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1.
ACS Infect Dis ; 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38920250

RESUMO

The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.

2.
Int J Parasitol Drugs Drug Resist ; 25: 100536, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38663046

RESUMO

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC50 = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit ß-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.

4.
PLoS Pathog ; 19(7): e1011517, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37471441

RESUMO

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.


Assuntos
Parasitos , Toxoplasma , Toxoplasmose , Animais , Humanos , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons , Toxoplasmose/parasitologia , Plasmodium falciparum
5.
Artigo em Inglês | MEDLINE | ID: mdl-37004488

RESUMO

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.


Assuntos
Parasitos , Toxoplasma , Toxoplasmose , Tuberculose , Humanos , Animais , Toxoplasma/genética , Toxoplasmose/tratamento farmacológico , Coenzima A
6.
Angew Chem Int Ed Engl ; 62(17): e202300221, 2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-36757665

RESUMO

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.


Assuntos
Mycobacterium tuberculosis , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Nucleotidiltransferases/metabolismo , Antituberculosos/farmacologia
7.
Angew Chem Weinheim Bergstr Ger ; 135(17): e202300221, 2023 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38515507

RESUMO

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

8.
PLoS Pathog ; 17(7): e1009797, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34324601

RESUMO

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmodium falciparum/enzimologia , Toxoplasma/enzimologia , Isoenzimas , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
9.
ACS Infect Dis ; 7(6): 1666-1679, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33939919

RESUMO

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.


Assuntos
Mycobacterium tuberculosis , Peptídeo Sintases/antagonistas & inibidores , Coenzima A , Cisteína/análogos & derivados , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Ácido Pantotênico/análogos & derivados , Peptídeo Sintases/genética
10.
J Med Chem ; 64(8): 4478-4497, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33792339

RESUMO

Malaria-causing Plasmodium parasites are developing resistance to antimalarial drugs, providing the impetus for new antiplasmodials. Although pantothenamides show potent antiplasmodial activity, hydrolysis by pantetheinases/vanins present in blood rapidly inactivates them. We herein report the facile synthesis and biological activity of a small library of pantothenamide analogues in which the labile amide group is replaced with a heteroaromatic ring. Several of these analogues display nanomolar antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase. Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound profile likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.


Assuntos
Antimaláricos/química , Isoxazóis/química , Ácido Pantotênico/análogos & derivados , Tiadiazóis/química , Triazóis/química , Animais , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Estabilidade de Medicamentos , Eritrócitos/citologia , Eritrócitos/parasitologia , Feminino , Meia-Vida , Humanos , Malária Falciparum/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Ácido Pantotênico/química , Ácido Pantotênico/metabolismo , Ácido Pantotênico/farmacologia , Ácido Pantotênico/uso terapêutico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium knowlesi/efeitos dos fármacos , Relação Estrutura-Atividade
11.
Nat Commun ; 12(1): 143, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420031

RESUMO

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Carboxiliases/antagonistas & inibidores , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Peptídeo Sintases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Carboxiliases/genética , Carboxiliases/metabolismo , Carboxiliases/ultraestrutura , Coenzima A/biossíntese , Cristalografia por Raios X , Ensaios Enzimáticos , Técnicas de Silenciamento de Genes , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeo Sintases/ultraestrutura , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
12.
ACS Infect Dis ; 6(7): 1844-1854, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32375471

RESUMO

Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the ß-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure-activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2'R-diastereomer is far more potent than the 2R,2'R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2'R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2'R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.


Assuntos
Antimaláricos , Antimaláricos/farmacologia , Coenzima A/metabolismo , Humanos , Ácido Pantotênico/análogos & derivados , Relação Estrutura-Atividade
13.
Biochem J ; 476(21): 3125-3139, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31488574

RESUMO

CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility-mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.


