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1.
Science ; 373(6551): 231-236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244417

RESUMO

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.


Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Interferência de RNA , Vírus de RNA/fisiologia , RNA Viral/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células-Tronco/enzimologia , Células-Tronco/virologia , Processamento Alternativo , Animais , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , RNA Helicases DEAD-box/química , Humanos , Imunidade Inata , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Organoides/enzimologia , Organoides/virologia , Infecções por Vírus de RNA/enzimologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Vírus de RNA/imunologia , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , Ribonuclease III/química , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Replicação Viral , Zika virus/genética , Zika virus/imunologia , Zika virus/fisiologia , Infecção por Zika virus/enzimologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
2.
Gene ; 793: 145745, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34077774

RESUMO

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.


Assuntos
Aspergillus oryzae/enzimologia , Ácidos Graxos/biossíntese , Proteínas Fúngicas/genética , Lipase/genética , Esporos Fúngicos/enzimologia , Triglicerídeos/biossíntese , Aspergillus oryzae/classificação , Aspergillus oryzae/genética , Sistemas CRISPR-Cas , Ácidos Graxos/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Humanos , Microbiologia Industrial , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lipase/metabolismo , Metabolismo dos Lipídeos/genética , Micélio/enzimologia , Micélio/genética , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética , Triglicerídeos/genética
3.
J Med Chem ; 64(11): 7760-7777, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34019417

RESUMO

N-Phenylpropenoyl-l-amino acids (NPAs) are inducible nitric oxide synthase (iNOS) inhibitors possessing preventive effects for Parkinson's disease (PD). Here, structural modifications for improving the iNOS inhibitory activity and blood-brain barrier (BBB) permeability of NPAs were conducted, leading to 20 optimized NPA derivatives (1-20). Compound 18, with the most potent activity (IC50 = 74 nM), high BBB permeability (Pe = 19.1 × 10-6 cm/s), and high selectivity over other NOS isoforms, was selected as the lead compound. Further studies demonstrated that 18 directly binds to iNOS. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced acute PD model, the oral administration of 18 (1 and 2 mg/kg) exerted preventive effects by alleviating the loss of dopaminergic (DAergic) neurons. Notably, in the MPTP-/probenecid-induced chronic PD model, the same dose of 18 also displayed a therapeutic effect by repairing the damaged DAergic neurons. Finally, good pharmacokinetic properties and low toxicity made 18 a promising candidate for the treatment of PD.


Assuntos
Aminoácidos/química , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Propanóis/química , Aminoácidos/metabolismo , Aminoácidos/farmacologia , Animais , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/patologia , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Simulação de Acoplamento Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Relação Estrutura-Atividade
4.
J Med Chem ; 64(11): 7341-7358, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34027661

RESUMO

Recently, irreversible inhibitors have attracted great interest in antitumors due to their advantages of forming covalent bonds to target proteins. Herein, some benzothiazepinone compounds (BTZs) have been designed and synthesized as novel covalent GSK-3ß inhibitors with high selectivity for the kinase panel. The irreversible covalent binding mode was identified by kinetics and mass spectrometry, and the main labeled residue was confirmed to be the unique Cys14 that exists only in GSK-3ß. The candidate 4-3 (IC50 = 6.6 µM) showed good proliferation inhibition and apoptosis-inducing ability to leukemia cell lines, low cytotoxicity on normal cell lines, and no hERG inhibition, which hinted the potential efficacy and safety. Furthermore, 4-3 exhibited decent pharmacokinetic properties in vivo and remarkably inhibited tumor growth in the acute promyelocytic leukemia (APL) mouse model. All the results suggest that these newly irreversible BTZ compounds might be useful in the treatment of cancer such as APL.


Assuntos
Benzotiazóis/química , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Animais , Apoptose/efeitos dos fármacos , Benzotiazóis/metabolismo , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Glicogênio Sintase Quinase 3 beta/metabolismo , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , Leucemia Promielocítica Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Molecules ; 26(7)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800655

RESUMO

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.


