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1.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670179

RESUMO

Fumarylacetoacetate hydrolase (FAH) is the fifth enzyme in the tyrosine catabolism pathway. A deficiency in human FAH leads to hereditary tyrosinemia type I (HT1), an autosomal recessive disorder that results in the accumulation of toxic metabolites such as succinylacetone, maleylacetoacetate, and fumarylacetoacetate in the liver and kidney, among other tissues. The disease is severe and, when untreated, it can lead to death. A low tyrosine diet combined with the herbicidal nitisinone constitutes the only available therapy, but this treatment is not devoid of secondary effects and long-term complications. In this study, we targeted FAH for the first-time to discover new chemical modulators that act as pharmacological chaperones, directly associating with this enzyme. After screening several thousand compounds and subsequent chemical redesign, we found a set of reversible inhibitors that associate with FAH close to the active site and stabilize the (active) dimeric species, as demonstrated by NMR spectroscopy. Importantly, the inhibitors are also able to partially restore the normal phenotype in a newly developed cellular model of HT1.


Assuntos
Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Tirosinemias/tratamento farmacológico , Tirosinemias/enzimologia , Animais , Domínio Catalítico , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Hidrolases/genética , Camundongos , Tirosinemias/genética
2.
Clin Exp Dermatol ; 45(2): 194-201, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31419330

RESUMO

BACKGROUND: The protein kynureninase (KYNU) has recently been reported to participate in the pathological processes of various diseases. AIM: To explore the expression and the biological function of KYNU in cutaneous squamous cell carcinoma (cSCC). METHODS: Expression of KYNU in cSCC cell lines and tissues was firstly evaluated based on the Gene Expression Omnibus and the Oncomine databases. Quantitative reverse transcription-PCR was performed to determine the mRNA expression of KYNU in cSCC cell lines. Small interfering RNA (siRNA) was used for silencing KYNU. The effect of KYNU on the growth and motility of cSCC cells was determined by cell counting kit-8, wound-healing and Transwell assays, and western blotting was used to determine the protein expression of KYNU, AKT, phosphoinositide 3-kinase (PI3K), phosphorylated (p)-AKT and p-PI3K. RESULTS: KYNU was significantly upregulated in cSCC tissues and cell lines. Knockdown of KYNU using siRNA noticeably suppressed the proliferation, migration and invasion ability of SCL-1 cells (P < 0.01). Western blotting revealed that phosphorylation of AKT and PI3K was markedly inhibited after silencing KYNU. The ratios of p-AKT/AKT and p-PI3K/PI3K were significantly decreased in the si-KYNU group compared with the control group. CONCLUSION: Depletion of KYNU could inhibit the growth of cSCC cells, possibly through modulating PI3K/AKT pathway. These data indicate that KYNU takes a key part in the malignant progression of cSCC, and could be considered as a promising therapeutic target for cSCC treatment.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Hidrolases/metabolismo , Neoplasias Cutâneas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo , Inativação Gênica , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Neoplasias Cutâneas/patologia
3.
Sci Rep ; 9(1): 17532, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772212

RESUMO

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/ß hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.


Assuntos
Eritrócitos/parasitologia , Hidrolases/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Biotina/análogos & derivados , Humanos , Hidrolases/antagonistas & inibidores , Estágios do Ciclo de Vida , Lipólise , Plasmodium falciparum/crescimento & desenvolvimento , Proteômica , Serina/metabolismo
4.
Br J Pharmacol ; 176 Suppl 1: S297-S396, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31710714

RESUMO

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14752. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.


Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Isomerases/antagonistas & inibidores , Ligases/antagonistas & inibidores , Liases/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Transferases/antagonistas & inibidores , Animais , Bases de Dados de Produtos Farmacêuticos , Inibidores Enzimáticos/química , Humanos , Hidrolases/química , Hidrolases/metabolismo , Isomerases/química , Isomerases/metabolismo , Ligantes , Ligases/química , Ligases/metabolismo , Liases/química , Liases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Transferases/química , Transferases/metabolismo
5.
ACS Chem Biol ; 14(10): 2295-2304, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31525885

RESUMO

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,ß-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.