Assuntos
Carboxiliases/química , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Espectrometria de Massas/métodos , Complexos Multienzimáticos/química , Peptídeo Sintases/química , Carboxiliases/antagonistas & inibidores , Carboxiliases/metabolismo , Dimerização , Inibidores Enzimáticos/química , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Cinética , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/metabolismo , Domínios Proteicos
14.
ChemMedChem ; 13(24): 2677-2683, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30370998

RESUMO

Pantothenamides are potent growth inhibitors of the malaria parasite Plasmodium falciparum. Their clinical use is, however, hindered due to the ubiquitous presence of pantetheinases in human serum, which rapidly degrade pantothenamides into pantothenate and the corresponding amine. We previously reported that replacement of the labile amide bond with a triazole ring not only imparts stability toward pantetheinases, but also improves activity against P. falciparum. A small library of new triazole derivatives was synthesized, and their use in establishing structure-activity relationships relevant to antiplasmodial activity of this family of compounds is discussed herein. Overall it was observed that 1,4-substitution on the triazole ring and use of an unbranched, one-carbon linker between the pantoyl group and the triazole are optimal for inhibition of intraerythrocytic P. falciparum growth. Our results imply that the triazole ring may mimic the amide bond with an orientation different from what was previously suggested for this amide bioisostere.


Assuntos
Amidas/síntese química , Antimaláricos/síntese química , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/síntese química , Plasmodium falciparum/efeitos dos fármacos , Triazóis/síntese química , Amidas/farmacologia , Antimaláricos/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Ácido Pantotênico/farmacologia , Relação Estrutura-Atividade , Triazóis/farmacologia
15.
PLoS Pathog ; 14(4): e1006918, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29614109

RESUMO

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Malária/tratamento farmacológico , Mutação , Ácido Pantotênico/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmodium falciparum/efeitos dos fármacos , Animais , Coenzima A/biossíntese , Eritrócitos/parasitologia , Malária/parasitologia , Ácido Pantotênico/farmacologia , Testes de Sensibilidade Parasitária , Fosforilação , Proteínas de Protozoários/genética
16.
Eur J Med Chem ; 143: 1139-1147, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29233590

RESUMO

Survival of the human malaria parasite Plasmodium falciparum is dependent on pantothenate (vitamin B5), a precursor of the fundamental enzyme cofactor coenzyme A. CJ-15,801, an enamide analogue of pantothenate isolated from the fungus Seimatosporium sp. CL28611, was previously shown to inhibit P. falciparum proliferation in vitro by targeting pantothenate utilization. To inform the design of next generation analogues, we set out to synthesize and test a series of synthetic enamide-bearing pantothenate analogues. We demonstrate that conservation of the R-pantoyl moiety and the trans-substituted double bond of CJ-15,801 is important for the selective, on-target antiplasmodial effect, while replacement of the carboxyl group is permitted, and, in one case, favored. Additionally, we show that the antiplasmodial potency of CJ-15,801 analogues that retain the R-pantoyl and trans-substituted enamide moieties correlates with inhibition of P. falciparum pantothenate kinase (PfPanK)-catalyzed pantothenate phosphorylation, implicating the interaction with PfPanK as a key determinant of antiplasmodial activity.


Assuntos
Antimaláricos/farmacologia , Ácido Pantotênico/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/síntese química , Antimaláricos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Ácido Pantotênico/síntese química , Ácido Pantotênico/química , Ácido Pantotênico/farmacologia , Testes de Sensibilidade Parasitária , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmodium falciparum/enzimologia , Relação Estrutura-Atividade
17.
ACS Infect Dis ; 3(7): 527-541, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28437604

RESUMO

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural ß-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites and PanAm phosphorylation by PfPanK present in parasite lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the ß-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.