Assuntos
Antineoplásicos/uso terapêutico , Digitoxina/uso terapêutico , Digoxina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Ouabaína/uso terapêutico , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Ensaios Clínicos como Assunto , Digitoxina/química , Digoxina/química , Reposicionamento de Medicamentos , Inibidores Enzimáticos/química , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Modelos Moleculares , Ouabaína/química , Ligação Proteica , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo
6.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919829

RESUMO

The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in "physiological hypoxia" and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1-/-, Hif-p4h-2 hypomorph and Hif-p4h-3-/- mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.


Assuntos
Regulação Enzimológica da Expressão Gênica , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Hipóxia/enzimologia , Hipóxia/genética , Mucosa Intestinal/enzimologia , Envelhecimento/metabolismo , Animais , Ceco/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Isoenzimas/metabolismo , Jejuno/metabolismo , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
7.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805821

RESUMO

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.


Assuntos
Ácido Abscísico/metabolismo , Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Endonucleases/genética , Regulação da Expressão Gênica de Plantas , Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/agonistas , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Ciclopentanos/farmacologia , Citoplasma/metabolismo , Secas , Endonucleases/antagonistas & inibidores , Endonucleases/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Oxilipinas/metabolismo , Oxilipinas/farmacologia , Células Vegetais/efeitos dos fármacos , Células Vegetais/enzimologia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/enzimologia , Estômatos de Plantas/genética , Plantas Geneticamente Modificadas , Prolina/metabolismo , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Água/metabolismo
8.
J Med Chem ; 64(11): 7331-7340, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33876637

RESUMO

Aberrant activation of the PI3K pathway has been intensively targeted for cancer therapeutics for decades, leading to more than 40 PI3K inhibitors advanced into clinical trials. However, it is increasingly noticed that PI3K inhibitors often showed limited efficacy as well as a number of serious on-target adverse effects during the clinical development. In this work, we designed and synthesized a novel photocaged PI3K inhibitor 1, which could be readily activated by UV irradiation to release a highly potent PI3K inhibitor 2. Upon UV irradiation, the photocaged inhibitor 1 demonstrated remarkably enhanced antiproliferative activity against multiple cancer cell lines and significant efficacy in the patient-derived tumor organoid model. Furthermore, 1 also showed favorable anticancer activity in an in vivo zebrafish xenograft model. Taken together, the photocaged PI3K inhibitor 1 represents a promising avenue for novel therapeutics toward precise cancer treatment.


Assuntos
Desenho de Fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Animais , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Cães , Estabilidade de Medicamentos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Transdução de Sinais/efeitos dos fármacos , Raios Ultravioleta , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/metabolismo
9.
J Biosci Bioeng ; 131(6): 605-612, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33814275

RESUMO

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (ß/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.


Assuntos
Aspergillus oryzae/enzimologia , alfa-Amilases/química , Amilases/metabolismo , Animais , Aspergillus oryzae/metabolismo , Bacillus subtilis/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Hordeum/enzimologia , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , alfa-Amilases Pancreáticas/química , Conformação Proteica , Estrutura Terciária de Proteína , Suínos/metabolismo , alfa-Amilases/metabolismo
10.
Biomed Pharmacother ; 139: 111525, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33882412

RESUMO

3-Hydroxytyrosol (HXT) is a natural polyphenol present in extra virgin olive oil. It is a key component of Mediterranean diet and is known for its strong antioxidant activity. The present study evaluated the potential of HXT as an anti-parkinsonian molecule in terms of its ability to inhibit MAO-B and thereby maintaining dopamine (DA) levels in Parkinson's disease (PD). In-silico molecular docking study followed by MMGBSA binding free energy calculation revealed that HXT has a strong binding affinity for MAO-B in comparison to MAO-A. Moreover, rasagiline and HXT interacted with the similar binding sites and modes of interactions. Additionally, molecular dynamics simulation studies revealed stable nature of HXT-MAO-B interaction and also provided information about the amino acid residues involved in binding. Moreover, in vitro studies revealed that HXT inhibited MAO-B in human platelets with IC50 value of 7.78 µM. In vivo studies using MPTP-induced mouse model of PD revealed increase in DA levels with concomitant decrease in DA metabolites (DOPAC and HVA) on HXT treatment. Furthermore, MAO-B activity was also inhibited on HXT administration to PD mice. In addition, HXT treatment prevented MPTP-induced loss of DA neurons in substantia nigra and their nerve terminals in the striatum. HXT also attenuated motor impairments in PD mice assessed by catalepsy bar, narrow beam walk and open field tests. Thus, the present findings reveal HXT as a potential inhibitor of MAO-B, which may be used as a lead molecule for the development of therapeutics for PD.