Assuntos
Reação Acrossômica/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Pirrolidinas/química , Pirrolidinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
6.
Carbohydr Res ; 484: 107776, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421353

RESUMO

Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-ß-d-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553T, inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-ß-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-ß-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-ß-d-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Flavobacteriaceae/enzimologia , Poríferos/química , Esteroides/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavobacteriaceae/efeitos dos fármacos , Fucus/microbiologia , Hidrolases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Estrutura Molecular , Polissacarídeo-Liase/antagonistas & inibidores , Polissacarídeos/química , Esteroides/química , Sulfatos/química
7.
J Biol Chem ; 294(28): 11047-11053, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31177094

RESUMO

A key metabolic adaptation of some species that face hypoxia as part of their life cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and most details of this pathway have remained elusive. Here, we used Caenorhabditis elegans as a model animal to elucidate key steps in RQ biosynthesis. Using RNAi and a series of C. elegans mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo Deletion of kynu-1, encoding a kynureninase that converts l-kynurenine (KYN) to anthranilic acid (AA) and 3-hydroxykynurenine (3HKYN) to 3-hydroxyanthranilic acid (3HAA), completely abolished RQ biosynthesis but did not affect Q levels. Deletion of kmo-1, which encodes a kynurenine 3-monooxygenase that converts KYN to 3HKYN, drastically reduced RQ but not Q levels. Knockdown of the Q biosynthetic genes coq-5 and coq-6 affected both Q and RQ levels, indicating that both biosynthetic pathways share common enzymes. Our study reveals that two pathways for RQ biosynthesis have independently evolved. Unlike in bacteria, where amination is the last step in RQ biosynthesis, in worms the pathway begins with the arylamine precursor AA or 3HAA. Because RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have potential utility for targeting parasitic infections that cause important neglected tropical diseases.


Assuntos
Caenorhabditis elegans/metabolismo , Cinurenina/metabolismo , Ubiquinona/análogos & derivados , Animais , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrolases/antagonistas & inibidores , Hidrolases/genética , Hidrolases/metabolismo , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Quinurenina 3-Mono-Oxigenase/genética , Quinurenina 3-Mono-Oxigenase/metabolismo , Espectrometria de Massas , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Tela Subcutânea/metabolismo , Ubiquinona/análise , Ubiquinona/biossíntese , Ubiquinona/metabolismo
8.
Neuropharmacology ; 157: 107673, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31233825

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by several behavioral disturbances, especially cognitive decline and deficits in social competence. Previous studies revealed that decreased social activity would accelerate AD progression, whereas enhanced social interaction could rescue AD-induced memory impairment. Collapsin response mediator protein 5 (CRMP5), which belongs to a family of cytosolic proteins, is abundantly expressed in the brain and is involved in the regulation of neurodevelopment and the pathology of several neuropsychiatric diseases. However, the functions of CRMP5 in AD are still unclear. Here, we demonstrated that 9-month-old 3xTg-AD mice exhibited social behavioral deficits and increased hippocampal CRMP5 levels compared to control (B6129S) mice. Knockdown of CRMP5 reversed the social deficits in 9-month-old 3xTg-AD mice, whereas CRMP5 overexpression decreased social interaction in both 3xTg-AD and control mice at 6 months of age. Interestingly, decreased expression of CRMP5 rescued AD-induced memory impairment, but overexpression of CRMP5 accelerated memory loss only in 3xTg-AD mice. In addition, we found that CRMP5 could regulate surface GluA2 and GluA2 S880 phosphorylation. These results suggest that CRMP5 regulates social behavior via modulation of surface GluA2 trafficking and affects memory performance in 3xTg-AD mice.


