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1.
Proc Natl Acad Sci U S A ; 119(51): e2213116119, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36512492

RESUMO

New antimicrobials are needed for the treatment of extensively drug-resistant Acinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that A. baumannii DHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on AbDHODH. The most potent (DSM186, DHODH IC50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse A. baumannii strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of AbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of AbDHODH as a drug target for the development of antimicrobials for the treatment of A. baumannii and potentially other high-risk bacterial infections.


Assuntos
Acinetobacter baumannii , Humanos , Camundongos , Animais , Di-Hidro-Orotato Desidrogenase , Testes de Sensibilidade Microbiana , Meropeném , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico
2.
Antimicrob Agents Chemother ; 68(2): e0068423, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38193705

RESUMO

Due to the spread of resistance to front-line artemisinin derivatives worldwide, there is a need for new antimalarials. Tartrolon E (TrtE), a secondary metabolite of a symbiotic bacterium of marine bivalve mollusks, is a promising antimalarial because it inhibits the growth of sexual and asexual blood stages of Plasmodium falciparum at sub-nanomolar levels. The potency of TrtE warrants further investigation into its mechanism of action, cytotoxicity, and ease with which parasites may evolve resistance to it.


Assuntos
Antimaláricos , Artemisininas , Lactonas , Malária Falciparum , Humanos , Plasmodium falciparum , Artemisininas/farmacologia , Antimaláricos/farmacologia , Malária Falciparum/parasitologia
3.
Antimicrob Agents Chemother ; 65(9): e0058621, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34152814

RESUMO

Malaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase (PfGyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear-encoded PfGyrA was disrupted using CRISPR/Cas9 gene editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of PfΔGyrA was possible in the presence of IPP. PfGyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis, but there is a need for a more detailed description of the mode of action of ciprofloxacin in malaria parasites. As predicted, PfΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin but not to the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued PfΔGyrA, possibly suggesting that ciprofloxacin may have an additional nonapicoplast target in P. falciparum. Overall, we confirm that PfGyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.


Assuntos
Antimaláricos , Apicoplastos , Apicoplastos/genética , DNA Girase/genética , Humanos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética
4.
J Biol Chem ; 290(33): 20313-24, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26055707

RESUMO

Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of bacterial infections and cancer, but the corresponding enzymes from malaria parasites remain understudied. This is due to the general challenges of producing malaria proteins in functional forms in heterologous expression systems. Here, we express full-length Plasmodium falciparum topoisomerase II (PfTopoII) in a wheat germ cell-free transcription-translation system. Functional activity of soluble PfTopoII from the translation lysates was confirmed through both a plasmid relaxation and a DNA decatenation activity that was dependent on magnesium and ATP. To facilitate future drug discovery, a convenient and sensitive fluorescence assay was established to follow DNA decatenation, and a stable, truncated PfTopoII was engineered for high level enzyme production. PfTopoII was purified using a DNA affinity column. Existing TopoII inhibitors previously developed for other non-malaria indications inhibited PfTopoII, as well as malaria parasites in culture at submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin, and etoposide exhibited 15-, 57-, and 3-fold selectivity for the malarial enzyme over human TopoII. Finally, it was possible to use the purified PfTopoII to dissect the different modes by which these varying classes of TopoII inhibitors could trap partially processed DNA. The present biochemical advancements will allow high throughput chemical screening of compound libraries and lead optimization to develop new lines of antimalarials.


Assuntos
DNA Topoisomerases Tipo II/metabolismo , Plasmodium falciparum/enzimologia , Inibidores da Topoisomerase II/farmacologia , Sequência de Aminoácidos , Animais , Sistema Livre de Células , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/efeitos dos fármacos , DNA Topoisomerases Tipo II/isolamento & purificação , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Triticum/genética
5.
J Med Chem ; 64(9): 6085-6136, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876936

RESUMO

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 (1) suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity in vitro against blood and liver schizont stages and show good efficacy in Plasmodium falciparum SCID mouse models. They are equally active against P. falciparum and Plasmodium vivax field isolates and are selective for Plasmodium DHODHs versus mammalian enzymes.


Assuntos
Antimaláricos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirróis/farmacologia , Animais , Antimaláricos/química , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/química , Camundongos , Plasmodium falciparum/efeitos dos fármacos , Pirróis/química , Relação Estrutura-Atividade
6.
J Med Chem ; 63(9): 4929-4956, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32248693

RESUMO

Malaria puts at risk nearly half the world's population and causes high mortality in sub-Saharan Africa, while drug resistance threatens current therapies. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated target for malaria treatment based on our finding that triazolopyrimidine DSM265 (1) showed efficacy in clinical studies. Herein, we describe optimization of a pyrrole-based series identified using a target-based DHODH screen. Compounds with nanomolar potency versus Plasmodium DHODH and Plasmodium parasites were identified with good pharmacological properties. X-ray studies showed that the pyrroles bind an alternative enzyme conformation from 1 leading to improved species selectivity versus mammalian enzymes and equivalent activity on Plasmodium falciparum and Plasmodium vivax DHODH. The best lead DSM502 (37) showed in vivo efficacy at similar levels of blood exposure to 1, although metabolic stability was reduced. Overall, the pyrrole-based DHODH inhibitors provide an attractive alternative scaffold for the development of new antimalarial compounds.


Assuntos
Antimaláricos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Malária Falciparum/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirróis/uso terapêutico , Animais , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimaláricos/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Masculino , Camundongos SCID , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/enzimologia , Ligação Proteica , Pirróis/síntese química , Pirróis/metabolismo , Pirróis/farmacocinética , Ratos , Relação Estrutura-Atividade
7.
ACS Omega ; 3(8): 9227-9240, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30197997

RESUMO

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.