Assuntos
Antiparkinsonianos/farmacologia , Intoxicação por MPTP/tratamento farmacológico , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/tratamento farmacológico , Álcool Feniletílico/análogos & derivados , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Simulação por Computador , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monoaminoxidase/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/patologia , Azeite de Oliva/química , Doença de Parkinson Secundária/patologia , Álcool Feniletílico/farmacologia , Substância Negra/efeitos dos fármacos , Substância Negra/patologia
11.
J Med Chem ; 64(8): 4462-4477, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33793216

RESUMO

A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activity lacking COX2 inhibitory activity. Compound d12 was the most potent with an IC50 of 1 nM, which was three times more potent than sildenafil and more selective with a selectivity index of >10,000-fold against all other PDE isozymes. Sildenafil inhibited the full-length and catalytic fragment of PDE5, while compound d12 only inhibited the full-length enzyme, suggesting a mechanism of enzyme inhibition distinct from sildenafil. The PDE5 inhibitory activity of compound d12 was confirmed in cells using a cGMP biosensor assay. Oral administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.


Assuntos
Celecoxib/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Inibidores da Fosfodiesterase 5/química , Pirazóis/química , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Celecoxib/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Inibidores da Fosfodiesterase 5/metabolismo , Ligação Proteica , Pirazóis/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Estereoisomerismo , Relação Estrutura-Atividade
12.
Nat Commun ; 12(1): 1944, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782402

RESUMO

CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.


Assuntos
Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Genoma de Planta , Oryza/genética , Agrobacterium tumefaciens , Alelos , Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Produtos Agrícolas , Endodesoxirribonucleases/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Oryza/metabolismo , Plantas Geneticamente Modificadas , RNA Guia/genética , RNA Guia/metabolismo , Alinhamento de Sequência
13.
Plant Sci ; 306: 110876, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33775371

RESUMO

Acid rain, as a typical abiotic stress, damages plant growth and production. Calcium (Ca) mediates plant growth and links the signal transduction in plants for adapting to abiotic stresses. To understand the effect of Ca2+ on plant adaptable response to acid rain, we investigated changes in activities and gene expression of antioxidative enzymes and fatty acid composition of membrane lipid in rice seedlings treated with exogenous Ca2+ (5 mM) or/and simulated acid rain (SAR, pH 3.5 / 2.5). Exogenous Ca2+ enhanced activities of superoxide dismutase, catalase and peroxidase isozymes in rice leaves under SAR stress by promoting activation of existing isoforms and up-regulation of Cu/Zn-SOD1, Cu/Zn-SOD2, Cu/Zn-SOD3, CAT1, CAT2 and POD1. Compared to SAR treatment alone, exogenous Ca2+ alleviated SAR-induced oxidative damage to cell membrane by enhancing antioxidative capacity, as shown by the decrease in concentrations of H2O2, O2- and malondialdehyde in rice leaves. Meanwhile, Ca2+ alleviated SAR-induced decrease in unsaturation of membrane lipid for maintaining membrane fluidity. Finally, exogenous Ca2+ alleviated SAR-induced inhibition on relative growth rate of rice. Therefore, Ca2+ could play a role in regulating activities of antioxidative enzymes as well as maintaining unsaturation of membrane lipid for enhancing tolerance in rice seedlings to acid rain stress.


