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1.
Sci Rep ; 11(1): 17810, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34497279

RESUMO

Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antivirais/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Comorbidade , Reposicionamento de Medicamentos , Humanos , Fígado/metabolismo , Fígado/patologia , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Lopinavir/química , Lopinavir/metabolismo , Lopinavir/farmacologia , Lopinavir/uso terapêutico , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Ritonavir/química , Ritonavir/metabolismo , Ritonavir/farmacologia , Ritonavir/uso terapêutico , SARS-CoV-2/isolamento & purificação , Especificidade por Substrato
2.
Science ; 373(6553): 413-419, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437114

RESUMO

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas
3.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
4.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209803

RESUMO

AMPylation is a prevalent posttranslational modification that involves the addition of adenosine monophosphate (AMP) to proteins. Exactly how Huntingtin-associated yeast-interacting protein E (HYPE), as the first human protein, is involved in the transformation of the AMP moiety to its substrate target protein (the endoplasmic reticulum chaperone binding to immunoglobulin protein (BiP)) is still an open question. Additionally, a conserved glutamine plays a vital key role in the AMPylation reaction in most filamentation processes induced by the cAMP (Fic) protein. In the present work, the detailed catalytic AMPylation mechanisms in HYPE were determined based on the density functional theory (DFT) method. Molecular dynamics (MD) simulations were further used to investigate the exact role of the inhibitory glutamate. The metal center, Mg2+, in HYPE has been examined in various coordination configurations, including 4-coordrinated, 5-coordinated and 6-coordinated. DFT calculations revealed that the transformation of the AMP moiety of HYPE with BiP followed a sequential pathway. The model with a 4-coordinated metal center had a barrier of 14.7 kcal/mol, which was consistent with the experimental value and lower than the 38.7 kcal/mol barrier of the model with a 6-coordinated metal center and the 31.1 kcal/mol barrier of the model with a 5-coordinated metal center. Furthermore, DFT results indicated that Thr518 residue oxygen directly attacks the phosphorus, while the His363 residue acts as H-bond acceptor. At the same time, an MD study indicated that Glu234 played an inhibitory role in the α-inhibition helix by regulating the hydrogen bond interaction between Arg374 and the Pγ of the ATP molecule. The revealed sequential pathway and the inhibitory role of Glu234 in HYPE were inspirational for understanding the catalytic and inhibitory mechanisms of Fic-mediated AMP transfer, paving the way for further studies on the physiological role of Fic enzymes.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Cristalografia por Raios X , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/química , Redes e Vias Metabólicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas
5.
Appl Environ Microbiol ; 87(18): e0069021, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34260306

RESUMO

Aryl coenzyme A (CoA) ligases belong to class I of the adenylate-forming enzyme superfamily (ANL superfamily). They catalyze the formation of thioester bonds between aromatic compounds and CoA and occur in nearly all forms of life. These ligases are involved in various metabolic pathways degrading benzene, toluene, ethylbenzene, and xylene (BTEX) or polycyclic aromatic hydrocarbons (PAHs). They are often necessary to produce the central intermediate benzoyl-CoA that occurs in various anaerobic pathways. The substrate specificity is very diverse between enzymes within the same class, while the dependency on Mg2+, ATP, and CoA as well as oxygen insensitivity are characteristics shared by the whole enzyme class. Some organisms employ the same aryl-CoA ligase when growing aerobically and anaerobically, while others induce different enzymes depending on the environmental conditions. Aryl-CoA ligases can be divided into two major groups, benzoate:CoA ligase-like enzymes and phenylacetate:CoA ligase-like enzymes. They are widely distributed between the phylogenetic clades of the ANL superfamily and show closer relationships within the subfamilies than to other aryl-CoA ligases. This, together with residual CoA ligase activity in various other enzymes of the ANL superfamily, leads to the conclusion that CoA ligases might be the ancestral proteins from which all other ANL superfamily enzymes developed.


