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1.
Nat Commun ; 11(1): 4333, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859933

RESUMO

Diarrhoea is one of the most burdensome and common adverse events of chemotherapeutics, and has no standardised therapy to date. Increasing evidence suggests that the gut microbiome can influence the development of chemotherapy-induced diarrhoea. Here we report findings from a randomised clinical trial of faecal microbiota transplantation (FMT) to treat diarrhoea induced by tyrosine kinase inhibitors (TKI) in patients with metastatic renal cell carcinoma (ClinicalTrials.gov number: NCT04040712). The primary outcome is the resolution of diarrhoea four weeks after the end of treatments. Twenty patients are randomised to receive FMT from healthy donors or placebo FMT (vehicle only). Donor FMT is more effective than placebo FMT in treating TKI-induced diarrhoea, and a successful engraftment is observed in subjects receiving donor faeces. No serious adverse events are observed in both treatment arms. The trial meets pre-specified endpoints. Our findings suggest that the therapeutic manipulation of gut microbiota may become a promising treatment option to manage TKI-dependent diarrhoea.


Assuntos
Carcinoma de Células Renais/complicações , Diarreia/terapia , Inibidores Enzimáticos/metabolismo , Transplante de Microbiota Fecal/métodos , Neoplasias Renais/complicações , Tirosina/metabolismo , Idoso , Método Duplo-Cego , Tratamento Farmacológico , Disbiose , Fezes , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Pessoa de Meia-Idade , Doadores de Tecidos
2.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas não Estruturais Virais/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
3.
Life Sci ; 256: 117914, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512010

RESUMO

Hyperglycemia and oxidative stress are the primary stressors that elicit mitochondria specific cell stress in diabetes. Here we hypothesized that elevated level of ROS in high glucose (HG) environment, trigger mitochondrial stress by damaging mitochondrial DNA (mtDNA), altering inflammatory mediators, and neurodegenerative markers via stress signalling pathway in retinal ganglion cells (RGC-5). Mechanistically, our findings illustrated that the HG environment increases the ROS production in retinal cells leading to the disruption of antioxidant defence mechanism, and altering mitochondrial machinery such as an increase in loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, and increase in mtDNA fragmentation. Furthermore, fragmented mtDNA escape from mitochondria into the cytosol, where it engaged with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN gene (STING) phosphorylation and activate interferon regulatory factor 3 (IRF3) via ERK1/2-Akt-tuberin-mTOR dependent pathways. Our results further indicate that siRNA-mediated gene silencing of tuberin suppresses the strong downregulation of tuberin-mTOR-IRF3 activation. HG environment resulted in activation of IRF3, coinciding with the increased expression of inflammatory mediators and neurodegenerative markers. Pre-treatment of N-acetyl-l-cysteine (NAC) or ERK1/2 or phosphoinositide3-kinase (PI3-K)/Akt inhibitors in RGC-5 cells significantly reduced the HG-induced IRF3 expression and declined the expression of neurodegenerative markers. Collectively, our results demonstrates that HG-induced over production of ROS, disrupts the antioxidant defence mechanism and mitochondrial dysfunction, leading to alterations of inflammatory mediators and neurodegenerative markers through the ERK1/2-Akt-tuberin-mTOR dependent signalling pathway in RGC-5 cells.


Assuntos
Inibidores Enzimáticos/metabolismo , Glucose/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Ganglionares da Retina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Acetilcisteína/metabolismo , Animais , DNA Mitocondrial , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/metabolismo , Inflamação/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , NADPH Oxidases/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
4.
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
5.
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
6.
Nat Chem Biol ; 16(6): 667-675, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393901

RESUMO

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.


