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1.
Nat Commun ; 12(1): 4195, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234119

RESUMO

SARS-CoV-2 can infect many domestic animals, including dogs. Herein, we show that dog angiotensin-converting enzyme 2 (dACE2) can bind to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD), and that both pseudotyped and authentic SARS-CoV-2 can infect dACE2-expressing cells. We solved the crystal structure of RBD in complex with dACE2 and found that the total number of contact residues, contact atoms, hydrogen bonds and salt bridges at the binding interface in this complex are slightly fewer than those in the complex of the RBD and human ACE2 (hACE2). This result is consistent with the fact that the binding affinity of RBD to dACE2 is lower than that of hACE2. We further show that a few important mutations in the RBD binding interface play a pivotal role in the binding affinity of RBD to both dACE2 and hACE2. Our work reveals a molecular basis for cross-species transmission and potential animal spread of SARS-CoV-2, and provides new clues to block the potential transmission chains of this virus.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Animais , Sítios de Ligação , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Cães , Células HeLa , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
2.
Virology ; 561: 107-116, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34217923

RESUMO

The fall of 2020 brought several new variants of SARS-CoV-2 circulating across the globe, and the steadily increasing COVID-19 cases are responsible for the emergence of these variants. All the SARS-CoV-2 variants reported to date have multiple mutations in the spike (S) protein, specifically in the receptor-binding domain (RBD). Here, we employed an integrated computational approach involving structure and sequence based predictions to study the effect of naturally occurring variations in the S-RBD on its stability and ACE2 binding affinity. The hotspot stabilizing residue mutations N501I, N501Y, Q493L, Q493H and K417R, strengthen the RBD-ACE2 complex by modulating the interaction statistics at the interface. Thus, we report here some critical mutations that could increase the binding affinity of the SARS-CoV-2 RBD with ACE2, increasing the viral infectivity and pathogenicity. Understanding the effect of these mutations will help in developing potential vaccines and therapeutics.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Sítios de Ligação , Mutação , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
3.
Nat Commun ; 12(1): 4108, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226550

RESUMO

DNA glycosylases must distinguish the sparse damaged sites from the vast expanse of normal DNA bases. However, our understanding of the nature of nucleobase interrogation is still limited. Here, we show that hNEIL1 (human endonuclease VIII-like 1) captures base lesions via two competing states of interaction: an activated state that commits catalysis and base excision repair, and a quarantine state that temporarily separates and protects the flipped base via auto-inhibition. The relative dominance of the two states depends on key residues of hNEIL1 and chemical properties (e.g. aromaticity and hydrophilicity) of flipped bases. Such a DNA repair mechanism allows hNEIL1 to recognize a broad spectrum of DNA damage while keeps potential gratuitous repair in check. We further reveal the molecular basis of hNEIL1 activity regulation mediated by post-transcriptional modifications and provide an example of how exquisite structural dynamics serves for orchestrated enzyme functions.


Assuntos
DNA Glicosilases/química , DNA Glicosilases/metabolismo , Reparo do DNA/fisiologia , Triagem , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , DNA/química , Dano ao DNA , DNA Glicosilases/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Especificidade por Substrato
4.
Nat Commun ; 12(1): 4151, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230484

RESUMO

The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of Gi1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation.


Assuntos
Quimiocinas/química , Quimiocinas/metabolismo , Receptores CCR5/química , Receptores CCR5/metabolismo , Sítios de Ligação , Quimiocina CCL3/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Microscopia Crioeletrônica , Ligantes , Modelos Moleculares , Conformação Proteica , Receptores CCR5/genética
5.
Cell Rep ; 36(2): 109353, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34237283

RESUMO

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Sítios de Ligação , Linhagem Celular , Reações Cruzadas , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Testes de Neutralização , Ligação Proteica/imunologia , Domínios Proteicos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química
6.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199119

RESUMO

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.


Assuntos
Sítios de Ligação , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Ligantes , Conformação Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutagênese , Ligação Proteica , Transporte Proteico , Ratos , Relação Estrutura-Atividade , Especificidade por Substrato
7.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199200

RESUMO

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV-N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV-N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV-N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV-N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.