Assuntos
Antimaláricos/farmacologia , Coenzima A/antagonistas & inibidores , Ácido Pantotênico/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , beta-Alanina/farmacologia , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimetabólitos/metabolismo , Antimetabólitos/farmacologia , Biotransformação , Radioisótopos de Carbono , Coenzima A/biossíntese , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Cinética , Modelos Moleculares , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/metabolismo , Testes de Sensibilidade Parasitária , Fosforilação , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , beta-Alanina/análogos & derivados , beta-Alanina/metabolismo
18.
Biochem Soc Trans ; 42(4): 1087-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25110007

RESUMO

Malaria kills more than half a million people each year. There is no vaccine, and recent reports suggest that resistance is developing to the antimalarial regimes currently recommended by the World Health Organization. New drugs are therefore needed to ensure malaria treatment options continue to be available. The intra-erythrocytic stage of the malaria parasite's life cycle is dependent on an extracellular supply of pantothenate (vitamin B5), the precursor of CoA (coenzyme A). It has been known for many years that proliferation of the parasite during this stage of its life cycle can be inhibited with pantothenate analogues. We have shown recently that pantothenamides, a class of pantothenate analogues with antibacterial activity, inhibit parasite proliferation at submicromolar concentrations and do so competitively with pantothenate. These compounds, however, are degraded, and therefore rendered inactive, by the enzyme pantetheinase (vanin), which is present in serum. In the present mini-review, we discuss the two strategies that have been put forward to overcome pantetheinase-mediated degradation of pantothenamides. The strategies effectively provide an opportunity for pantothenamides to be tested in vivo. We also put forward our 'blueprint' for the further development of pantothenamides (and other pantothenate analogues) as potential antimalarials.


Assuntos
Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Amidoidrolases/metabolismo , Animais , Coenzima A/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Malária/tratamento farmacológico , Malária/metabolismo , Ácido Pantotênico/metabolismo , Plasmodium falciparum/efeitos dos fármacos
19.
Anal Biochem ; 451: 76-8, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24333332

RESUMO

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.


Assuntos
Miniaturização , Fósforo/análise , Radiometria , Compostos de Bário/química , Precipitação Química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Ácido Pantotênico/química , Ácido Pantotênico/metabolismo , Fósforo/química , Radioisótopos de Fósforo/química , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sulfato de Zinco/química
20.
PLoS One ; 8(2): e54974, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23405100

RESUMO

Growth of the virulent human malaria parasite Plasmodium falciparum is dependent on an extracellular supply of pantothenate (vitamin B(5)) and is susceptible to inhibition by pantothenate analogues that hinder pantothenate utilization. In this study, on the hunt for pantothenate analogues with increased potency relative to those reported previously, we screened a series of pantothenamides (amide analogues of pantothenate) against P. falciparum and show for the first time that analogues of this type possess antiplasmodial activity. Although the active pantothenamides in this series exhibit only modest potency under standard in vitro culture conditions, we show that the potency of pantothenamides is selectively enhanced when the parasite culture medium is pre-incubated at 37°C for a prolonged period. We present evidence that this finding is linked to the presence in Albumax II (a serum-substitute routinely used for in vitro cultivation of P. falciparum) of pantetheinase activity: the activity of an enzyme that hydrolyzes the pantothenate metabolite pantetheine, for which pantothenamides also serve as substrates. Pantetheinase activity, and thereby pantothenamide degradation, is reduced following incubation of Albumax II-containing culture medium for a prolonged period at 37°C, revealing the true, sub-micromolar potency of pantothenamides. Importantly we show that the potent antiplasmodial effect of pantothenamides is attenuated with pantothenate, consistent with the compounds inhibiting parasite proliferation specifically by inhibiting pantothenate and/or CoA utilization. Additionally, we show that the pantothenamides interact with P. falciparum pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A. This is the first demonstration of on-target antiplasmodial pantothenate analogues with sub-micromolar potency, and highlights the potential of pantetheinase-resistant pantothenamides as antimalarial agents.


Assuntos
Amidas/farmacologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Antimaláricos/farmacologia , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Amidoidrolases/farmacologia , Células Cultivadas , Coenzima A/metabolismo , Eritrócitos/parasitologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/farmacologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes/farmacologia
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