Assuntos
Doença de Alzheimer , Hidrolases/fisiologia , Transtornos da Memória/fisiopatologia , Proteínas Associadas aos Microtúbulos/fisiologia , Comportamento Social , Doença de Alzheimer/genética , Animais , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Hidrolases/antagonistas & inibidores , Hidrolases/biossíntese , Masculino , Transtornos da Memória/prevenção & controle , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/biossíntese , Fosforilação/fisiologia , Receptores de AMPA/metabolismo
9.
Science ; 363(6426)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30705156

RESUMO

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Coenzima A/metabolismo , Guanidina/análogos & derivados , Hidrolases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Ureia/análogos & derivados , Proteína de Transporte de Acila/metabolismo , Animais , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Feminino , Guanidina/farmacologia , Hidrolases/genética , Metabolismo dos Lipídeos , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/genética , Óperon , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas , Ureia/farmacologia
10.
EMBO Rep ; 19(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30021834

RESUMO

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub-Saharan Africa. For germination, Striga seeds require host-released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X-100 inhibits S. hermonthica seed germination without affecting host plants. High-resolution X-ray structures reveal that Triton X-100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7-specific inhibition by Triton X-100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga-specific herbicides.


Assuntos
Germinação/efeitos dos fármacos , Herbicidas/química , Hidrolases/química , Octoxinol/química , Proteínas de Plantas/química , Plantas Daninhas/química , Striga/enzimologia , Controle de Plantas Daninhas , Cristalografia por Raios X , Herbicidas/farmacologia , Hidrolases/antagonistas & inibidores , Octoxinol/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Striga/efeitos dos fármacos , Striga/fisiologia
11.
Food Chem ; 258: 111-117, 2018 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-29655711

RESUMO

The acorns of Quercus fabri Hance tree, or the mandarin duck fruits have been consumed by locals in the form of noodle, yet there is little scientific study. We found that they have much lower digestibility compared with that of rice flour. Solvent extraction using hexane, dichloromethane, and acetone-ethanol-water (AEW) mixture yielded three fractions but the starch hydrolase inhibitors were only found in the AEW fraction. Using assay-guided fractionation of the extracts, we were able to further separate the active compounds by using Sephadex LH-20 column and characterize the inhibitor chemical identities by LC-MS(n). The main active compounds are ellagitannins including pedunculagin, punicalagin, castalagin, and eucalbanin. Our results indicate that the acorns from Quercus fabri Hance have potential for preparation of low glycemic index foods due to the endogenous starch hydrolase inhibitors.


Assuntos
Hidrolases/antagonistas & inibidores , Quercus/química , Amido/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrolases/metabolismo , Taninos Hidrolisáveis/análise , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/isolamento & purificação , Taninos Hidrolisáveis/metabolismo , Cinética , Extração Líquido-Líquido , Quercus/metabolismo , Solventes/química , Espectrometria de Massas em Tandem , Taninos/química , Taninos/isolamento & purificação , Taninos/metabolismo
12.
Ann Med ; 50(4): 323-332, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29537306

RESUMO

OBJECTIVES: Psoriasis vulgaris is a chronic inflammatory and immune-mediated skin disease. 44 metabonomics biomarkers were identified by high-throughput liquid chromatography coupled to mass spectrometry in our previous work, but the roles of metabonomics biomarkers in the pathogenesis of psoriasis is unclear. METHODS: The metabonomics biomarker-enzyme network was constructed. The key metabonomics biomarkers and enzymes were screened out by network analysis. The binding affinity between each metabonomics biomarker and target was calculated by molecular docking. A binding energy-weighted polypharmacological index was introduced to evaluate the importance of target-related pathways. RESULTS: Long-chain fatty acids, phospholipids, Estradiol and NADH were the most important metabonomics biomarkers. Most key enzymes belonged hydrolase, thioesterase and acyltransferase. Six proteins (TNF-alpha, MAPK3, iNOS, eNOS, COX2 and mTOR) were extensively involved in inflammatory reaction, immune response and cell proliferation, and might be drug targets for psoriasis. PI3K-Akt signaling pathway and five other pathways had close correlation with the pathogenesis of psoriasis and could deserve further research. CONCLUSIONS: The inflammatory reaction, immune response and cell proliferation are mainly involved in psoriasis. Network pharmacology provide a new insight into the relationships between metabonomics biomarkers and the pathogenesis of psoriasis. KEY MESSAGES • Network pharmacology was adopted to identify key metabonomics biomarkers and enzymes. • Six proteins were screened out as important drug targets for psoriasis. • A binding energy-weighted polypharmacological index was introduced to evaluate the importance of target-related pathways.