Assuntos
Proteínas de Bactérias , Coenzima A Ligases , Monofosfato de Adenosina/metabolismo , Aerobiose , Anaerobiose , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Especificidade por Substrato
6.
Eur Rev Med Pharmacol Sci ; 25(10): 3923-3932, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34109607

RESUMO

Angiotensin converting enzyme 2 (ACE2) has potentially conflicting roles in health and disease. COVID-19 coronavirus binds to human cells via ACE2 receptor, which is expressed on almost all body organs. Boosting the ACE2 receptor levels on heart and lung cells may provide more cellular enter to virus thereby worsening the infection. Therefore, among the drug targets, ACE2 is suggested as a vital target of COVID-19 therapy. This hypothesis is based on the protective role of the drugs acting on ACE2. Therefore, this review discusses the impact and challenges of using ACE2 as a target in the current therapy of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Antivirais/química , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/uso terapêutico , Enzima de Conversão de Angiotensina 2/metabolismo , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/uso terapêutico , Antivirais/metabolismo , Antivirais/uso terapêutico , Azitromicina/química , Azitromicina/metabolismo , Azitromicina/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/metabolismo , Hidroxicloroquina/uso terapêutico , SARS-CoV-2/isolamento & purificação , Vitamina D/química , Vitamina D/metabolismo , Vitamina D/uso terapêutico
7.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065600

RESUMO

Curcumin is a natural bioactive component derived from the turmeric plant Curcuma longa, which exhibits a range of beneficial activities on human cells. Previously, an inhibitory effect of curcumin on platelets was demonstrated. However, it is unknown whether this inhibitory effect is due to platelet apoptosis or procoagulant platelet formation. In this study, curcumin did not activate caspase 3-dependent apoptosis of human platelets, but rather induced the formation of procoagulant platelets. Interestingly, curcumin at low concentration (5 µM) potentiated, and at high concentration (50 µM) inhibited ABT-737-induced platelet apoptosis, which was accompanied by inhibition of ABT-737-mediated thrombin generation. Platelet viability was not affected by curcumin at low concentration and was reduced by 17% at high concentration. Furthermore, curcumin-induced autophagy in human platelets via increased translocation of LC3I to LC3II, which was associated with activation of adenosine monophosphate (AMP) kinase and inhibition of protein kinase B activity. Because curcumin inhibits P-glycoprotein (P-gp) in cancer cells and contributes to overcoming multidrug resistance, we showed that curcumin similarly inhibited platelet P-gp activity. Our results revealed that the platelet inhibitory effect of curcumin is mediated by complex processes, including procoagulant platelet formation. Thus, curcumin may protect against or enhance caspase-dependent apoptosis in platelets under certain conditions.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Plaquetas/efeitos dos fármacos , Curcumina/farmacologia , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Monofosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Curcuma/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Piperazinas/farmacologia , Extratos Vegetais/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
8.
J Biol Chem ; 297(1): 100868, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34119520

RESUMO

In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ-which normally uses W-C base pairing for DNA synthesis-in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ's role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.


Assuntos
Monofosfato de Adenosina/análogos & derivados , DNA Polimerase beta/metabolismo , Replicação do DNA , Monofosfato de Adenosina/metabolismo , Pareamento de Bases , Linhagem Celular , Adutos de DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Mutação
9.
Biochemistry ; 60(24): 1869-1875, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34110129

RESUMO

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro, and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Ebolavirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Alanina/genética , Alanina/metabolismo , Antivirais/metabolismo , Pareamento de Bases , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo
10.
Biomed Res Int ; 2021: 6687551, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34104650

RESUMO

In the present study, we examined the synergetic effect of forskolin and mevastatin administration on lipid profile and lipid metabolism in omental adipose tissue in dyslipidemic rats. The study was conducted on forty male albino rats. The rats were randomly classified into four main groups of ten animals in each group as follows: group A, served as control nontreated; group B, rats that received Triton WR 1339 (500 mg/kg); group C, rats that received Triton WR 1339 with forskolin (100% FSK extract 0.5 mg/kg/day) for four weeks; and group D, dyslipidemic rats received both mevastatin and forskolin. At the end of the experimental period, blood and omental adipose tissue samples were collected, preserved, and used for biochemical determination of lipid profile and mRNA expression profile of adenylate cyclase (AC), hormone-sensitive lipase, respectively (HSL), and adenosine monophosphate-activated protein kinase (AMPK). The results showed a significant decline in the serum concentration of total cholesterol, LDL-cholesterol, and triglycerides, although there was a significant increase in serum levels of HDL-cholesterol and glycerol in rats received forskolin alone or with mevastatin when compared with control and dyslipidemic groups. The mRNA expression levels of AC, HSL, and AMPK were significantly increased in omental adipose tissue of rats received forskolin when compared with other groups. In conclusion, forskolin acts synergistically with mevastatin to lower lipid profile and improve lipid metabolism in dyslipidemic rats through upregulation of AMPK expression.


