The Structures of Human Dihydroorotate Dehydrogenase with and without Inhibitor Reveal Conformational Flexibility in the Inhibitor and Substrate Binding Sites

Walse, Björn; Dufe, Veronica Tamu; Svensson, Bo; Fritzson, Ingela; Dahlberg, Leif; Khairoullina, Alfia; Wellmar, Ulf; Al-Karadaghi, Salam

doi:10.1021/bi8003318

Cited by 69 publications

(71 citation statements)

References 46 publications

(64 reference statements)

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“…For example, brequinar is an inhibitor of dihydrooratate dehydrogenase (DHODH), an enzyme component of the de novo pyrimidine biosynthesis pathway. 15 Clustering analysis identified a compound with an extremely similar activity profile, terprenin, that is described in the literature as simply an ''immunosuppressive,'' with no mention as to its cellular target 16 (Fig. 7A).…”

Section: Applicationdrelating Activity Profiles To Cellular Moasmentioning

confidence: 97%

Pathway Reporter Assays Reveal Small Molecule Mechanisms of Action

King

Selinger

Mapa

et al. 2009

JALA: Journal of the Association for Laboratory Automation

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C ell-based, phenotypic screening of small molecules often identifies compounds with provocative biological properties. However, determining the cellular target(s) and/or mechanism of action (MoA) of lead compounds remains an extremely challenging and timeconsuming exercise. To provide insights into a compound's cellular action and greatly reduce the time required for MoA determination, we have developed a screening platform consisting of an extensive series of reporter gene assays (RGAs). A collection of O11,000 compounds of known MoA (e.g., World Drug Index entries) were screened against the entire panel. The output provided evidence that an RGA signature could be ascribed to numerous, biologically diverse MoAs. The reference database generated suggested novel biological activity for particular compounds. For example, the profiling data led to the prediction that the cellular target of the natural product terprenin was dihydroorotate dehydrogenase (DHODH), which was confirmed experimentally. The screening methodology developed for this endeavor renders it amenable to the future examination of compounds with unknown MoA, in an automated, inexpensive, and time-efficient manner. ( JALA 2009;14:374-82)

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Section: Applicationdrelating Activity Profiles To Cellular Moasmentioning

confidence: 97%

Pathway Reporter Assays Reveal Small Molecule Mechanisms of Action

King

Selinger

Mapa

et al. 2009

JALA: Journal of the Association for Laboratory Automation

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“…Subsequently a number of additional X-ray structures of the human enzyme bound to various inhibitors have been reported (e.g. [63–65]), and the X-ray structure of Pf DHODH has been solved bound to both A77 1726 [59] and to novel malarial inhibitors from the triazolopyrimidine series [66], the latter of which will be discussed in detail below. The central structural element of DHODH from all class types is the core β/α-barrel domain.…”

Section: Structural Analysis Of Dhodhmentioning

confidence: 99%

Plasmodium Dihydroorotate Dehydrogenase: A Promising Target for Novel Anti-Malarial Chemotherapy

Phillips¹,

Rathod²

2010

IDDT

192

149

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Malaria remains a globally prevalent infectious disease that leads to significant morbidity and mortality. While there are a number of drugs approved for its treatment, drug resistance has compromised most of them, making the development of new drugs for the treatment and prevention of malaria essential. The completion of the Plasmodium falciparum genome and a growing understanding of parasite biology are fueling the search for novel drug targets. Despite this, few targets have been chemically validated in vivo. The pyrimidine biosynthetic pathway illustrates one of the best examples of successful identification of anti-malarial drug targets. This review focuses on recent studies to exploit the fourth enzyme in the de novo pyrimidine biosynthetic pathway of P. falciparum, dihydroorotate dehydrogenase (PfDHODH), as a new target for drug discovery. Several chemical scaffolds have been identified by high throughput screening as potent inhibitors of PfDHODH and these show strong selectivity for the malarial enzyme over that from the human host. Potent activity against parasites in whole cell models with good correlation between activity on the enzyme and the parasite have also been observed for a number of the identified series. Lead optimization of a triazolopyrimidine-based series has identified an analog with prolonged plasma exposure, that is orally bioavailable, and which shows good efficacy against the in vivo mouse model of the disease. These data provide strong evidence that PfDHODH is a validated target for the identification of new antimalarial chemotherapy. The challenge remains to identify compounds with the necessary combination of potency and metabolic stability to allow identification of a clinical candidate. KeywordsMalaria; Plasmodium; pyrimidine biosynthesis; dihydroorotate dehydrogenase; drug discovery Malaria the disease, and the current state of chemotherapyMalaria is a global disease caused by protozoan Apicomplexan parasites of the Plasmodium species and is transmitted by the Anopheles mosquito [1,2]. It is endemic in 90 countries, putting over 2 billion people at risk and leading to the deaths of 1-2 million people annually. Pregnant woman and children are the most susceptible to severe disease. Until the early 1950s malaria was endemic in parts of the US and Europe, but it was eliminated through extensive control programs. It is now found primarily in tropical and subtropical countries with the African population most at risk. A number of anti-malarial agents are in clinical use, however the development of resistance to both chloroquine and sulfadoxine-pyrimethamine led to the * Author to whom all correspondence should be addressed. margaret.phillips@UTSouthwestern.edu; Tel: (214) 645-6164. NIH Public Access Author ManuscriptInfect Disord Drug Targets. Author manuscript; available in PMC 2010 June 10. Published in final edited form as:Infect Disord Drug Targets. 2010 June ; 10(3): 226-239. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript rapid increase in the...

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“…After the release of orotate, ubiquinone binds to a second site and receives an electron from the co-substrate. The orotate synthesized by DHODH is converted into UMP (uridine monophosphate) by the enzyme complex UMPS (UMP synthase) [9,10]. …”

Section: Introductionmentioning

confidence: 99%

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients

et al. 2012

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Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.

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The Structures of Human Dihydroorotate Dehydrogenase with and without Inhibitor Reveal Conformational Flexibility in the Inhibitor and Substrate Binding Sites

Cited by 69 publications

References 46 publications

Pathway Reporter Assays Reveal Small Molecule Mechanisms of Action

Pathway Reporter Assays Reveal Small Molecule Mechanisms of Action

Plasmodium Dihydroorotate Dehydrogenase: A Promising Target for Novel Anti-Malarial Chemotherapy

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients

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