Assuntos
Fármacos Dermatológicos/farmacologia , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Psoríase/tratamento farmacológico , Pele/enzimologia , Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Conjuntos de Dados como Assunto , Fármacos Dermatológicos/uso terapêutico , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular/métodos , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Ligação Proteica/efeitos dos fármacos , Psoríase/imunologia , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia
13.
Kidney Int ; 93(2): 365-374, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29061334

RESUMO

Ischemia/reperfusion is a common cause of acute kidney injury (AKI). However, mechanisms underlying the sudden loss in kidney function and tissue injury remain to be fully elucidated. Here, we investigated the role of peptidyl arginine deiminase-4 (PAD4), which converts arginine to citrulline and plays a role in epigenetic regulation and inflammation, in renal ischemia/reperfusion injury. PAD4 expression was highly induced in infiltrating leukocytes 24 hours following renal ischemia and reperfusion. This induction was accompanied by citrullination of histone H3 and formation of neutrophil extracellular traps in kidneys of wild-type mice. By contrast, PAD4-deficient mice did not form neutrophil extracellular traps, expressed lower levels of pro-inflammatory cytokines and were partially protected from renal ischemia/reperfusion-induced AKI. Furthermore, PAD4-deficient mice recovered kidney function 48 hours after ischemia/reperfusion, whereas kidney function in the wild-type mice progressively worsened. Administration of DNase I, which degrades neutrophil extracellular traps or the PAD-specific inhibitor YW3-56 before ischemia, partially prevented renal ischemia/reperfusion-induced AKI. Notably, transfer of neutrophils from wild-type, but not from PAD4-deficient mice, was sufficient to restore renal neutrophil extracellular trap formation and impair kidney function following renal ischemia/reperfusion. Thus, neutrophil PAD4 plays a pivotal role in renal ischemia/reperfusion-induced AKI.


Assuntos
Lesão Renal Aguda/enzimologia , Armadilhas Extracelulares/enzimologia , Hidrolases/metabolismo , Rim/enzimologia , Neutrófilos/enzimologia , Traumatismo por Reperfusão/enzimologia , Lesão Renal Aguda/patologia , Lesão Renal Aguda/fisiopatologia , Lesão Renal Aguda/prevenção & controle , Animais , Citrulinação , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Histiócitos/metabolismo , Hidrolases/antagonistas & inibidores , Hidrolases/deficiência , Hidrolases/genética , Mediadores da Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Neutrófilos/transplante , Proteína-Arginina Desiminase do Tipo 4 , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Traumatismo por Reperfusão/prevenção & controle
14.
Biochemistry ; 56(51): 6734-6742, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29190068

