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1.
PLoS One ; 15(9): e0236081, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960890

RESUMO

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.


Assuntos
Cinamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutaratos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ácido Succínico/farmacologia , Animais , Linhagem Celular Tumoral , Cinamatos/química , Inibidores Enzimáticos/química , Glucose/metabolismo , Glutaratos/química , Glicogênio Fosforilase/metabolismo , Glicólise/efeitos dos fármacos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Metilação , Camundongos , Ácido Succínico/química
2.
Nat Commun ; 11(1): 3862, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737291

RESUMO

Allostery in proteins influences various biological processes such as regulation of gene transcription and activities of enzymes and cell signaling. Computational approaches for analysis of allosteric coupling provide inexpensive opportunities to predict mutations and to design small-molecule agents to control protein function and cellular activity. We develop a computationally efficient network-based method, Ohm, to identify and characterize allosteric communication networks within proteins. Unlike previously developed simulation-based approaches, Ohm relies solely on the structure of the protein of interest. We use Ohm to map allosteric networks in a dataset composed of 20 proteins experimentally identified to be allosterically regulated. Further, the Ohm allostery prediction for the protein CheY correlates well with NMR CHESCA studies. Our webserver, Ohm.dokhlab.org, automatically determines allosteric network architecture and identifies critical coupled residues within this network.


Assuntos
Algoritmos , Proteínas Quimiotáticas Aceptoras de Metil/química , Mapeamento de Interação de Proteínas/estatística & dados numéricos , Software , Regulação Alostérica , Sítio Alostérico , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Internet , Proteínas Quimiotáticas Aceptoras de Metil/antagonistas & inibidores , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
3.
Gene ; 763: 144997, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32783992

RESUMO

The CRISPR-Cas system currently stands as one of the best multifaceted tools for site-specific genome engineering in mammals. An important aspect of research in this field focusses on improving the specificity and efficacy of precise genome editing in multiple model systems. The cornerstone of this mini-review is one of the extensively investigated small molecule inhibitor, SCR7, which abrogates NHEJ, a Ligase IV-dependent DSB repair pathway, thus guiding integration of the foreign DNA fragment via the more precise homology directed repair during genome editing. One of our recent studies sheds light on properties of different forms of SCR7. Here, we give a succinct account on the use of SCR7 and its different forms in CRISPR-Cas system, highlighting their chemical properties and biological relevance as potent efficiency-enhancing CRISPR tools.


Assuntos
Sistemas CRISPR-Cas , Inibidores Enzimáticos/farmacologia , Edição de Genes/métodos , Pirimidinas/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacos , Bases de Schiff/farmacologia , Animais , DNA Ligase Dependente de ATP/antagonistas & inibidores , Inibidores Enzimáticos/química , Humanos , Pirimidinas/química , Bases de Schiff/química
4.
Life Sci ; 259: 118169, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738360

RESUMO

AIMS: The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. MATERIALS AND METHODS: The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2'OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2'OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2'OMTase followed by re-docking analysis. The apo and ligand-bound 2'OMTase were further validated and analyzed by using molecular dynamics simulation. KEY FINDINGS: The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2'OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2'OMTase. SIGNIFICANCE: The results suggested that these drugs may be used as potential inhibitors for 2'OMTase for combating the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/virologia , Homologia de Sequência de Aminoácidos
5.
Biomolecules ; 10(7)2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32679775

RESUMO

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600-1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Amidas/antagonistas & inibidores , Amidas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Mutação , Pandemias , Proteoma/antagonistas & inibidores , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismo
6.
Nucleic Acids Res ; 48(14): 7914-7923, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652039

RESUMO

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.


Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fator sigma/antagonistas & inibidores , Fator sigma/química , Tiofenos/química
7.
J Comput Aided Mol Des ; 34(10): 1091-1103, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32601839