8.
J Med Chem ; 59(11): 5416-31, 2016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-27127993

RESUMO

Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.


Assuntos
Antimaláricos/farmacologia , Inibidores Enzimáticos/farmacologia , Malária Falciparum/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Pirimidinas/farmacologia , Triazóis/farmacologia , Animais , Antimaláricos/síntese química , Antimaláricos/química , Di-Hidro-Orotato Desidrogenase , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos SCID , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química
9.
ACS Infect Dis ; 2(12): 945-957, 2016 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-27641613

RESUMO

The emergence of drug-resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria, and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF5-aniline moiety of DSM265 with a series of CF3-pyridinyls while maintaining the core triazolopyrimidine scaffold. This effort led to the identification of DSM421, which has improved solubility, lower intrinsic clearance, and increased plasma exposure after oral dosing compared to DSM265, while maintaining a long predicted human half-life. Its improved physical and chemical properties will allow it to be formulated more readily than DSM265. DSM421 showed excellent efficacy in the SCID mouse model of P. falciparum malaria that supports the prediction of a low human dose (<200 mg). Importantly DSM421 showed equal activity against both P. falciparum and P. vivax field isolates, while DSM265 was more active on P. falciparum. DSM421 has the potential to be developed as a single-dose cure or once-weekly chemopreventative for both P. falciparum and P. vivax malaria, leading to its advancement as a preclinical development candidate.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Malária Falciparum/prevenção & controle , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Antimaláricos/química , Antimaláricos/farmacocinética , Di-Hidro-Orotato Desidrogenase , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pirimidinas/química , Ratos , Relação Estrutura-Atividade
10.
Sci Transl Med ; 7(296): 296ra111, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26180101

RESUMO

Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.


Assuntos
Antimaláricos/química , Inibidores Enzimáticos/química , Malária Falciparum/tratamento farmacológico , Malária Falciparum/prevenção & controle , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirimidinas/química , Triazóis/química , Administração Oral , Animais , Antimaláricos/farmacocinética , Área Sob a Curva , Células CACO-2 , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Plasmodium falciparum , Pirimidinas/farmacocinética , Coelhos , Especificidade por Substrato , Triazóis/farmacocinética
11.
J Med Chem ; 57(12): 5381-94, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24801997

RESUMO

Malaria is one of the most serious global infectious diseases. The pyrimidine biosynthetic enzyme Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is an important target for antimalarial chemotherapy. We describe a detailed analysis of protein-ligand interactions between DHODH and a triazolopyrimidine-based inhibitor series to explore the effects of fluorine on affinity and species selectivity. We show that increasing fluorination dramatically increases binding to mammalian DHODHs, leading to a loss of species selectivity. Triazolopyrimidines bind Plasmodium and mammalian DHODHs in overlapping but distinct binding sites. Key hydrogen-bond and stacking interactions underlying strong binding to PfDHODH are absent in the mammalian enzymes. Increasing fluorine substitution leads to an increase in the entropic contribution to binding, suggesting that strong binding to mammalian DHODH is a consequence of an enhanced hydrophobic effect upon binding to an apolar pocket. We conclude that hydrophobic interactions between fluorine and hydrocarbons provide significant binding energy to protein-ligand interactions. Our studies define the requirements for species-selective binding to PfDHODH and show that the triazolopyrimidine scaffold can alternatively be tuned to inhibit human DHODH, an important target for autoimmune diseases.


Assuntos
Antimaláricos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Pirimidinas/química , Tiazóis/química , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Cães , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Plasmodium falciparum/efeitos dos fármacos , Pirimidinas/síntese química , Pirimidinas/farmacologia , Ratos , Especificidade da Espécie , Relação Estrutura-Atividade , Termodinâmica , Tiazóis/síntese química , Tiazóis/farmacologia
12.
J Med Chem ; 55(17): 7425-36, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22877245

RESUMO

Plasmodium falciparum causes approximately 1 million deaths annually. However, increasing resistance imposes a continuous threat to existing drug therapies. We previously reported a number of potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase that inhibit parasite in vitro growth with similar activity. Lead optimization of this series led to the recent identification of a preclinical candidate, showing good activity against P. falciparum in mice. As part of a backup program around this scaffold, we explored heteroatom rearrangement and substitution in the triazolopyrimidine ring and have identified several other ring configurations that are active as PfDHODH inhibitors. The imidazo[1,2-a]pyrimidines were shown to bind somewhat more potently than the triazolopyrimidines depending on the nature of the amino aniline substitution. DSM151, the best candidate in this series, binds with 4-fold better affinity (PfDHODH IC(50) = 0.077 µM) than the equivalent triazolopyrimidine and suppresses parasites in vivo in the Plasmodium berghei model.


Assuntos
Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Pirimidinas/farmacologia , Animais , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/química , Camundongos , Mimetismo Molecular , Plasmodium berghei/efeitos dos fármacos , Pirimidinas/química , Relação Estrutura-Atividade
13.
J Med Chem ; 54(15): 5540-61, 2011 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-21696174

RESUMO

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential antimalarials, we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound toward clinical candidate status.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirimidinas/química , Triazóis/química , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Fenômenos Químicos , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Resistência a Medicamentos , Humanos , Camundongos , Plasmodium falciparum/enzimologia , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/farmacocinética , Triazóis/farmacologia
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