanism for the development of antimicrobial agents selective for H. pylori.

Section: Methodsmentioning

confidence: 99%

Helicobacter pylori-selective Antibacterials Based on Inhibition of Pyrimidine Biosynthesis

Copeland¹,

Marcinkeviciene²,

Haque³

et al. 2000

Self Cite

“…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).…”

mentioning

confidence: 97%

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

Baldwin

Michnoff

Malmquist

et al. 2005

188

210

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.

“…In addition to A77 1726, a number of high affinity inhibitors of human DHODH have been reported along with extensive structure-activity analysis (22)(23)(24)(25). Furthermore, high affinity inhibitors of Helicobacter pylori DHODH and the Escherichia coli enzyme have also been described that display considerable species selectivity (26,27). Thus, the known species differences in DHODH inhibitor binding, and the established clinical pharmacology of DHODH inhibitors in humans, suggest that selective DHODH inhibitors may also be developed for malaria chemotherapy.…”

mentioning

confidence: 99%

Malarial Dihydroorotate Dehydrogenase

Baldwin¹,

Farajallah²,

Malmquist³

et al. 2002

104

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k cat 8 s ؊1 , K m app DHO (40 -80 M)). The substrate specificity of the ubiquinone cofactor (CoQ n ) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ n was a determinant of reaction efficiency; CoQ 4 , CoQ 6 and decylubiquinone (CoQ D ) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ 0 and vitamin K 3 ) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K m app . Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC 50 's ranging from 0.1-1.0 mM. The IC 50 values for inhibition of the malarial enzyme are 10 2 -10 4 -fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.Malaria afflicts between 500 and 900 million people worldwide and causes greater than 2 million deaths per year (1, 2), making this parasitic disease an enormous public health problem throughout the developing world. Currently, several medicinal therapies are available for use as prophylaxis and treatment for at-risk or infected individuals. However, widespread drug resistance against these agents (e.g. chloroquine (3), atovaquone (4), pyrimethamine (5), and sulfadoxine (6)), has compromised the effectiveness of these treatments resulting in the pressing need for the development of new anti-malarial compounds. The identification of targets that exploit the unique biology of the parasite is an essential step in the development of new therapeutics.Pyrimidines are essential metabolites in all cells. They are required not only for DNA and RNA biosynthesis, but also for the biosynthesis of phospholipids and glycoproteins. The de novo pyrimidine biosynthetic pathway is intact in most organisms, including Plasmodium (7-9). However, unlike mammalian cells, the human malaria parasite, Plasmodium falciparum, cannot salvage preformed pyrimidine bases or nucleosides and utilizes de n...