Assuntos
Proteínas de Bactérias/metabolismo , Ebolavirus/metabolismo , HIV-1/metabolismo , Orthomyxoviridae/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Ebolavirus/imunologia , Ebolavirus/isolamento & purificação , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Polissacarídeos/imunologia , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/imunologia
8.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200244

RESUMO

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Bactérias/fisiologia , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica/métodos , Proteínas de Escherichia coli/genética , Modelos Moleculares , Ligação Proteica , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/ultraestrutura
9.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202053

RESUMO

Our understanding of the structure-function relationships of biomolecules and thereby applying it to drug discovery programs are substantially dependent on the availability of the structural information of ligand-protein complexes. However, the correct interpretation of the electron density of a small molecule bound to a crystal structure of a macromolecule is not trivial. Our analysis involving quality assessment of ~0.28 million small molecule-protein binding site pairs derived from crystal structures corresponding to ~66,000 PDB entries indicates that the majority (65%) of the pairs might need little (54%) or no (11%) attention. Out of the remaining 35% of pairs that need attention, 11% of the pairs (including structures with high/moderate resolution) pose serious concerns. Unfortunately, most users of crystal structures lack the training to evaluate the quality of a crystal structure against its experimental data and, in general, rely on the resolution as a 'gold standard' quality metric. Our work aims to sensitize the non-crystallographers that resolution, which is a global quality metric, need not be an accurate indicator of local structural quality. In this article, we demonstrate the use of several freely available tools that quantify local structural quality and are easy to use from a non-crystallographer's perspective. We further propose a few solutions for consideration by the scientific community to promote quality research in structural biology and applied areas.


Assuntos
Sítios de Ligação , Ligantes , Substâncias Macromoleculares/química , Modelos Moleculares , Proteínas/química , Cristalografia por Raios X , Substâncias Macromoleculares/metabolismo , Conformação Molecular , Ligação Proteica , Proteínas/metabolismo
10.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202294

RESUMO

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Di-Hidro-Orotase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Naftoquinonas/farmacologia , Pirimidinas/biossíntese , Antineoplásicos Fitogênicos/química , Sítios de Ligação , Produtos Biológicos/química , Domínio Catalítico , Di-Hidro-Orotase/química , Di-Hidro-Orotase/genética , Inibidores Enzimáticos/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Mutação , Naftoquinonas/química , Ligação Proteica , Relação Estrutura-Atividade
11.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202520

RESUMO

Trypanosoma brucei (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against T. brucei.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/classificação , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/metabolismo , Citosol/metabolismo , Imunofluorescência , Proteínas de Choque Térmico HSP70/química , Azul de Metileno/química , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Quercetina/química , Coloração e Rotulagem , Relação Estrutura-Atividade
12.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202606

RESUMO

Cancer is heterogeneous among patients, requiring a thorough understanding of molecular subtypes and the establishment of therapeutic strategies based on its behavior. Gastric cancer (GC) is adenocarcinoma with marked heterogeneity leading to different prognoses. As an effort, we previously identified a stem-like subtype, which is prone to metastasis, with the worst prognosis. Here, we propose FNBP1 as a key to high-level cell motility, present only in aggressive GC cells. FNBP1 is also up-regulated in both the GS subtype from the TCGA project and the EMT subtype from the ACRG study, which include high portions of diffuse histologic type. Ablation of FNBP1 in the EMT-type GC cell line brought changes in the cell periphery in transcriptomic analysis. Indeed, loss of FNBP1 resulted in the loss of invasive ability, especially in a three-dimensional culture system. Live imaging indicated active movement of actin in FNBP1-overexpressed cells cultured in an extracellular matrix dome. To find the transcription factor which drives FNBP1 expression in an EMT-type GC cell line, the FNBP1 promoter region and DNA binding motifs were analyzed. Interestingly, the Sp1 motif was abundant in the promoter, and pharmacological inhibition and knockdown of Sp1 down-regulated FNBP1 promoter activity and the transcription level, respectively. Taken together, our results propose Sp1-driven FNBP1 as a key molecule explaining aggressiveness in EMT-type GC cells.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas de Ligação a Ácido Graxo/genética , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição Sp1/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Matriz Extracelular , Proteínas de Ligação a Ácido Graxo/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Motivos de Nucleotídeos , Neoplasias Gástricas/patologia
13.
Nat Commun ; 12(1): 4249, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253716

