Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protist<i>Trypanosoma cruzi</i>

Matoba, Kazuaki; Nara, Takeshi; Aoki, Takashi; Honma, Teruki; Tanaka, Akiko; Inoue, M.; Matsuoka, Shigeru; Inaoka, Daniel Ken; Kita, Kiyoshi; Harada, Shigeharu

doi:10.1107/s1744309109031959

Cited by 7 publications

(6 citation statements)

References 27 publications

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“…The kinetic properties of PfATC-Met3 as well as PfATC-RK were investigated according to Mü ller et al (2010), Motomizu et al (1983) and Matoba et al (2009) with minor modifications. Briefly, the reaction was carried out at room temperature in a total volume of 160 ml of 14 mM l-aspartate and 1 mM carbamoyl phosphate (CP) saturated substrate solution in 200 mM Tris-acetate buffer pH 8.…”

Section: Activity Assaysmentioning

confidence: 99%

Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

et al. 2016

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The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5 Å resolution crystal structure of PfATC is reported. The space group of the PfATC crystals was determined to be monoclinic P21, with unit-cell parameters a = 87.0, b = 103.8, c = 87.1 Å, α = 90.0, β = 117.7, γ = 90.0°. The presented PfATC model shares a high degree of homology with the catalytic domain of Escherichia coli ATC. There is as yet no evidence of the existence of a regulatory domain in PfATC. Similarly to E. coli ATC, PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.

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Section: Activity Assaysmentioning

confidence: 99%

Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

et al. 2016

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“…Homotrimeric structure of TcATCase has been shown by its crystal structure which diffracted at 2.8 Å and is also supported by gel filtration chromatography and dynamic light scattering studies. In the crystal structure of TcATCase the substrate (carbamoyl phosphate) was bound to loop residues (Cys85-Thr95) which otherwise exhibited a disordered arrangement in the ligand free state [ 26 ].…”

Section: Enzymes Of the Pyrimidine Pathway Of Trypanosomatidsmentioning

confidence: 99%

Fresh insights into the pyrimidine metabolism in the trypanosomatids

Tiwari

Dubey

2018

Parasites Vectors

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The trypanosomatid parasites continue their killing spree resulting in significant annual mortality due to the lack of effective treatments and the prominence of these diseases in poorer countries. These dimorphic parasites thrive unchecked in the host system, outsmarting the immune mechanisms. An understanding of biology of these parasitic forms will help in the management and elimination of these fatal diseases. Investigation of various metabolic pathways in these parasites has shed light in the understanding of the unique biology of the trypansomatids. An understanding of these pathways have helped in tracing the soft targets in the metabolic pathways, which could be used as effective drug targets which would further impact the therupeutic implications. Pyrimidine pathway is a vital metabolic pathway which yields in the formation of pyrimidines, which are then integrated in nucleic acids (DNA and RNA) in sugars (UDP sugars) and lipids (CDP lipids). A wealth of data and information has been generated in the past decades by in-depth analyses of pyrimidine pathway in the trypanosomatid parasites, which can aid in the identification of anomalies between the parasitic and host counterpart which could be further harnessed to develop therapeutic interventions for the treatment of parasitic diseases. This review presents an updated and comprehensive detailing of the pyrimidine metabolism in the trypansomatids, their uniqueness and their distinctions, and its possible outcomes that would aid in the eradication of these parasitic diseases.

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“…In this regard, one of the most relevant goals to design new drugs is selecting targets and mechanistic pathways other than those described for current drugs, thereby limiting resistance. Three strategies have been proposed for the development of new drugs used for the treatment of neglected diseases: the first strategy is based on the development of compounds that interfere with or modulate key, vital proteins or enzymes for parasites, such as peptidases [16,17], enzymes that catalyze purines and pyrimidines such as aspartate transcarbamoylase [18], trypanothione reductase (TR) and the iron-containing superoxide dismutase (Fe-SOD), which protect the parasite against oxidative damage by reactive oxygen species [19]. Additionally, human proteins that have low identity with their orthologous proteins in parasites are the subject of studies intended to carry out screening through docking and molecular dynamics of modulator compounds of the activity of such proteins [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

In Silico, In Vitro, and Pharmacokinetic Studies of UBMC-4, a Potential Novel Compound for Treating against Trypanosoma cruzi

et al. 2022

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The lack of therapeutic alternatives for the treatment of Chagas disease, a neglected disease, drives the discovery of new drugs with trypanocidal activity. Consequently, we conducted in vitro studies using UBMC-4, a potential Trypanosoma cruzi AKT-like pleckstrin homology (PH) domain inhibitory compound found using bioinformatics tools. The half effective concentration (EC50) on intracellular amastigotes was determined at 1.85 ± 1 μM showing low cytotoxicity (LC50) > 40 μM on human cell lines tested. In order to study the lethal effect caused by the compound on epimastigotes, morphological changes were assessed by scanning and transmission electron microscopy. Progressive alterations such as flagellum inactivation, cell size reduction, nuclear structure alteration, condensation of chromatin towards the nuclear periphery, vacuole formation, and mitochondrial swelling with kinetoplast integrity loss were evidenced. In addition, apoptosis-like markers in T. cruzi were assessed by flow cytometry, demonstrating that the effect of UBMC-4 on T. cruzi AKT-like kinase reduced the tolerance to nutritional stress-triggered, apoptosis-like events, including DNA fragmentation, mitochondrial damage, and loss of plasma membrane integrity. After this, UBMC-4 was formulated for oral administration and pharmacokinetics were analyzed in a mouse model. Finally, upon oral administration of 200 mg/kg in mice, we found that a UBMC-4 plasma concentration remaining in circulation beyond 24 h after administration is well described by the two-compartment model. We conclude that UBMC-4 has an effective trypanocidal activity in vitro at low concentrations and this effect is evident in T. cruzi cell structures. In mice, UBMC-4 was well absorbed and reached plasma concentrations higher than the EC50, showing features that would aid in developing a new drug to treat Chagas disease.

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Crystallization and preliminary X-ray analysis of aspartate transcarbamoylase from the parasitic protistTrypanosoma cruzi

Cited by 7 publications

References 27 publications

Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

Crystal structure of truncated aspartate transcarbamoylase fromPlasmodium falciparum

Fresh insights into the pyrimidine metabolism in the trypanosomatids

In Silico, In Vitro, and Pharmacokinetic Studies of UBMC-4, a Potential Novel Compound for Treating against Trypanosoma cruzi

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