Assuntos
Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de Sinais
7.
Xenobiotica ; 50(10): 1170-1179, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32367776

RESUMO

Herbs are often administered in combination with therapeutic drugs, raising the possibility for herb-drug interactions (HDIs). Furoquinoline alkaloids are found in Rutaceae plants, which are structurally similar and have many medicinal properties. This study aims to investigate the inhibition of four furoquinoline alkaloids on the activity of UDP-glucuronosyltransferases (UGTs).The recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition type and parameters were determined, and in silico docking was employed to elucidate the inhibition difference of furoquinoline alkaloids towards UGTs.Dictamine, haplopine, γ-fagarine and skimmianine strongly inhibited UGT1A3, UGT1A7, UGT1A9 and UGT2B4, respectively. Among them, dictamnine inhibited more than 70% of the four UGTs. Inhibition kinetics determination showed that they all exerted competitive inhibition, and the inhibition kinetic constant (Ki) was determined to be 8.3, 7.2, 3.7 and 33.9 µM, respectively. In vitro-in vivo extrapolation (IVIVE) was employed to demonstrate the inhibition possibility for four alkaloids. Skimmianine was proved to be more suitable for clinical application. In silico docking study indicated that the hydrophobic interactions played a key role in the inhibition of furoquinoline alkaloids towards three of the four UGTs. In conclusion, monitoring the interactions between furoquinoline alkaloids and drugs mainly undergoing UGTs-catalyzed metabolism is necessary.


Assuntos
Inibidores Enzimáticos/metabolismo , Glucuronosiltransferase/metabolismo , Himecromona/metabolismo , Alcaloides , Simulação por Computador , Interações Ervas-Drogas , Humanos , Simulação de Acoplamento Molecular , Quinolinas
8.
Science ; 368(6498): 1499-1504, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32358203

RESUMO

The pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global crisis. Replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp) enzyme, a target of the antiviral drug remdesivir. Here we report the cryo-electron microscopy structure of the SARS-CoV-2 RdRp, both in the apo form at 2.8-angstrom resolution and in complex with a 50-base template-primer RNA and remdesivir at 2.5-angstrom resolution. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp, where remdesivir is covalently incorporated into the primer strand at the first replicated base pair, and terminates chain elongation. Our structures provide insights into the mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/química , Betacoronavirus/enzimologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Antivirais/metabolismo , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Domínio Catalítico , Microscopia Crioeletrônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , RNA Replicase/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
9.
Arch Biochem Biophys ; 687: 108369, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32335047

RESUMO

A neutral tea polysaccharide (TPSN) was isolated from green tea. Gas chromatography analysis showed that TPSN was composed of d-glucose, l-arabinose and d-galactose residues at a molar ratio of 90.0: 9.1: 0.9. The weight-averaged molecular weight of TPSN was determined as about 2.0 × 105 g mol-1 using static light scattering analysis. The result of nuclear magnetic resonance (NMR) spectroscopy indicated that TPSN and water-soluble starch had similar structures. TPSN exhibited inhibitory activity towards α-amylase through the noncompetitive inhibition mechanism, but the tertiary structure of α-amylase related to enzymatic activity, analyzed using circular dichroism spectroscopy, was not affected by TPSN. Meanwhile, TPSN exhibited hydrolysis properties catalyzed by α-amylase. Molecular docking analysis revealed that the various behaviors of TPSN to α-amylase could be attributed to that the different chain segments of TPSN combined with different amino acid residues of α-amylase.


Assuntos
Inibidores Enzimáticos/química , Polissacarídeos/química , Chá/química , alfa-Amilases/antagonistas & inibidores , Animais , Camellia sinensis/química , Ensaios Enzimáticos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Peso Molecular , Polissacarídeos/isolamento & purificação , Polissacarídeos/metabolismo , Ligação Proteica , Suínos , alfa-Amilases/metabolismo
10.
PLoS One ; 15(4): e0224853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298262

RESUMO

Diets rich in flavonoids have been related with low obesity rates, which could be related with their potential to inhibit pancreatic lipase, the main enzyme of fat assimilation. Some flavonoids can aggregate in aqueous medium suggesting that the inhibition mechanism could occur on both molecular and colloidal levels. This study investigates the interaction of two flavonoid aggregates, quercetin and epigallocatechin gallate (EGCG), with pancreatic lipase under simplified intestinal conditions. The stability and the morphology of these flavonoid aggregates were studied in four different solutions: Control (water), salt, low lipase concentration and high lipase concentration. Particles were found by optical microscopy in almost all the solutions tested, except EGCG-control. The results show that the precipitation rate decreases for quercetin and increases for EGCG in salt solution and that lipase stabilize quercetin aggregates. In addition, both flavonoids were shown to precipitate together with pancreatic lipase resulting in a sequestering of the enzyme.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Mucosa Intestinal/metabolismo , Lipase/metabolismo , Quercetina/farmacologia , Animais , Antioxidantes/metabolismo , Catequina/metabolismo , Catequina/farmacologia , Dimerização , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Lipase/antagonistas & inibidores , Quercetina/metabolismo , Suínos
11.
Chem Biol Interact ; 322: 109053, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198085