RESUMO

Herein, the LASSBio Chemical Library is presented as a valuable source of compounds for screening to identify hits suitable for subsequent hit-to-lead optimization stages. A feature of the LASSBio Chemical Library worth highlighting is the fact that it is a smart library designed by medicinal chemists with pharmacological activity as the main priority. The great majority of the compounds part of this library have shown in vivo activity in animal models, which is an indication that they possess overall favorable bioavailability properties and, hence, adequate pharmacokinetic profiles. This, in turn, is supported by the fact that approximately 85% of the compounds are compliant with Lipinski's rule of five and ca. 95% are compliant with Veber's rules, two important guidelines for oral bioavailability. In this work it is presented a virtual screening methodology combining a pharmacophore-based model and an empirical Gibbs free energy-based model for the ligand-protein interaction to explore the LASSBio Chemical Library as a source of new hits for the inhibition of the phosphatidylinositol 4-kinase IIIß (PI4KIIIß) enzyme, which is related to the development of viral infections (including enteroviruses, SARS coronavirus, and hepatitis C virus), cancers and neurological diseases. The approach resulted in the identification of two hits, LASSBio-1799 (7) and LASSBio-1814 (10), which inhibited the target enzyme with IC50 values of 3.66 µM and IC50 and 6.09 µM, respectively. This study also enabled the determination of the structural requirements for interactions with the active site of PI4KIIIß, demonstrating the importance of both acceptor and donor hydrogen bonding groups for forming interactions with binding site residues Val598 and Lys549.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sítios de Ligação , Domínio Catalítico , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
8.
PLoS One ; 15(7): e0235483, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697773

RESUMO

A series of potent HIV-1 protease inhibitors, containing diverse piperidine analogues as the P2-ligands, 4-substituted phenylsulfonamides as the P2'-ligands and a hydrophobic cyclopropyl group as the P1'-ligand, were designed, synthesized and evaluated in this work. Among these twenty-four target compounds, many of them exhibited excellent activity against HIV-1 protease with half maximal inhibitory concentration (IC50) values below 20 nM. Particularly, compound 22a containing a (R)-piperidine-3-carboxamide as the P2-ligand and a 4-methoxylphenylsulfonamide as the P2'-ligand exhibited the most effective inhibitory activity with an IC50 value of 3.61 nM. More importantly, 22a exhibited activity with inhibition of 42% and 26% against wild-type and Darunavir (DRV)-resistant HIV-1 variants, respectively. Additionally, the molecular docking of 22a with HIV-1 protease provided insight into the ligand-binding properties, which was of great value for further study.


Assuntos
Inibidores Enzimáticos/química , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , HIV-1/efeitos dos fármacos , Piperidinas/farmacologia , Cristalografia por Raios X , Darunavir/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Infecções por HIV/virologia , Protease de HIV/química , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , HIV-1/química , HIV-1/patogenicidade , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologia
9.
J Chromatogr A ; 1625: 461294, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709337

RESUMO

It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 µM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.


Assuntos
Corydalis/química , Distribuição Contracorrente/métodos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Histona Desmetilases/antagonistas & inibidores , Interface Usuário-Computador , Bioensaio , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Histona Desmetilases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Solventes , Espectrometria de Massas em Tandem
10.
Food Chem ; 333: 127418, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32653680

RESUMO

Fermented camel milk possesses a weak (liquid-like) gel structure. We aimed to 1) investigate the characteristics, bioactivities and rheological properties of the exopolysaccharide (EPS) produced by Lactococcus garvieae-C47 (exopolysaccharide-C47 product), a potential probiotic bacterium, on milk extracted from camels and 2) examine the rheological properties of the fermented camel milk produced by L. garvieae-C47. Exopolysaccharide-C47 product (molecular weight: 7.3 × 106 Da) was composed of the following monosaccharides: glucose (82.51%), arabinose (5.32%) and xylose (12.17%). The antioxidant, antitumor and α-amylase inhibitory activities of exopolysaccharide-C47 product reached up to 67.52, 59.35 and 91.0%, respectively. The apparent viscosity of exopolysaccharide-C47 product decreased with the increase in shear rate and declined by increasing the temperature up to 50 °C. The rheological properties of exopolysaccharide-C47 product are influenced by the salt type and pH value. The exopolysaccharide product produced by L. garvieae C47 possesses valuable health benefits and has the ability to improve the weak structure of fermented camel milk.