RESUMO

5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that regulates gene expression. Charting the landscape of 5hmC in human tissues is fundamental to understanding its regulatory functions. Here, we systematically profiled the whole-genome 5hmC landscape at single-base resolution for 19 types of human tissues. We found that 5hmC preferentially decorates gene bodies and outperforms gene body 5mC in reflecting gene expression. Approximately one-third of 5hmC peaks are tissue-specific differentially-hydroxymethylated regions (tsDhMRs), which are deposited in regions that potentially regulate the expression of nearby tissue-specific functional genes. In addition, tsDhMRs are enriched with tissue-specific transcription factors and may rewire tissue-specific gene expression networks. Moreover, tsDhMRs are associated with single-nucleotide polymorphisms identified by genome-wide association studies and are linked to tissue-specific phenotypes and diseases. Collectively, our results show the tissue-specific 5hmC landscape of the human genome and demonstrate that 5hmC serves as a fundamental regulatory element affecting tissue-specific gene expression programs and functions.


Assuntos
5-Metilcitosina/análogos & derivados , Genoma Humano , Especificidade de Órgãos/genética , 5-Metilcitosina/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Metilação de DNA/genética , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Fatores de Transcrição/metabolismo
14.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199659

RESUMO

Herein we describe a combined experimental and in silico study of the interaction of a series of pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives (PBTs) with parallel G-quadruplex (GQ) DNA aimed at correlating their previously reported anticancer activities and the stabilizing effects observed by us on c-myc oncogene promoter GQ structure. Circular dichroism (CD) melting experiments were performed to characterize the effect of the studied PBTs on the GQ thermal stability. CD measurements indicate that two out of the eight compounds under investigation induced a slight stabilizing effect (2-4 °C) on GQ depending on the nature and position of the substituents. Molecular docking results allowed us to verify the modes of interaction of the ligands with the GQ and estimate the binding affinities. The highest binding affinity was observed for ligands with the experimental melting temperatures (Tms). However, both stabilizing and destabilizing ligands showed similar scores, whilst Molecular Dynamics (MD) simulations, performed across a wide range of temperatures on the GQ in water solution, either unliganded or complexed with two model PBT ligands with the opposite effect on the Tms, consistently confirmed their stabilizing or destabilizing ability ascertained by CD. Clues about a relation between the reported anticancer activity of some PBTs and their ability to stabilize the GQ structure of c-myc emerged from our study. Furthermore, Molecular Dynamics simulations at high temperatures are herein proposed for the first time as a means to verify the stabilizing or destabilizing effect of ligands on the GQ, also disclosing predictive potential in GQ-targeting drug discovery.


Assuntos
DNA/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/química , Telômero/química , Sítios de Ligação/efeitos dos fármacos , Dicroísmo Circular , Simulação por Computador , DNA/química , DNA/ultraestrutura , Humanos , Ligantes , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/ultraestrutura , Telômero/efeitos dos fármacos , Telômero/genética
15.
Molecules ; 26(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205704

