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1.
Biochemistry ; 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38306231

RESUMO

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

2.
Am J Hum Genet ; 104(4): 625-637, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30879639

RESUMO

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.


Assuntos
Doença de Fabry/genética , Doença de Fabry/terapia , RNA Mensageiro/uso terapêutico , alfa-Galactosidase/genética , Animais , Modelos Animais de Doenças , Endocitose , Terapia de Reposição de Enzimas , Terapia Genética , Humanos , Lipídeos/química , Lisossomos/metabolismo , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/farmacocinética , Distribuição Tecidual , Triexosilceramidas/metabolismo
3.
Bioorg Med Chem Lett ; 41: 127974, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771585

RESUMO

Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.


Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Éteres/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Compostos de Anilina/química , Antineoplásicos/química , Linhagem Celular Tumoral , Éteres/química , Humanos , L-Lactato Desidrogenase/química
4.
5.
Nat Commun ; 11(1): 5339, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087718

RESUMO

Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.


Assuntos
Terapia de Reposição de Enzimas/métodos , Acidemia Propiônica/terapia , RNA Mensageiro/uso terapêutico , Animais , Modelos Animais de Doenças , Glutamatos/uso terapêutico , Humanos , Cinética , Lipídeos/química , Fígado/enzimologia , Metilmalonil-CoA Descarboxilase/química , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/enzimologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Acidemia Propiônica/genética , Acidemia Propiônica/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética
6.
EBioMedicine ; 45: 519-528, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303505

RESUMO

BACKGROUND: Isolated methylmalonic acidemia/aciduria (MMA) is an ultra-rare, serious, inherited metabolic disorder with significant morbidity and mortality. Exogenously delivered mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, is a potential therapy to produce functional MUT enzyme in liver. METHODS: Two 12-week repeat-dose studies were conducted to evaluate the efficacy and safety of intravenously-administered hMUT mRNA encapsulated in lipid nanoparticles in two murine models of MMA. FINDINGS: In MMA hypomorphic mice, hMUT mRNA treatment resulted in dose-dependent and reproducible biomarker responses after each dose. Enzymatically-active MUT protein was produced in liver in a dose-dependent manner. hMUT mRNA was well-tolerated with no adverse effects, as indicated by the lack of clinical observations, minimal changes in clinical chemistry parameters, and histopathology examination across all tissues. In severe MMA mice, hMUT mRNA led to substantially improved survival and growth and ameliorated biochemical abnormalities, all of which are cardinal clinical manifestations in severely affected patients. INTERPRETATION: These data demonstrate durable functional benefit of hMUT mRNA and support development of this new class of therapy for a devastating, pediatric disorder. FUND: This work was funded by Moderna, Inc.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Fígado/metabolismo , Metilmalonil-CoA Mutase/farmacologia , RNA Mensageiro/farmacologia , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Criança , Modelos Animais de Doenças , Humanos , Lipídeos/genética , Fígado/efeitos dos fármacos , Fígado/patologia , Metilmalonil-CoA Mutase/genética , Camundongos , RNA Mensageiro/genética
7.
Nat Med ; 24(12): 1899-1909, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30297912

RESUMO

Acute intermittent porphyria (AIP) results from haploinsufficiency of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthesis pathway. Patients with AIP have neurovisceral attacks associated with increased hepatic heme demand. Phenobarbital-challenged mice with AIP recapitulate the biochemical and clinical characteristics of patients with AIP, including hepatic overproduction of the potentially neurotoxic porphyrin precursors. Here we show that intravenous administration of human PBGD (hPBGD) mRNA (encoded by the gene HMBS) encapsulated in lipid nanoparticles induces dose-dependent protein expression in mouse hepatocytes, rapidly normalizing urine porphyrin precursor excretion in ongoing attacks. Furthermore, hPBGD mRNA protected against mitochondrial dysfunction, hypertension, pain and motor impairment. Repeat dosing in AIP mice showed sustained efficacy and therapeutic improvement without evidence of hepatotoxicity. Finally, multiple administrations to nonhuman primates confirmed safety and translatability. These data provide proof-of-concept for systemic hPBGD mRNA as a potential therapy for AIP.