RESUMO

Campylobacter jejuni is the most common bacterial cause of gastroenteritis and a major contributor to infant mortality in the developing world. The increasing incidence of antibiotic-resistant C. jejuni only adds to the urgency to develop effective therapies. Because of the essential role that polyamines play, particularly in protection from oxidative stress, enzymes involved in the biosynthesis of these metabolites are emerging as promising antibiotic targets. The recent description of an alternative pathway for polyamine synthesis, distinct from that in human cells, in C. jejuni suggests this pathway could be a target for novel therapies. To that end, we determined X-ray crystal structures of C. jejuni agmatine deiminase (CjADI) and demonstrated that loss of CjADI function contributes to antibiotic sensitivity, likely because of polyamine starvation. The structures provide details of key molecular features of the active site of this protein. Comparison of the unliganded structure (2.1 Å resolution) to that of the CjADI-agmatine complex (2.5 Å) reveals significant structural rearrangements that occur upon substrate binding. The shift of two helical regions of the protein and a large conformational change in a loop near the active site generate a narrow binding pocket around the bound substrate. This change optimally positions the substrate for catalysis. In addition, kinetic analysis of this enzyme demonstrates that CjADI is an iminohydrolase that effectively deiminates agmatine. Our data suggest that C. jejuni agmatine deiminase is a potentially important target for combatting antibiotic resistance, and these results provide a valuable framework for guiding future drug development.


Assuntos
Campylobacter jejuni/enzimologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Hidrolases/antagonistas & inibidores , Aminoglicosídeos/farmacologia , Campylobacter jejuni/efeitos dos fármacos , Domínio Catalítico , Cristalografia por Raios X , Hidrolases/química , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Conformação Proteica
15.
Antivir Chem Chemother ; 25(3): 90-93, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29172646

RESUMO

Objective To synthesize 3,7-dideazaneplanocin and evaluate its antiviral potential. Methods The target 3,7-dideazaneplanocin has been prepared in five steps from a readily available cyclopentenol. A thorough in vitro antiviral analysis was conducted versus both DNA and RNA viruses. Results A rational synthesis of 3,7-dideazaneplanocin was conceived and successfully pursued in such a way that it can be adapted to various analogs of 3,7-dideazaneplanocin. Using standard antiviral assays, no activity for 3,7-dideazaneplanocn was found. Conclusion Two structural features are necessary for adenine-based carbocyclic nucleosides (like neplanocin) for potential antiviral properties: (i) inhibition of S-adenosylhomocysteine hydrolase and/or (ii) C-5' activation via the mono-nucleotide. These two requisite adenine structural features to fit these criteria are not present in in the target 3,7-dideazaneplanocin: (i) an N-7 is necessary for inhibition of the hydrolase and the N-3 is claimed to be essential for phosphorylation at C-5'. Thus, it is not surprising that 3,7-dideazaneplaoncin lacked antiviral properties.


Assuntos
Antivirais/farmacologia , Vírus de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Piridonas/farmacologia , Vírus de RNA/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular , Piridonas/síntese química , Piridonas/química
16.
Expert Opin Ther Pat ; 27(12): 1291-1304, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28870136

RESUMO

INTRODUCTION: Ectonucleotidases are a broad family of metallo-ectoenzymes that are responsible for hydrolysing a variety of nucleotides to nucleosides, hence orchestrating the activation of P1 and P2 cell receptors via controlled release of nucleotides and nucleosides. Many disorders such as impaired calcification including aortic calcification, neurological and immunological disorders, platelet aggregation, cell proliferation and metastasis. are characterized by an increase in expression of these ectonucleotidases. Consequently, selective inhibitors of ectonucleotidases are required for therapeutic intervention. Area covered: Several classes of compounds such as purine, nucleotide derivatives (e.g., ARL67156) and monoclonal antibodies, have shown promising ectonucleotidase inhibitory potential. This review discusses chemistry and therapeutic applications of ectonucleotidase inhibitors patented from 2011 to 2016. Expert opinion: All eukaryotic cells express nucleotide and nucleoside receptors on their cell surface and are capable of releasing extracellular nucleotides. Ectonucleotidases are a broad family of metallo-ectoenzymes that hydrolyze a variety of nucleotides to nucleosides. These extracellular nucleotides and nucleosides are important cell signalling molecules and mediate a variety of (patho)physiological processes by acting upon their respective P1 and/or P2 receptors. Discovery of molecules that can selectively inhibit or activate ectonucleotidases is crucial from therapeutic point of view, since it allows human intervention into purinergic cell signalling, thereby allowing us to modulate related (patho)physiological processes as desired.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Animais , Humanos , Hidrolases/metabolismo , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Patentes como Assunto , Transdução de Sinais/efeitos dos fármacos
17.
Bioorg Med Chem Lett ; 27(19): 4544-4547, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28882482

