scholarly journals Pyrimidine Pathway-Dependent and -Independent Functions of the Toxoplasma gondii Mitochondrial Dihydroorotate Dehydrogenase

2016 ◽  
Vol 84 (10) ◽  
pp. 2974-2981 ◽  
Author(s):  
Miryam Andrea Hortua Triana ◽  
Daniela Cajiao Herrera ◽  
Barbara H. Zimmermann ◽  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Target ◽  
De Novo ◽  
Mutant Gene ◽  
Pyrimidine Biosynthesis ◽  
Dihydroorotate Dehydrogenase ◽  
Content Type ◽  
Independent Functions ◽  
Fourth Step ◽  
Essential Function

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step ofde novopyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. InToxoplasma gondii, disruption of the first, fifth, or sixth step ofde novopyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrion-associated DHODH ofT. gondii(TgDHODH) failed. To further address the essentiality ofTgDHODH, mutant gene alleles deficient inTgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenousDHODHgene with catalytically deficientDHODHgene alleles induced uracil auxotrophy. Catalytically deficientTgDHODH localized to the mitochondria, and parasites retained mitochondrial membrane potential. These results show thatTgDHODH is essential for the synthesis of pyrimidines and suggest thatTgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis.

scholarly journals Identification of 3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

2021 ◽  
Vol 22 (13) ◽  
pp. 7236
Author(s):  
Endah Dwi Hartuti ◽  
Takaya Sakura ◽  
Mohammed S. O. Tagod ◽  
Eri Yoshida ◽  
Xinying Wang ◽  
...  
Keyword(s):  
Drug Target ◽  
De Novo ◽  
Dihydroorotate Dehydrogenase ◽  
Chemical Library ◽  
New Class ◽  
De Novo Biosynthesis ◽  
Starting Point ◽  
Novel Drugs ◽  
Low Toxicity ◽  
Fourth Step

Plasmodium falciparum’s resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development.

scholarly journals Pantothenic Acid Biosynthesis in the Parasite Toxoplasma gondii: a Target for Chemotherapy

2014 ◽  
Vol 58 (11) ◽  
pp. 6345-6353 ◽  
Author(s):  
Sarmad N. Mageed ◽  
Fraser Cunningham ◽  
Alvin Wei Hung ◽  
Hernani Leonardo Silvestre ◽  
Shijun Wen ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Target ◽  
Parasitic Infection ◽  
Pantothenic Acid ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Pantothenate Synthetase ◽  
Genes Encoding ◽  
Range Of Values ◽  
Important Parasite

ABSTRACTToxoplasma gondiiis a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis inT. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed inT. gondii. A panel of inhibitors developed to targetMycobacterium tuberculosispantothenate synthetase were tested and found to exhibit a range of values for inhibition ofT. gondiigrowth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence,T. gondiiencodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target.

scholarly journals TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Joseph M. Varberg ◽  
Leah R. Padgett ◽  
Gustavo Arrizabalaga ◽  
William J. Sullivan
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Target ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
World Population ◽  
Content Type ◽  
Tubulin Acetylation ◽  
Parasite Replication ◽  
New Treatments

ABSTRACT Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target. Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

scholarly journals Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa

Reproduction ◽  
2002 ◽  
pp. 757-768 ◽  
Author(s):  
EA Carrey ◽  
C Dietz ◽  
DM Glubb ◽  
M Loffler ◽  
JM Lucocq ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Dihydroorotate Dehydrogenase ◽  
Aspartate Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Pyrimidine Nucleotides ◽  
Carbamyl Phosphate Synthetase ◽  
De Novo Biosynthesis ◽  
Ump Synthase ◽  
Mammalian Spermatozoa

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

scholarly journals DHODH and cancer: promising prospects to be explored

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yue Zhou ◽  
Lei Tao ◽  
Xia Zhou ◽  
Zeping Zuo ◽  
Jin Gong ◽  
...  
Keyword(s):  
Biological Function ◽  
De Novo ◽  
Mitochondrial Respiratory Chain ◽  
Cancer Therapies ◽  
Dihydroorotate Dehydrogenase ◽  
Specific Mechanism ◽  
Pyrimidine Synthesis ◽  
The Past ◽  
De Novo Pyrimidine Synthesis ◽  
Fourth Step

AbstractHuman dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme catalyzing the fourth step in the de novo pyrimidine synthesis pathway. It is originally a target for the treatment of the non-neoplastic diseases involving in rheumatoid arthritis and multiple sclerosis, and is re-emerging as a validated therapeutic target for cancer therapy. In this review, we mainly unravel the biological function of DHODH in tumor progression, including its crucial role in de novo pyrimidine synthesis and mitochondrial respiratory chain in cancer cells. Moreover, various DHODH inhibitors developing in the past decades are also been displayed, and the specific mechanism between DHODH and its additional effects are illustrated. Collectively, we detailly discuss the association between DHODH and tumors in recent years here, and believe it will provide significant evidences and potential strategies for utilizing DHODH as a potential target in preclinical and clinical cancer therapies.

scholarly journals De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii

Nature ◽  
2002 ◽  
Vol 415 (6874) ◽  
pp. 926-929 ◽  
Author(s):  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Pyrimidine Biosynthesis

scholarly journals Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1468
Author(s):  
Dan Sato ◽  
Endah Dwi Hartuti ◽  
Daniel Ken Inaoka ◽  
Takaya Sakura ◽  
Eri Amalia ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Drug Target ◽  
De Novo ◽  
Binding Pocket ◽  
Severe Form ◽  
Eimeria Tenella ◽  
Potential Drug Target ◽  
Dihydroorotate Dehydrogenase ◽  
Potential Drug ◽  
De Novo Biosynthesis

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

scholarly journals Antiviral Activity of Inhibitors of Pyrimidine De-Novo Biosynthesis

1996 ◽  
Vol 7 (1) ◽  
pp. 7-13 ◽  
Author(s):  
M. Wachsman ◽  
F. M. Hamzeh ◽  
N. B. Assadi ◽  
P. S. Lietman
Keyword(s):  
Antiviral Activity ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
De Novo ◽  
Antiviral Effect ◽  
Pyrimidine Biosynthesis ◽  
Dihydroorotate Dehydrogenase ◽  
Dot Blot ◽  
De Novo Biosynthesis ◽  
Biosynthesis Inhibitors

Evaluation of the elevation of host cell biosynthesis of deoxynucleoside triphosphates (dNTP's) induced by human cytomegalovirus (HCMV) infection as a target for antiviral therapeutics was carried out. The concentrations of all four intracellular dNTP's rose rapidly following HCMV infection, and were markedly above baseline by 8 h post infection (p.i.). All four deoxynucleoside triphosphates remained elevated above baseline for at least 72 h p.i. The effects of inhibitors of the de-novo pathway of pyrimidine biosynthesis on HCMV viral replication-were quantified by DNA dot blot. All pyrimidine biosynthesis inhibitors examined inhibited the HCMV DNA replication at concentrations that were non-toxic to the cell. These drugs were also more effective against HCMV, which is highly dependent on host denovo synthesis, than against HSV-1 which encodes enzymes capable of increasing the supply of dNTP's. The antiviral effect of brequinar, an inhibitor of one of the enzymes of the de-novo pathway (dihydroorotate dehydrogenase), was examined to determine if it coincided with a decrease in dNTP's. HCMV-infected fibroblasts and uninfected control cells were treated with a concentration of brequinar able to inhibit HCMV DNA levels 90%. It was found that brequinar markedly lowered the levels of dTTP found in treated cells compared to untreated cells in both HCMV-infected and uninfected cells.

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