Assuntos
Monofosfato de Adenosina/metabolismo , Colforsina/farmacologia , Dislipidemias/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/fisiologia , Lovastatina/análogos & derivados , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , HDL-Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Dislipidemias/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lovastatina/farmacologia , Masculino , Ratos , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacos
11.
J Pharm Pharm Sci ; 24: 227-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048668

RESUMO

PURPOSE: Remdesivir and its active metabolite are predominantly eliminated via renal route; however, information regarding renal uptake transporters is limited. In the present study, the interaction of remdesivir and its nucleoside analog GS-441524 with OATP4C1 was evaluated to provide the detailed information about its renal handling. METHODS: We used HK-2 cells, a proximal tubular cell line derived from normal kidney, to confirm the transport of remdesivir and GS-441524. To assess the involvement of OATP4C1 in handling remdesivir and GS-441524, the uptake study of remdesivir and GS-441524 was performed by using OATP4C1-overexpressing Madin-Darby canine kidney II (MDCKII) cells. Moreover, we also evaluated the IC50 and Ki value of remdesivir. RESULTS: The time-dependent remdesivir uptake in HK-2 cells was observed. The results of inhibition study using OATs and OCT2 inhibitors and OATP4C1 knockdown suggested the involvement of renal drug transporter OATP4C1. Remdesivir was taken up by OATP4C1/MDCKII cells. OATP4C1-mediated uptake of remdesivir increased linearly up to 10 min and reached a steady state at 30 min. Remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner with the IC50 and apparent Ki values of 42 ± 7.8 µM and 37 ± 6.9 µM, respectively. CONCLUSIONS: We have provided novel information about renal handling of remdesivir. Furthermore, we evaluated the potential drug interaction via OATP4C1 by calculating the Ki value of remdesivir. OATP4C1 may play a pivotal role in remdesivir therapy for COVID-19, particularly in patients with kidney injury.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/metabolismo , COVID-19/tratamento farmacológico , Furanos/metabolismo , Túbulos Renais Proximais/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Pirróis/metabolismo , Triazinas/metabolismo , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/uso terapêutico , Alanina/metabolismo , Alanina/uso terapêutico , Animais , Antivirais/uso terapêutico , COVID-19/metabolismo , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Aprovação de Drogas , Furanos/uso terapêutico , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Células Madin Darby de Rim Canino , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Pirróis/uso terapêutico , Triazinas/uso terapêutico
13.
Bioorg Chem ; 112: 104967, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33975232

RESUMO

Nowadays, over 200 countries face a wellbeing emergency because of epidemiological disease COVID-19 caused by the SARS-CoV-2 virus. It will cause a very high effect on the world's economy and the worldwide health sector. The present work is an investigation of the newly synthesized 4-benzyl-1-(2,4,6-trimethyl-benzyl)-piperidine (M1BZP) molecule's inhibitory potential against important protein targets of SARS-CoV-2 using computational approaches. M1BZP crystallizes in monoclinic type with P1211 space group. For the title compound M1BZP, spectroscopic characterization like 1H NMR, 13C NMR, FTIR, were carried out. The geometry of the compound had been optimized by the DFT method and its results were compared with the X-ray diffraction data. The calculated energies for the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) showed the stability and reactivity of the title compound. Intermolecular interactions in the crystal network were determined using Hirshfeld surface analyses. The molecular electrostatic potential (MEP) picture was drawn using the same level of theory to visualize the chemical reactivity and charge distribution on the molecule. Molecular docking study performed for the synthesized compound revealed an efficient interaction with the COVID-19 protease and resulted in good activities. We hope the present study would help workers in the field to develop potential vaccines and therapeutics against the novel coronavirus. Virtual ADME studies were carried out as well and a relationship between biological, electronic, and physicochemical qualifications of the target compound was determined. Toxicity prediction by computational technique for the title compound was also carried out.