RESUMO

M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Ésteres/farmacologia , Etambutol/farmacologia , Hidrolases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ésteres/síntese química , Ésteres/química , Etambutol/síntese química , Etambutol/química , Hidrolases/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade
18.
J Enzyme Inhib Med Chem ; 32(1): 1203-1208, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28933232

RESUMO

Protein citrullination catalysed by peptidylarginine deiminase (PAD) may play an important pathogenic role in several chronic inflammatory diseases and malignancies. PAD2, PAD4, and citrullinated proteins are found in the synovium of rheumatoid arthritis patients. PAD activity is dependent on calcium and reducing conditions. However, reactive oxygen species (ROS) have been shown to induce citrullination of histones in granulocytes. Here we examine the ability of H2O2 and leukocyte-derived ROS to regulate PAD activity using citrullination of fibrinogen as read-out. H2O2 at concentrations above 40 µM inhibited the catalytic activity of PAD2 and PAD4 in a dose-dependent manner. PMA-stimulated leukocytes citrullinated fibrinogen and this citrullination was markedly enhanced when ROS formation was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI). In contrast, PAD released from stimulated leukocytes was unaffected by exogenously added H2O2 at concentrations up to 1000 µM. The role of ROS in regulating PAD activity may play an important part in preventing hypercitrullination of proteins.


Assuntos
Biocatálise , Hidrolases/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Dose-Resposta a Droga , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Hidrolases/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Estrutura Molecular , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
19.
FASEB J ; 31(12): 5577-5591, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842426

RESUMO

Exaggerated maternal immune responses must be strictly controlled to ensure a successful pregnancy. Neutrophil extracellular traps (NETs) have recently been implicated as a potential mechanism for promoting inflammation in pregnancy-related disorders. In this study, we demonstrated that NETs play a key role in the pathogenesis of sphingosine kinase (Sphk)-mediated pregnancy loss. Perturbing the sphingolipid pathway by disrupting Sphk genes during pregnancy led to excessive NET formation exclusively at the fetomaternal interface and early fetal death. Neutrophils that formed NETs were characterized by histone hypercitrullination and peptidylarginine deiminase 4 (PAD4) overexpression. In addition, thrombus formation was enhanced in the decidua, but not in the plasma, of Sphk-deficient mice. Blocking NET formation with a PAD4 inhibitor protected Sphk-deficient mice from pregnancy loss. The PAD4 inhibition significantly reduced the expression of hypercitrullinated histone in neutrophils and ameliorated vascular injury in the decidua of Sphk-deficient mice. Moreover, NET formation was induced in human neutrophils stimulated with Sphk-deficient human decidual cells. Together, these findings indicate that targeting NETs might be a novel therapeutic strategy to treat idiopathic pregnancy loss in humans.-Mizugishi, K., Yamashita, K. Neutrophil extracellular traps are critical for pregnancy loss in sphingosine kinase-deficient mice on 129Sv/C57BL/6 background.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Citrulinação/efeitos dos fármacos , Armadilhas Extracelulares/genética , Feminino , Hidrolases/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ornitina/análogos & derivados , Ornitina/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Gravidez , Complicações na Gravidez , Proteína-Arginina Desiminase do Tipo 4 , Protrombina/metabolismo , Trombina/metabolismo , Trombomodulina/sangue , Útero/efeitos dos fármacos , Útero/metabolismo
20.
Cancer Lett ; 409: 30-41, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-28844713

RESUMO

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.


Assuntos
Hidrolases/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Células HEK293 , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/biossíntese , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ornitina/análogos & derivados , Ornitina/farmacologia , Proteína-Arginina Desiminase do Tipo 1 , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto
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