Assuntos
Terapia Genética , Hidroximetilbilano Sintase/genética , Porfiria Aguda Intermitente/terapia , RNA Mensageiro/administração & dosagem , Animais , Modelos Animais de Doenças , Feminino , Haploinsuficiência/genética , Heme/genética , Heme/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Hidroximetilbilano Sintase/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Porfiria Aguda Intermitente/genética , Porfiria Aguda Intermitente/patologia , RNA Mensageiro/genética
8.
Cell Rep ; 21(12): 3548-3558, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29262333

RESUMO

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/terapia , Terapia Genética/métodos , Metilmalonil-CoA Mutase/genética , Nanopartículas/administração & dosagem , RNA Mensageiro/genética , Administração Intravenosa , Animais , Feminino , Humanos , Lipídeos/química , Fígado/metabolismo , Masculino , Metilmalonil-CoA Mutase/metabolismo , Camundongos , Nanopartículas/química , RNA Mensageiro/metabolismo
9.
J Med Chem ; 60(22): 9184-9204, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29120638

RESUMO

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.


Assuntos
Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Pirazóis/farmacologia , Tiazóis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Membranas Artificiais , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Permeabilidade , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacocinética , Ratos , Solubilidade , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Tiazóis/farmacocinética
10.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 5): 566-71, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25945710

RESUMO

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.


Assuntos
Aspartato Aminotransferases/química , Giardia lamblia/química , Leishmania major/química , Trypanosoma brucei brucei/química , Cristalização , Giardia lamblia/enzimologia , Leishmania major/enzimologia , Estrutura Secundária de Proteína , Trypanosoma brucei brucei/enzimologia
12.
J Med Chem ; 56(20): 7772-87, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24083782

RESUMO

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.


Assuntos
Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Alanina/análogos & derivados , Alanina/síntese química , Alanina/farmacocinética , Alanina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Compostos Heterocíclicos/farmacocinética , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Nus , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Oxazepinas/síntese química , Oxazepinas/farmacocinética , Oxazepinas/farmacologia , Estrutura Terciária de Proteína , Ratos , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Med Chem ; 56(20): 7788-803, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24093940

RESUMO

The IAPs are key regulators of the apoptotic pathways and are commonly overexpressed in many cancer cells. IAPs contain one to three BIR domains that are crucial for their inhibitory function. The pro-survival properties of XIAP come from binding of the BIR domains to the pro-apoptotic caspases. The BIR3 domain of XIAP binds and inhibits caspase 9, while the BIR2 domain binds and inhibits the terminal caspases 3 and 7. While XIAP BIR3 inhibitors have previously been reported, they also inhibit cIAP1/2 and promote the release of TNFα, potentially limiting their therapeutic utility. This paper will focus on the optimization of selective XIAP BIR2 inhibitors leading to the discovery of highly potent benzodiazepinone 36 (IC50 = 45 nM), which has high levels of selectivity over XIAP BIR3 and cIAP1 BIR2/3 and shows efficacy in a xenograft pharmacodynamic model monitoring caspase activity while not promoting the release of TNFα in vitro.


Assuntos
Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Alanina/análogos & derivados , Alanina/síntese química , Alanina/farmacocinética , Alanina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Benzodiazepinonas/síntese química , Benzodiazepinonas/farmacocinética , Benzodiazepinonas/farmacologia , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Compostos Heterocíclicos/farmacocinética , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Camundongos Nus , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
14.
ACS Med Chem Lett ; 3(9): 764-8, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-24900545

RESUMO

3-[4-((1S,2S,3R,5S,7S)-5-Hydroxyadamantan-2-ylcarbamoyl)benzyl]-4-oxo-1-phenyl-1,4-dihydro-[1,8]naphthyridine-2-carboxylic acid methyl ester (4) was identified as a novel, druglike and selective quinolone pan JNK inhibitor. In this communication, some of the structure-activity relationship of the azaquinolone analogues leading to 4 is discussed. The focus is on how changes at the amide functionality affected the biochemical potency, cellular potency, metabolic properties, and solubility of this class of JNK inhibitors. Optimization of these properties led to the identification of the adamantyl analogue, 4. 4 achieved proof of mechanism in both rat and mouse TNF-α challenge models.

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