Assuntos
Antivirais/metabolismo , Piperidinas/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Antivirais/síntese química , Antivirais/química , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Cristalografia por Raios X , Teoria da Densidade Funcional , Meia-Vida , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Piperidinas/síntese química , Piperidinas/metabolismo , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo
14.
Nat Commun ; 12(1): 2426, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893288

RESUMO

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Repetições de Tetratricopeptídeos , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico/química , Homeostase , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
15.
Acta Biochim Biophys Sin (Shanghai) ; 53(6): 775-783, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33891090

RESUMO

Resveratrol, a natural compound extracted from the skins of grapes, berries, or other fruits, has been shown to have anti-tumor effects against multiple myeloma (MM) via promoting apoptosis and inhibiting cell viability. In addition to apoptosis, autophagy also plays a significant role in anti-tumor effects. However, whether autophagy is involved in anti-MM activity of resveratrol remains unclear. In this study, human MM cell lines U266, RPMI-8226, and NCI-H929 were treated with resveratrol. Cell Counting Kit-8 assay and colony formation assay were used to measure cell viability. Western blot analysis was used to detect apoptosis- and autophagy-associated proteins. 3-Methyladenine (3-MA) was applied to inhibit autophagy. Results showed that resveratrol inhibited cell viability and colony formation via promoting apoptosis and autophagy in MM cell lines U266, RPMI-8226, and NCI-H929. Resveratrol promoted apoptosis-related proteins, Caspase-3 activating poly-ADP-ribose polymerase and Caspase-3 cleavage, and decreased the protein level of Survivin in a dose-dependent manner. Additionally, resveratrol upregulated the levels of LC3 and Beclin1 in a dose-dependent way, indicating that autophagy might be implicated in anti-MM effect of resveratrol. Furthermore, 3-MA relieved the cytotoxicity of resveratrol by blocking the autophagic flux. Resveratrol increased the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase and decreased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream substrates p70S6K and 4EBP1 in a dose-dependent manner, leading to autophagy. Therefore, our results suggest that resveratrol exerts anti-MM effects through apoptosis and autophagy, which can be used as a new therapeutic strategy for MM in clinic.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mieloma Múltiplo/metabolismo , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Monofosfato de Adenosina/metabolismo , Proteína Beclina-1/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mieloma Múltiplo/patologia , Fosforilação/efeitos dos fármacos
16.
FEBS J ; 288(12): 3746-3771, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33825330

RESUMO

Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor activated during energy stress that plays a key role in maintaining energy homeostasis. This ubiquitous signaling pathway has been implicated in multiple functions including mitochondrial biogenesis, redox regulation, cell growth and proliferation, cell autophagy and inflammation. The protective role of AMPK in cardiovascular function and the involvement of dysfunctional AMPK in the pathogenesis of cardiovascular disease have been highlighted in recent years. In this review, we summarize and discuss the role of AMPK in the regulation of blood flow in response to metabolic demand and the basis of the AMPK physiological anticontractile, antioxidant, anti-inflammatory, and antiatherogenic actions in the vascular system. Investigations by others and us have demonstrated the key role of vascular AMPK in the regulation of endothelial function, redox homeostasis, and inflammation, in addition to its protective role in the hypoxia and ischemia/reperfusion injury. The pathophysiological implications of AMPK involvement in vascular function with regard to the vascular complications of metabolic disease and the therapeutic potential of AMPK activators are also discussed.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Vasos Sanguíneos/metabolismo , Doenças Cardiovasculares/metabolismo , Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Metabólicas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/metabolismo , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Vasos Sanguíneos/patologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Hipóxia/genética , Hipóxia/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos/genética , Doenças Metabólicas/genética , Doenças Metabólicas/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estresse Fisiológico
17.
Phys Chem Chem Phys ; 23(10): 5852-5863, 2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33688867