RESUMO

The discovery of drugs capable of inhibiting SARS-CoV-2 is a priority for human beings due to the severity of the global health pandemic caused by COVID-19. To this end, repurposing of FDA-approved drugs such as NSAIDs against COVID-19 can provide therapeutic alternatives that could be utilized as an effective safe treatment for COVID-19. The anti-inflammatory activity of NSAIDs is also advantageous in the treatment of COVID-19, as it was found that SARS-CoV-2 is responsible for provoking inflammatory cytokine storms resulting in lung damage. In this study, 40 FDA-approved NSAIDs were evaluated through molecular docking against the main protease of SARS-CoV-2. Among the tested compounds, sulfinpyrazone 2, indomethacin 3, and auranofin 4 were proposed as potential antagonists of COVID-19 main protease. Molecular dynamics simulations were also carried out for the most promising members of the screened NSAID candidates (2, 3, and 4) to unravel the dynamic properties of NSAIDs at the target receptor. The conducted quantum mechanical study revealed that the hybrid functional B3PW91 provides a good description of the spatial parameters of auranofin 4. Interestingly, a promising structure-activity relationship (SAR) was concluded from our study that could help in the future design of potential SARS-CoV-2 main protease inhibitors with expected anti-inflammatory effects as well. NSAIDs may be used by medicinal chemists as lead compounds for the development of potent SARS-CoV-2 (Mpro) inhibitors. In addition, some NSAIDs can be selectively designated for treatment of inflammation resulting from COVID-19.


Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Anti-Inflamatórios não Esteroides/metabolismo , Antivirais/química , Antivirais/farmacologia , Auranofina/química , Auranofina/farmacologia , Sítios de Ligação , COVID-19/complicações , Biologia Computacional , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/etiologia , Bases de Dados de Compostos Químicos , Humanos , Indometacina/química , Indometacina/farmacologia , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfimpirazona/química , Sulfimpirazona/farmacologia , Estados Unidos , United States Food and Drug Administration
16.
Molecules ; 26(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205768

RESUMO

Since December 2019, novel coronavirus disease 2019 (COVID-19) pandemic has caused tremendous economic loss and serious health problems worldwide. In this study, we investigated 14 natural compounds isolated from Amphimedon sp. via a molecular docking study, to examine their ability to act as anti-COVID-19 agents. Moreover, the pharmacokinetic properties of the most promising compounds were studied. The docking study showed that virtually screened compounds were effective against the new coronavirus via dual inhibition of SARS-CoV-2 RdRp and the 3CL main protease. In particular, nakinadine B (1), 20-hepacosenoic acid (11) and amphimedoside C (12) were the most promising compounds, as they demonstrated good interactions with the pockets of both enzymes. Based on the analysis of the molecular docking results, compounds 1 and 12 were selected for molecular dynamics simulation studies. Our results showed Amphimedon sp. to be a rich source for anti-COVID-19 metabolites.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Proteases 3C de Coronavírus/química , Poríferos/química , Poríferos/metabolismo , RNA Polimerase Dependente de RNA/química , SARS-CoV-2/efeitos dos fármacos , Amino Açúcares/química , Amino Açúcares/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Sítios de Ligação , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacocinética , COVID-19/tratamento farmacológico , Biologia Computacional , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Piridinas/química , Piridinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/metabolismo
17.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208159

RESUMO

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V-FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.


Assuntos
Apoptose/genética , Peixes-Gato/genética , Citocromos c/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Citocromos c/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência
18.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208199

RESUMO

Thus far, many hypotheses have been proposed explaining the cause of depression. Among the most popular of these are: monoamine, neurogenesis, neurobiology, inflammation and stress hypotheses. Many studies have proven that neurogenesis in the brains of adult mammals occurs throughout life. The generation of new neurons persists throughout adulthood in the mammalian brain due to the proliferation and differentiation of adult neural stem cells. For this reason, the search for drugs acting in this mechanism seems to be a priority for modern pharmacotherapy. Paroxetine is one of the most commonly used antidepressants. However, the exact mechanism of its action is not fully understood. The fact that the therapeutic effect after the administration of paroxetine occurs after a few weeks, even if the levels of monoamine are rapidly increased (within a few minutes), allows us to assume a neurogenic mechanism of action. Due to the confirmed dependence of depression on serotonin, norepinephrine, dopamine and γ-aminobutyric acid levels, studies have been undertaken into paroxetine interactions with these primary neurotransmitters using in silico and in vitro methods. We confirmed that paroxetine interacts most strongly with monoamine transporters and shows some interaction with γ-aminobutyric acid transporters. However, studies of the potency inhibitors and binding affinity values indicate that the neurogenic mechanism of paroxetine's action may be determined mainly by its interactions with serotonin transporters.


Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Paroxetina/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Animais , Sítios de Ligação , Células CHO , Cricetulus , Humanos , Simulação de Acoplamento Molecular , Neurotransmissores/química , Neurotransmissores/metabolismo , Paroxetina/química
19.
J Mol Model ; 27(8): 221, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236507

RESUMO

Natural products have served human life as medications for centuries. During the outbreak of COVID-19, a number of naturally derived compounds and extracts have been tested or used as potential remedies against COVID-19. Tetradenia riparia extract is one of the plant extracts that have been deployed and claimed to manage and control COVID-19 by some communities in Tanzania and other African countries. The active compounds isolated from T. riparia are known to possess various biological properties including antimalarial and antiviral. However, the underlying mechanism of the active compounds against SARS-CoV-2 remains unknown. Results in the present work have been interpreted from the view point of computational methods including molecular dynamics, free energy methods, and metadynamics to establish the related mechanism of action. Among the constituents of T. riparia studied, luteolin inhibited viral cell entry and was thermodynamically stable. The title compound exhibit residence time and unbinding kinetics of 68.86 ms and 0.014 /ms, respectively. The findings suggest that luteolin could be potent blocker of SARS-CoV-2 cell entry. The study shades lights towards identification of bioactive constituents from T. riparia against COVID-19, and thus bioassay can be carried out to further validate such observations.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Luteolina/farmacologia , Simulação de Dinâmica Molecular , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/isolamento & purificação , Antivirais/metabolismo , Sítios de Ligação , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Cinética , Lamiaceae/química , Luteolina/isolamento & purificação , Luteolina/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Virais/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo
20.
J Mol Model ; 27(8): 222, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236527

RESUMO

The crescent evolution of a global pandemic COVID-19 and its respiratory syndrome (SARS-Cov-2) has been a constant concern (Ghosh 2021; Khan et al. 2021; Alazmi and Motwalli 2020; Vargas et al. 2020). The absence of a proven and effective medication has compelled all the scientific community to search for a new drug. The use of known drugs is a faster way to develop new therapies. Molecular docking is a powerful tool (Gao et al. J Mol Model 10: 44-54, 2004; Singh et al. J Mol Model 18: 39-51, 2012; Schulz-Gasch and Stahl J Mol Model 9:47-57, 2003) to study the interaction of potential drugs with SARS-CoV-2, Alsalme et al. (2020) and Sanders et al. (2020) spike protein as a consequence the main goal of this article is to present the result of the study of an interaction between (R and S)-Linezolid with receptor-binding domain (RBD) of SARS-Cov-2 spike protein complexed with human Angiostensin-converting enzyme 2 (ACE2) (6vW1 - from PDB). The Linezolid enantiomers were optimized at B3LYP/6-311++G(2d,p) level of theory. Molecular docking of the system (S)-Linezolid⋯RBD⋯ACE2 and (R)-Linezolid⋯RBD⋯ACE2 was performed, the analysis was made using LigPlot+ and NCIplot software packages, to understand the intermolecular interactions. The UV-Vis and ECD of the complexes - (R and S)-Linezolid⋯RBD⋯ACE2 were performed in two layers with DFT/6-311++G(3df,2p) and DFT/6-31G(d), respectively. The results showed that only the (S)-Linezolid had a stable interaction with - 8.05 kcal.mol- 1, whereas all the R-enantiomeric configurations had positive values of binding energy. The (S)-Linezolid had the same interactions as in the (S)-Linezolid ⋯ Haluarcula morismortui Ribosomal system, where it is well-known the fact that the latter has biological activity. A specific interaction on the fluorine ring justified an attenuation on the ECD signal, in comparison to isolated species. Therefore, some biological activity of (S)-Linezolid with SARS-CoV-2 RBD was expected, indicated by the modification of its ECD signal and justified by a similar interaction in the S-Linezolid⋯Haluarcula marismortui Ribosomal system.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Linezolida/farmacologia , Simulação de Acoplamento Molecular , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/metabolismo , Sítios de Ligação , COVID-19/virologia , Interações Hospedeiro-Patógeno , Humanos , Cinética , Linezolida/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Virais/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade
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