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1.
ACS Infect Dis ; 6(5): 986-999, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32129597

RESUMO

Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.


Assuntos
Antimaláricos , Aspartato Carbamoiltransferase/genética , Naftiridinas/farmacologia , Plasmodium falciparum , Proteínas de Protozoários/genética , Antimaláricos/farmacologia , Ácido Aspártico , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética
2.
Expert Opin Drug Discov ; 15(2): 189-202, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31959021

RESUMO

Introduction: Malaria is one of the most prevalent human infections worldwide with over 40% of the world's population living in malaria-endemic areas. In the absence of an effective vaccine, emergence of drug-resistant strains requires urgent drug development. Current methods applied to drug target validation, a crucial step in drug discovery, possess limitations in malaria. These constraints require the development of techniques capable of simplifying the validation of Plasmodial targets.Areas covered: The authors review the current state of the art in techniques used to validate drug targets in malaria, including our contribution - the protein interference assay (PIA) - as an additional tool in rapid in vivo target validation.Expert opinion: Each technique in this review has advantages and disadvantages, implying that future validation efforts should not focus on a single approach, but integrate multiple approaches. PIA is a significant addition to the current toolset of antimalarial validation. Validation of aspartate metabolism as a druggable pathway provided proof of concept of how oligomeric interfaces can be exploited to control specific activity in vivo. PIA has the potential to be applied not only to other enzymes/pathways of the malaria parasite but could, in principle, be extrapolated to other infectious diseases.


Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas , Malária/tratamento farmacológico , Animais , Desenvolvimento de Medicamentos , Humanos , Malária/parasitologia , Plasmodium/isolamento & purificação , Plasmodium/parasitologia , Estudos de Validação como Assunto
3.
Microbiologyopen ; 8(7): e00779, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30821109

RESUMO

The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

4.
Biochem Biophys Res Commun ; 497(3): 835-842, 2018 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-29476738

RESUMO

Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.


Assuntos
Antiparasitários/química , Antiparasitários/farmacologia , Aspartato Carbamoiltransferase/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Aspartato Carbamoiltransferase/química , Aspartato Carbamoiltransferase/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Simulação de Acoplamento Molecular , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos
5.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 7): 523-33, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27380369

RESUMO

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 Šresolution 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, ß = 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.


Assuntos
Aspartato Carbamoiltransferase/química , Ácido Aspártico/química , Carbamoil-Fosfato/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Carbamoil-Fosfato/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Modelos Moleculares , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Plasmodium falciparum/enzimologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
Biomed Res Int ; 2015: 351289, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25722976

RESUMO

Apicomplexan parasites cause infectious diseases that are either a severe public health problem or an economic burden. In this paper we will shed light on how oxidative stress can influence the host-pathogen relationship by focusing on three major diseases: babesiosis, coccidiosis, and toxoplasmosis.


Assuntos
Babesia/metabolismo , Cryptosporidium/metabolismo , Estresse Oxidativo , Toxoplasma/metabolismo , Animais , Babesia/patogenicidade , Babesiose/metabolismo , Babesiose/parasitologia , Babesiose/patologia , Coccidiose/metabolismo , Coccidiose/parasitologia , Coccidiose/patologia , Cryptosporidium/patogenicidade , Interações Hospedeiro-Parasita , Humanos , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Toxoplasmose/patologia
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