Assuntos
Fermentação , Lactococcus/metabolismo , Leite/microbiologia , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/farmacologia , Probióticos/metabolismo , Reologia , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Camelus , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Peso Molecular , Polissacarídeos Bacterianos/biossíntese , Viscosidade , alfa-Amilases/antagonistas & inibidores
11.
Food Chem ; 331: 127240, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32585546

RESUMO

The present study focused on the phytochemical profiling along with evaluation of in vitro antioxidant, α-glucosidase and α-amylase inhibitory activities of various crudes and fractions obtained from Lepisanthes fruticosa (Roxb) Leenh fruit. Ethanolic seed crude extract exhibited the strongest radical scavenging, ß-carotene bleaching activity, α-glucosidase inhibition and the highest total phenolic content (TPC). Column chromatography afforded various fractions with fraction M4 being the most potent due to the strongest radical scavenging, ß-carotene bleaching, α-glucosidase inhibition and greatest amount of TPC. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of ethanolic seed crude extract and fraction M4 showed the presence of various phytochemicals with antioxidant and antidiabetic properties, which include mostly flavonoids and tannins. The results may suggest that the ethanolic crude seed extract and its fraction could be an excellent source of bioactive phytochemicals with antioxidant and antidiabetic potential.


Assuntos
Antioxidantes/química , Inibidores Enzimáticos/análise , Fenóis/análise , Extratos Vegetais/química , Sapindaceae/química , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Flavonoides/análise , Flavonoides/química , Frutas/química , Frutas/metabolismo , Fenóis/química , Sapindaceae/metabolismo , Espectrometria de Massas em Tandem , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo
12.
J Phys Chem B ; 124(32): 6955-6962, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32521159

RESUMO

Starting from late 2019, the coronavirus disease 2019 (COVID-19) has emerged as a once-in-a-century pandemic with deadly consequences, which urgently calls for new treatments, cures, and supporting apparatuses. Recently, because of its positive results in clinical trials, remdesivir was approved by the Food and Drug Administration to treat COVID-19 through Emergency Use Authorization. Here, we used molecular dynamics simulations and free energy perturbation methods to study the inhibition mechanism of remdesivir to its target SARS-CoV-2 virus RNA-dependent RNA polymerase (RdRp). We first constructed the homology model of this polymerase based on a previously available structure of SARS-CoV NSP12 RdRp (with a sequence identity of 95.8%). We then built a putative preinsertion binding structure by aligning the remdesivir + RdRp complex to the ATP bound poliovirus RdRp without the RNA template. The putative binding structure was further optimized with molecular dynamics simulations. The resulting stable preinsertion state of remdesivir appeared to form hydrogen bonds with the RNA template when aligned with the newly solved cryo-EM structure of SARS-CoV-2 RdRp. The relative binding free energy between remdesivir and ATP was calculated to be -2.80 ± 0.84 kcal/mol, where remdesivir bound much stronger to SARS-CoV-2 RdRp than the natural substrate ATP. The ∼100-fold improvement in the Kd from remdesivir over ATP indicates an effective replacement of ATP in blocking of the RdRp preinsertion site. Key residues D618, S549, and R555 are found to be the contributors to the binding affinity of remdesivir. These findings suggest that remdesivir can potentially act as a SARS-CoV-2 RNA-chain terminator, effectively stopping its RNA replication, with key residues also identified for future lead optimization and/or drug resistance studies.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/metabolismo , Betacoronavirus/enzimologia , Inibidores Enzimáticos/metabolismo , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Antivirais/química , Sítios de Ligação , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , RNA Replicase/química , Termodinâmica , Proteínas não Estruturais Virais/química
13.
Chem Biol Interact ; 327: 109148, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32511959

RESUMO

We investigated the inhibitory effects of 13 organophosphate esters (OPEs) and hydrolytic metabolites on the carboxylesterase activity of rat liver microsomes in vitro in order to examine whether there might be a potential impact on human health, and to elucidate the structure activity relationship. Among the test compounds, 2-ethylhexyl diphenyl phosphate (EDPhP) was the most potent inhibitor of carboxylesterase activity, as measured in terms of 4-nitrophenol acetate hydrolase activity, followed by tri-m-cresyl phosphate (TmCP), cresyl diphenyl phosphate (CDPhP) and triphenyl phosphate (TPhP). The IC50 values were as follows: EDPhP (IC50: 0.03 µM) > TmCP (0.4 µM) > CDPhP (0.8 µM) > TPhP (14 µM) > tris(1,3-dichloro-2-propyl) phosphate (17 µM) > tris(2-ethylhexyl) phosphate (77 µM) > tri-n-propyl phosphate (84 µM) > tris(2-chloroethyl) phosphate (104 µM) > tris(2-butoxyethyl) phosphate (124 µM) > tri-n-butyl phosphate (230 µM). The IC50 value of EDPhP was three orders of magnitude lower than that of bis(4-nitrophenyl) phosphate, which is widely used as an inhibitor of carboxylesterase. Trimethyl phosphate, triethyl phosphate and tris(2-chloroisopropyl) phosphate slightly inhibited the carboxylesterase activity; their IC50 values were above 300 µM. Lineweaver-Burk plots indicated that the inhibition by several OPEs was non-competitive. Diphenyl and monophenyl phosphates, which are metabolites of TPhP, showed weaker inhibitory effects than that of TPhP.