RESUMO

COVID-19 has recently caused a global health crisis and an effective interventional therapy is urgently needed. Remdesivir is one effective inhibitor for SARS-CoV-2 viral RNA replication. It supersedes other NTP analogues because it not only terminates the polymerization activity of RNA-dependent RNA polymerase (RdRp), but also inhibits the proofreading activity of intrinsic exoribonuclease (ExoN). Even though the static structure of Remdesivir binding to RdRp has been solved and biochemical experiments have suggested it to be a "delayed chain terminator", the underlying molecular mechanisms is not fully understood. Here, we performed all-atom molecular dynamics (MD) simulations with an accumulated simulation time of 24 microseconds to elucidate the inhibitory mechanism of Remdesivir on nucleotide addition and proofreading. We found that when Remdesivir locates at an upstream site in RdRp, the 1'-cyano group experiences electrostatic interactions with a salt bridge (Asp865-Lys593), which subsequently halts translocation. Our findings can supplement the current understanding of the delayed chain termination exerted by Remdesivir and provide an alternative molecular explanation about Remdesivir's inhibitory mechanism. Such inhibition also reduces the likelihood of Remdesivir to be cleaved by ExoN acting on 3'-terminal nucleotides. Furthermore, our study also suggests that Remdesivir's 1'-cyano group can disrupt the cleavage site of ExoN via steric interactions, leading to a further reduction in the cleavage efficiency. Our work provides plausible and novel mechanisms at the molecular level of how Remdesivir inhibits viral RNA replication, and our findings may guide rational design for new treatments of COVID-19 targeting viral replication.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Cianetos/química , Nucleotídeos/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/fisiologia , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Humanos , Simulação de Dinâmica Molecular , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Ribose/química , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Eletricidade Estática , Replicação Viral/efeitos dos fármacos
18.
J Tradit Chin Med ; 41(1): 107-116, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33522203

RESUMO

OBJECTIVE: To observe the therapeutic effect of Shenzhu Tiaopi granule (, STG) on insulin resistance (IR) in the liver of diabetic Goto-Kakizaki (GK) rat and investigate underlying mechanisms. METHODS: Ten 12-week-old male Wistar rats were assigned as normal control (NC) group, while 40 12-week-old male specific-pathogen-free GK rats were randomly divided into four experimental groups, 10 diabetic rats each. Animals were fed with a normal diet. Fasting blood glucose (FBG), water intake, and body weight were recorded during 6 weeks of daily single-dose treatment: STG low-dose group, 4.5 g/kg (STG-L); STG high-dose group,9 g/kg (STG-H); metformin group, 0.1 g/kg (MET); model control (MC) and NC groups, equal volume of 0.9% NaCl solution. The serum fasting insulin (FINS), C-Peptide and IR index (HOMA-IR) were detected every 2 weeks during treatment and glucose tolerance was measured in the 3rd day before the material was taken. After the 6-week STG treatment, Liver tissues were processed for hematoxylin-eosin staining to perform light microscopy analysis and for assessing expression and distribution of insulin receptor substrates (IRS-1) and glucose transporter (GLUT-4) by immunohistochemistry analysis. Expression levels of liver kinase B1 (LKB1) / adenosine 5'-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway proteins, including LKB1, phospho-AMPK (p-AMPK)/AMPK, phospho-mTOR (p-mTOR)/mTOR, and ribosomal protein S6 kinase polypeptide 1 (S6K1),were detected by Western blotting. RESULTS: STG significantly reduced the FBG level and liver fat deposition in diabetic GK rats. After STG treatment completion, FINS, HOMA-IR, C-Peptide and area under blood glucose curve (AUC) were lower in STG groups than in the MC group, indicating that IR was reduced and liver fat lesions were resolved. In liver tissues, STG groups displayed significantly higher IRS-1 and GLUT-4 expression than the MC group, along with increasedLKB1 and p-AMPK/AMPK expression and decreased p-mTOR/mTOR and phospho-S6K1expression, suggesting that STG stimulatedLKB1 activation of AMPK and suppressed them TOR/S6K1 downstream pathway. CONCLUSION: Growing GK rats developed hepatic IR, but STG treatment significantly improved hyperglycemia and IR and resolved hepatic fatty lesions. Interestingly, STG treatment stimulated the expression of IRS-1 and GLUT-4 in the liver of diabetic GK rats, indicating a potential involvement in the regulation of theLKB1/AMPK/mTOR signaling pathway.


Assuntos
Monofosfato de Adenosina/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Medicamentos de Ervas Chinesas/administração & dosagem , Resistência à Insulina , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Endogâmicos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
19.
Nat Commun ; 12(1): 460, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469029

RESUMO

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação/genética , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Guanosina Trifosfato/metabolismo , Humanos , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Fagocitose , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/isolamento & purificação , Proteínas rab1 de Ligação ao GTP/ultraestrutura
20.
Food Chem ; 347: 128973, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444888

RESUMO

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01-10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 µM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (-2.58 kcal·mol-1) and molecular distance (4.2 Å). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.


Assuntos
Citosol/enzimologia , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Vigna/enzimologia , Vigna/genética , Monofosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Frutose-Bifosfatase/química , Frutose-Bifosfatase/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Vigna/citologia
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