RESUMO

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 µM and 0.62 min-1, respectively. The calculated kobs at 10 µM was 0.29 min-1, above the 0.02 min-1 risk level. After incubation with NOT at 10 µM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.


Assuntos
Cumarínicos/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Ativação Metabólica , Apiaceae/química , Apiaceae/metabolismo , Cumarínicos/química , Citocromo P-450 CYP2D6/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
12.
J Med Chem ; 63(8): 3915-3934, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32212728

RESUMO

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Indazóis/química , Indazóis/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indazóis/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo
13.
J Med Chem ; 63(7): 3538-3551, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32134266

RESUMO

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.


Assuntos
Inibidores Enzimáticos/química , Glicogênio Sintase/antagonistas & inibidores , Imidazóis/química , Pirazóis/química , Animais , Caenorhabditis elegans/enzimologia , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/metabolismo , Cinética , Estrutura Molecular , Ligação Proteica , Pirazóis/síntese química , Pirazóis/metabolismo , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade
14.
J Med Chem ; 63(6): 3172-3187, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32125853

RESUMO

Drug resistance has been a major threat in cancer therapies that necessitates the development of new strategies to overcome this problem. We report here a cell-based high-throughput screen of a library containing two-million molecules for the compounds that inhibit the proliferation of non-small-cell lung cancer (NSCLC). Through the process of phenotypic screening, target deconvolution, and structure-activity relationship (SAR) analysis, a compound of furanonaphthoquinone-based small molecule, AS4583, was identified that exhibited potent activity in tyrosine kinase inhibitor (TKI)-sensitive and TKI-resistant NSCLC cells (IC50 = 77 nM) and in xenograft mice. The mechanistic studies revealed that AS4583 inhibited cell-cycle progression and reduced DNA replication by disrupting the formation of the minichromosomal maintenance protein (MCM) complex. Subsequent SAR study of AS4583 gave compound RJ-LC-07-48 which exhibited greater potency in drug-resistant NSCLC cells (IC50 = 17 nM) and in mice with H1975 xenograft tumor.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Furanos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Manutenção de Minicromossomo/metabolismo , Naftoquinonas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Furanos/síntese química , Furanos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Naftoquinonas/síntese química , Naftoquinonas/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Med Chem ; 63(6): 3215-3226, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32142284

RESUMO

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.


Assuntos
Inibidores Enzimáticos/química , Indóis/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Tiofenos/química , Sítio Alostérico , Cristalografia por Raios X , Descoberta de Drogas , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Indóis/síntese química , Indóis/metabolismo , Estrutura Molecular , NAD/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Espermidina/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(12): 6531-6539, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156733

RESUMO

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas , Inibidores Enzimáticos/metabolismo , Staphylococcus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sequência Conservada , DNA/metabolismo , Edição de Genes , Genoma Bacteriano/genética , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Staphylococcus/química , Staphylococcus aureus/enzimologia
17.
Food Chem ; 318: 126482, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32145543

RESUMO

We have examined the trans-resveratrol/lipase interaction by quantitative and qualitative analyses of fluorescence spectra, molecular docking and quantum-chemical calculations at DFT level. Interactions of CpLIP2 from C. parapsilosis CBS 604 and trans-resveratrol were confirmed with a major contribution of tryptophan residues to fluorescence quenching. A thermodynamic study across a wide temperature range was consistent with the presence of a single binding site with a binding free energy of -24 kJ/mol. Nevertheless, trans-resveratrol competitively inhibited CpLIP2 activity. Molecular docking and quantum-chemical calculations were consistent with a strong binding of trans-resveratrol to the CpLIP2 catalytic site via electrostatic and hydrophobic forces. The structural analysis quantitatively revealed an energy transfer from W51 and W350 to trans-resveratrol with a distance of 32 Å. Precise understanding of trans-resveratrol/CpLIP2 interactions has important implications on lipases for screening of stilbenoid.