Assuntos
Carboxilesterase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Organofosfatos/farmacologia , Animais , Carboxilesterase/química , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Cinética , Estrutura Molecular , Organofosfatos/química , Ratos , Relação Estrutura-Atividade
14.
Nucleic Acids Res ; 48(14): 7973-7980, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597964

RESUMO

Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.


Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Exodesoxirribonuclease V/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Benzimidazóis/química , Sítios de Ligação , Inibidores Enzimáticos/química , Exodesoxirribonuclease V/química , Exodesoxirribonuclease V/genética , Exodesoxirribonuclease V/metabolismo , Mutação
15.
Eur J Med Chem ; 200: 112412, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32502861

RESUMO

Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by trimethoprim, a compound with antibacterial activity. Previously, we reported a trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety demonstrating increased potency against S. aureus. We have expanded this series and assessed in vitro enzyme inhibition (Ki) and whole cell growth inhibition properties (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle, as well as simultaneous modification at positions on the dihydrophthalazine. MIC values increased from 0.0626-0.5 µg/mL into the 0.5-1 µg/mL range when the edge positions were modified with either methyl or methoxy groups. Changes at the chiral carbon affected Ki measurements but with little impact on MIC values. Our structural data revealed accommodation of predominantly the S-enantiomer of the inhibitors within the folate-binding pocket. Longer modifications at the chiral carbon, such as p-methylbenzyl, protrude from the pocket into solvent and result in poorer Ki values, as do modifications with greater torsional freedom, such as 1-ethylpropyl. The most efficacious Ki was 0.7 ± 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of the phthalazine moiety into a shallow surface at the edge of the site that can accommodate either enantiomer of the inhibitor. The current design, therefore, highlights how to engineer specific placement of the inhibitor within this alternative pocket, which in turn maximizes the enzyme inhibitory properties of racemic mixtures.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Staphylococcus aureus/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Sítios de Ligação , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade , Trimetoprima/análogos & derivados , Trimetoprima/química
16.
J Biosci Bioeng ; 130(3): 239-246, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32389468

RESUMO

This research first reports the tyrosinase inhibition and mechanism of Leucrocin I and its modified peptides (TILI-1 and TILI-2). Docking simulation showed that these peptides were predicted to bind and interact to active site of tyrosinase and exhibited the possibility to promote tyrosinase inhibition. Therefore, these peptides were synthesized, and their inhibitory activity was investigated. The results showed that the highest tyrosinase inhibition was achieved by TILI-2 followed by TILI-1 and Leucrocin I. A Lineweaver-Burk plot indicated that Leucrocin I exhibited mixed type characteristics, while its modified peptides exhibited competitive inhibition. Based on the greatest tyrosinase inhibition, TILI-2 was selected for further study. TILI-2 showed irreversible inhibition with two-step inactivation. Additionally, Leucrocin I and its modified peptides showed no toxicity toward B16F1 and HaCaT cells and decreased melanin and tyrosinase content in B16F1 cells. These results suggest that these peptides are promising peptides for the treatment of hyperpigmentation.