Assuntos
Chuva Ácida/efeitos adversos , Adaptação Fisiológica , Antioxidantes/metabolismo , Cálcio/metabolismo , Isoenzimas/metabolismo , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Estresse Fisiológico/fisiologia , Produtos Agrícolas/enzimologia , Produtos Agrícolas/crescimento & desenvolvimento
14.
Nat Commun ; 12(1): 1952, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782393

RESUMO

The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Canais de Cloreto/genética , Glutamato Descarboxilase/genética , Estômatos de Plantas/metabolismo , Transpiração Vegetal/genética , Água/metabolismo , Ácido gama-Aminobutírico/metabolismo , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Adaptação Fisiológica/genética , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hordeum/genética , Hordeum/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/genética , Transpiração Vegetal/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Soja/genética , Soja/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/metabolismo , Vicia faba/genética , Vicia faba/metabolismo
15.
Eur J Med Chem ; 216: 113283, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33667848

RESUMO

In the present study, we describe the design of different series of benzofuran-based derivatives as potential carbonic anhydrase inhibitors (CAIs). The adopted design is based on bioisosteric replacement for the p-fluorophenyl SLC-0111 tail with the lipophilic 2-methylbenzofuran or 5-bromobenzofuran tails to furnish the 2-methylbenzofuran (MBF) sulfonamides (MBFS; 9, 11 and 13) and 5-bromobenzofuran (BBF) sulfonamides (BBFS; 27a-b, 28a-b and 29a-c), respectively. Thereafter, the urea spacer was either elongated to furnish MBFS (17 and 19), and BBFS (30) series, or replaced by a carbamate one to afford MBFS (15). All the designed compounds were synthesized and evaluated for their inhibitory activities against four human (h) CA isoforms: hCA I, II, IX and XII. MBFS (11b and 17) and BBFS (28b, 29a and 30) efficiently inhibited the tumor-related CA IX isoform in the single-digit nanomolar range (KIs = 8.4, 7.6, 5.5, 7.1 and 1.8 nM, respectively). In particular, MBFS 11b and BBFS 28b exhibited good selectivity toward hCA IX isoform over the main off-target hCA II isoform (S.I. = 26.4 and 58.9, respectively). As a consequence, 11b and 28b were examined for their anticancer and pro-apoptotic activities toward MDA-MB-231 and MCF-7 cancer cell lines.


Assuntos
Benzofuranos/química , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/química , Antígenos de Neoplasias/metabolismo , Benzofuranos/metabolismo , Benzofuranos/farmacologia , Sítios de Ligação , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Simulação de Acoplamento Molecular , Compostos de Fenilureia , Relação Estrutura-Atividade , Sulfonamidas/química
16.
Eur J Med Chem ; 216: 113332, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33714914

RESUMO

Histone deacetylases (HDACs) have been implicated in a number of diseases including cancer, cardiovascular disorders, diabetes mellitus, neurodegenerative disorders and inflammation. For the treatment of epigenetically altered diseases such as cancer, HDAC inhibitors have made a significant progress in terms of development of isoform selective inhibitiors. Isoform specific HDAC inhibitors have less adverse events and better safety profile. A HDAC isoform i.e., HDAC2 demonstrated significant role in the development of variety of diseases, mainly involved in the cancer and neurodegenerative disorders. Discovery and development of selective HDAC2 inhibitors have a great potential for the treatment of target diseases. In the present compilation, we have reviewed the role of HDAC2 in progression of cancer and neurodegenerative disorders, and information on the drug development opportunities for selective HDAC2 inhibition.


Assuntos
Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/uso terapêutico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Desenho de Fármacos , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
17.
Mol Biol Rep ; 48(3): 2429-2436, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33761087

RESUMO

Prostate cancer (PCa) is a highly heterogeneous and unpredictable progressive disease. Sensitivity of PCa cells to androgens play a central role in tumor aggressiveness but biomarkers with high sensitivity and specificity that follow the progression of the disease has not yet been verified. The vitamin D endocrine system and its receptors, the Vitamin D Receptor (VDR) and the Protein Disulfide-Isomerase A3 (PDIA3), are related to anti-tumoral effects as well as carcinogenesis and have therefore been suggested as potential candidates for the prevention and therapy of several cancer forms, including PCa. In this study, we evaluated the mRNA expression of VDR and PDIA3 involved in vitamin D signaling in cell lines representing different stages of PCa (PNT2, P4E6, LNCaP, DU145 and PC3). This study further aimed to evaluate vitamin D receptors and their isoforms as potential markers for clinical diagnosis of PCa. A novel transcript isoform of PDIA3 (PDIA3N) was identified and found to be expressed in all PCa cell lines analyzed. Androgen-independent cell lines showed a higher mRNA expression ratio between PDIA3N/PDIA3 contrary to androgen-dependent cell lines that showed a lower mRNA expression ratio between PDIA3N/PDIA3. The structure of PDIA3N differed from PDIA3. PDIA3N was found to be a N-truncated isoform of PDIA3 and differences in protein structure suggests an altered protein function i.e. cell location, thioredoxin activity and affinity for 1,25(OH)2D3. Collectively, PDIA3 transcript isoforms, the ratio between PDIA3N/PDIA3 and especially PDIA3N, are proposed as candidate markers for future studies with different stages of PCa progression.