Assuntos
Candida parapsilosis/enzimologia , Lipase/metabolismo , Resveratrol/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Fluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Lipase/antagonistas & inibidores , Lipase/química , Simulação de Acoplamento Molecular , Resveratrol/química , Resveratrol/farmacocinética , Termodinâmica
18.
Phys Chem Chem Phys ; 22(10): 5487-5499, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32101223

RESUMO

Ubiquitin specific protease 7 (USP7) has attracted increasing attention because of its multifaceted roles in different tumor types. The crystal structures of USP7-inhibitor complexes resolved recently provide reliable models for computational structure-based drug design (SBDD) towards USP7. How to accurately estimate USP7-ligand binding affinity is quite critical to guarantee the reliability of SBDD. In this study, we assessed the reliability of multiple computational methods to the binding affinity prediction for a series of USP7 inhibitors with the pyrimidinone scaffold, including molecular docking scoring, MM/PB(GB)SA, and umbrella sampling (US). It was found that the accuracy of the evaluated computational methods for binding affinity prediction follows the order: US-based method > MM/PB(GB)SA > Glide XP scoring. The calculation results demonstrate that incorporating protein flexibility through induced-fit docking or ensemble docking cannot improve the performance of the Glide scoring based on rigid-receptor docking. For the MM/PB(GB)SA methods, the choice of the protein structure and the calculation procedure has a marked impact on the predictions. More importantly, we discovered for the first time that there are significant differences in the dissociation pathways of strong-binding inhibitors and weak-binding inhibitors of USP7, which may be used as a new criterion to judge whether an inhibitor is a strong binder or not. It is expected that our work can provide valuable guidance on the design and discovery of potent USP7 inhibitors.


Assuntos
Química Computacional , Inibidores Enzimáticos/metabolismo , Pirimidinonas/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Ligação Proteica
19.
Life Sci ; 253: 117360, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32001269

RESUMO

AIMS: Progesterone receptor membrane component 1 (PGRMC1) has been reported to mediate the neuroprotective effect of progesterone, but the exact mechanism has not been elucidated. Therefore, the purpose of this study was to investigate the signalling pathway downstream of PGRMC1 in progesterone-induced neuroprotection. Recognition of the mechanism of progesterone opens novel perspectives for the treatment of diseases of the nervous system. MAIN METHODS: The PGRMC1 protein level was knocked down in rat primary cortical neurons, and Aß25-35 was used to establish an Alzheimer's disease cell model. The neuroprotective effect of progesterone was assessed by Hoechst 33258 staining and a cell counting kit-8 (CCK-8) assay. Then, proteomic and bioinformatic methods were used to analyse the proteins altered in response to PGRMC1 silencing to identify target proteins and signalling pathways involved in PGRMC1-mediated progesterone-induced neuroprotection. These findings were further verified by using signalling pathway inhibitors and western blotting. KEY FINDINGS: The neuroprotective effect of progesterone was significantly attenuated with PGRMC1 silencing. The expression of many proteins in the Ras signalling pathway was significantly changed in response to PGRMC1 silencing. FTI-277 inhibited progesterone-induced neuroprotection. Progesterone increased the expression of total Ras and Grb2. SIGNIFICANCE: These findings provide new perspectives for understanding the mechanism of and role of PGRMC1 in progesterone-induced neuroprotection. The Ras signalling pathway is the signalling pathway downstream of PGRMC1 in the mediation of progesterone-induced neuroprotection.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Proteínas de Membrana/metabolismo , Neuroproteção/efeitos dos fármacos , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose , Sobrevivência Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2/metabolismo , Técnicas de Inativação de Genes/métodos , Inativação Gênica , Humanos , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteômica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Espectrometria de Massas em Tandem
20.
Biochemistry ; 59(7): 831-835, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32022543

RESUMO

Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.


Assuntos
Adenina/análogos & derivados , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/metabolismo , Adenina/química , Adenina/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Domínio Catalítico , Inibidores Enzimáticos/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ligação Proteica , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/química , Pirrolidinas/química
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