Assuntos
Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Camundongos , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/metabolismo , Peptídeos/metabolismo , Conformação Proteica
17.
Nat Commun ; 11(1): 2478, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424158

RESUMO

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage. SOAT1 is a target to treat several human diseases. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 Å resolution reveals that a small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design targeting hSOAT1 and other mammalian MBOAT family members.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Esterol O-Aciltransferase/química , Sítios de Ligação , Biocatálise , Células HEK293 , Humanos , Ligantes , Multimerização Proteica , Esterol O-Aciltransferase/ultraestrutura , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos
18.
PLoS One ; 15(5): e0233468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469975

RESUMO

Transcription disequilibria are characteristic of many neurodegenerative diseases. The activity-evoked transcription of immediate early genes (IEGs), important for neuronal plasticity, memory and behavior, is altered in CNS diseases and governed by epigenetic modulation. KDM1A, a histone 3 lysine 4 demethylase that forms part of transcription regulation complexes, has been implicated in the control of IEG transcription. Here we report the development of vafidemstat (ORY-2001), a brain penetrant inhibitor of KDM1A and MAOB. ORY-2001 efficiently inhibits brain KDM1A at doses suitable for long term treatment, and corrects memory deficit as assessed in the novel object recognition testing in the Senescence Accelerated Mouse Prone 8 (SAMP8) model for accelerated aging and Alzheimer's disease. Comparison with a selective KDM1A or MAOB inhibitor reveals that KDM1A inhibition is key for efficacy. ORY-2001 further corrects behavior alterations including aggression and social interaction deficits in SAMP8 mice and social avoidance in the rat rearing isolation model. ORY-2001 increases the responsiveness of IEGs, induces genes required for cognitive function and reduces a neuroinflammatory signature in SAMP8 mice. Multiple genes modulated by ORY-2001 are differentially expressed in Late Onset Alzheimer's Disease. Most strikingly, the amplifier of inflammation S100A9 is highly expressed in LOAD and in the hippocampus of SAMP8 mice, and down-regulated by ORY-2001. ORY-2001 is currently in multiple Phase IIa studies.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Transtornos da Memória/tratamento farmacológico , Inibidores da Monoaminoxidase/farmacologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/psicologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/psicologia , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Epigênese Genética/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Inibidores da Monoaminoxidase/química , Inibidores da Monoaminoxidase/farmacocinética , Ratos , Ratos Sprague-Dawley
19.
Food Chem ; 326: 126962, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32413753

RESUMO

A concise method of natural blue pigment from Vaccinium bracteatum thunb. leaves (VBTL) was proposed firstly in this research. The potential properties of new pigment source needed to be assessed for further application. During the shelf life and thermal-accelerated storage, the blue pigment was prone to stable in the mildly acidic system, which was observed retention of 35.85% for storage of 12 weeks in pH 4.0. The 65% of blue pigment retained after the in vitro simulated digestion. The blue pigment exhibited inhibition effect on the pancreatic α-amylase (IC50 = 2.915 mg/mL). The analysis of inhibition kinetics indicated the blue pigment was an uncompetitive inhibitor. Through fluorescence quenching and circular dichroism study, the blue pigment inhibited α-amylase activity via the structural unfolding of α-amylase molecule and changing the secondary structure. These findings provided the new evidence of the digestion resistibility of VBTL pigment and the functional food - "Wu mi".


Assuntos
Inibidores Enzimáticos/química , Pigmentos Biológicos/química , Extratos Vegetais/química , Vaccinium myrtillus/química , alfa-Amilases/antagonistas & inibidores , Folhas de Planta/química , alfa-Amilases/química
20.
Food Chem ; 326: 126968, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428854

RESUMO

An understanding of the antityrosinase capacity and polyphenols changes during hydrothermal treatments was crucial for application of asparagus. Therefore, asparagus extract was treated at a range of 80-160 °C for 30-150 min in a high temperature reactor. The results suggested that tyrosinase inhibition rate of untreated asparagus extract was recorded as 3.26% but significantly increased to 51.22% and 50.80% after heating for 90 min at 140 °C (lnR0 of 7.21) and 160 °C (lnR0 of 8.57), respectively. The generation and degradation of polyphenols followed the pseudo-first-order kinetic model. The coumaric acid content was increased from 35.03 µg/mL to 307.66 µg/mL at lnR0 of 8.16. The degradation of rutin in asparagus extract was far less compared to that of coumaric acid. Compounds formed were determined by UPLC-Q-TOF-MS yielding main fragments at m/z 451 and 601. In conclusion, hydrothermal treatment was a feasible method for increasing the antityrosinase capacity of asparagus.


Assuntos
Asparagus (Planta)/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Polifenóis/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indústria de Processamento de Alimentos/métodos , Cinética , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/química , Polifenóis/análise , Polifenóis/farmacologia , Rutina/química , Temperatura
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