Assuntos
Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Estadiamento de Neoplasias , Neoplasias da Próstata/genética , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
J Enzyme Inhib Med Chem ; 36(1): 719-726, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33648390

RESUMO

Carbonic anhydrases (CAs, EC 4.2.1.1) activators were shown to be involved in memory enhancement and learning in animal models of cognition. Here we investigated the CA activating effects of a large series of histamine based compounds, including histamine receptors (H1R - H4R) agonists, antagonists and other derivatives of this autacoid. CA activators may be thus useful for improving cognition as well as in diverse therapeutic areas (phobias, obsessive-compulsive disorder, generalised anxiety, post-traumatic stress disorders), for which activation of this enzyme was recently shown to be involved.


Assuntos
Anidrases Carbônicas/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Emoções/efeitos dos fármacos , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Memória/efeitos dos fármacos , Anidrases Carbônicas/genética , Agonistas dos Receptores Histamínicos/química , Antagonistas dos Receptores Histamínicos/química , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estrutura Molecular
19.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33649847

RESUMO

Diffuse large B­cell lymphoma (DLBCL) is a highly heterogeneous malignant tumor type, and epigenetic modifications such as acetylation or deacetylation serve vital roles in its development. Chidamide, a novel histone deacetylase inhibitor, exerts an anticancer effect against various types of cancer. The present study aimed to evaluate the cellular effect of chidamide on a number of DLBCL cell lines and to investigate its underlying mechanism. The results demonstrated that chidamide induced the death of these cells in a concentration­(0­30 µmol/l) and time­dependent (24­72 h) manner, as determined using the Cell Counting Kit­8 cell viability assay. Moreover, chidamide promoted cellular apoptosis, which was identified via flow cytometry and western blot analysis, with an increase in cleaved caspase­3 expression and a decrease in Bcl­2 expression. Chidamide treatment also decreased the expression level of STAT3 and its phosphorylation, which was accompanied by the downregulation of a class­I histone deacetylase (HDAC) inhibitor, chidamide. Collectively, these data suggested that chidamide can be a potent therapeutic agent to treat DLBCL by inducing the apoptotic death of DLBCL cells by inhibiting the HDACs/STAT3/Bcl­2 pathway.


Assuntos
Aminopiridinas/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Histona Desacetilases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Isoenzimas/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/genética
20.
Eur J Med Chem ; 217: 113351, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33744685

RESUMO

In this research, rational design, synthesis, carbonic anhydrases (CAs) inhibitory effects, and cytotoxicities of the 4-(3-(2-arylidenehydrazine-1-carbonyl)-5-(thiophen-2-yl)-1H-pyrazole-1-yl)benzenesulfonamides 1-20 were reported. Compound 18 (Ki = 7.0 nM) was approximately 127 times more selective cancer-associated hCA IX inhibitor over hCA I, while compound 17 (Ki = 10.6 nM) was 47 times more selective inhibitor of hCA XI over hCA II compared to the acetazolamide. Compounds 11 (CC50 = 5.2 µM) and 20 (CC50 = 1.6 µM) showed comparative tumor-specificity (TS= > 38.5; >128.2) with doxorubicin (TS > 43.0) towards HSC-2 cancer cell line. Western blot analysis demonstrated that 11 induced slightly apoptosis whereas 20 did not induce detectable apoptosis. A preliminary analysis showed that some correlation of tumor-specificity of 1-20 with the chemical descriptors that reflect hydrophobic volume, dipole moment, lowest hydrophilic energy, and topological structure. Molecular docking simulations were applied to the synthesized ligands to elucidate the predicted binding mode and selectivity profiles towards hCA I, hCA II, and hCA IX.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química
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