RESUMO
Diabetic neuropathy (DN) encompasses a group of clinical or subclinical manifestations involving a dysfunction in the peripheral nervous system. The cause of the dysfunction is the development of microvascular complications related to diabetes, a disease that affects about 381 million people worldwide. Approximately 50% of patients currently diagnosed with diabetes are expected to manifest DN in the next 10 years. The diagnosis can be made clinically by establishing a good patient history and delving into the symptoms to rule out other etiologies. Treatment of DN focuses on glycemic control and the use of medications to reduce pain, including NSAIDs, antidepressants and antiepileptic drugs. The pathogenesis is of multifactorial origin, associated with various metabolic, vascular, inflammatory and neurodegenerative disorders. The three fundamental cellular alterations participating in the development of DN are chronic inflammation, endothelial dysfunction and oxidative stress. Since the combination of all three is capable of giving rise to nerve ischemia and direct axonal injury, these factors play a key role in the development of polyneuropathy. However, neuronal and microvascular changes do not occur in the same way in all patients with DN, some of whom have no detectable blood abnormalities.
Assuntos
Diabetes Mellitus , Neuropatias Diabéticas , Neuropatias Diabéticas/diagnóstico , Neuropatias Diabéticas/tratamento farmacológico , Humanos , Inflamação/complicações , Estresse OxidativoRESUMO
Generation 4 of polyamidoamine dendrimer (G4-PAMAM) has several biological effects due to its tridimensional globular structure, repetitive branched amides, tertiary amines, and amino-terminal subunit groups liked to a common core. G4-PAMAM is cytotoxic due to its positive charges. However, its cytotoxicity could increase in cancer cells due to the excessive intracellular negative charges in these cells. Furthermore, this work reports G4-PAMAM chemical structural characterization using UHPLC-QTOF-MS/MS (LC-MS) by electrospray ionization to measure its population according to its positive charges. Additionally, the antiproliferative effects and intracellular localization were explored in the HMC-1 and K-562 cell lines by confocal microscopy. The LC-MS results show that G4-PAMAM generated multivalent mass spectrum values, and its protonated terminal amino groups produced numerous positive charges, which allowed us to determine its exact mass despite having a high molecular weight. Additionally, G4-PAMAM showed antiproliferative activity in the HMC-1 tumor cell line after 24 h (IC50 = 16.97 µM), 48 h (IC50 = 7.02 µM) and 72 h (IC50 = 5.98 µM) and in the K-562 cell line after 24 h (IC50 = 15.14 µM), 48 h (IC50 = 14.18 µM) and 72 h (IC50 = 9.91 µM). Finally, our results showed that the G4-PAMAM dendrimers were located in the cytoplasm and nucleus in both tumor cell lines studied.
Assuntos
Dendrímeros/farmacologia , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Nylons/farmacologia , Antineoplásicos/análise , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida , Dendrímeros/análise , Dendrímeros/farmacocinética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Concentração Inibidora 50 , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Células K562 , Leucemia/patologia , Nylons/análise , Nylons/farmacocinética , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
BACKGROUND: Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer activity, but is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties against HeLa, rhabdomyosarcoma and breast cancer cell cultures. OBJECTIVE: We studied the epigenetic mechanism of o-OH-VPA as an HDACI and evaluated whether it was toxic to normal cells. METHODS: HeLa cells and primary human fibroblasts were used for this study as carcinogenic and normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and doubling time were determined by the Trypan-blue method. HDAC activity was tested using the colorimetric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cytometry and caspase-3 colorimetric assay, respectively. RESULTS: o-OH-VPA (IC50 = 0.1 mM) was fifty-eight times more effective than VPA (IC50 = 5.8 mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affecting the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the increase in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity toward normal cells. CONCLUSION: Our results revealed that o-OH-VPA is an HDACI with a selective effect against HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.
Assuntos
Epigênese Genética , Ácido Valproico , Amidas , Linhagem Celular Tumoral , Células HeLa , Histona Desacetilases/metabolismo , Humanos , Pentanos , Proteínas Repressoras/metabolismo , Ácido Valproico/farmacologiaRESUMO
Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.
Assuntos
Antineoplásicos , Glutamina , Ácido Valproico , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Glutaminase/antagonistas & inibidores , Glutaminase/química , Glutamina/química , Glutamina/farmacologia , Células HeLa , Humanos , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Metabolômica , Modelos Moleculares , Ácido Valproico/química , Ácido Valproico/farmacologiaRESUMO
The fucosterol has been reported numerous biological activities. In this study, the activity in vitro of the fucosterol from Sargassum horridum as potential human acetylcholinesterase inhibitor was evaluated. The structural identification was obtained by nuclear magnetic resonance (NMR) spectroscopy and based on experimental data, we combined docking and molecular dynamics simulations coupled to the molecular-mechanics-generalized-born-surface-area approach to evaluating the structural and energetic basis for the molecular recognition of fucosterol and neostigmine at the binding site of acetylcholinesterase (AChE). In addition, the Lineweaver-Burk plot showed the nature of a non-competitive inhibition. The maximum velocity (Vmax) and the constant of Michaelis-Menten (Km) estimated for fucosterol (0.006 µM) were 0.015 1/Vo (ΔA/h and 6.399 1/[ACh] mM-1, respectively. While, for neostigmine (0.14 µM), the Vmax was 0.022 1/Vo (ΔA/h) and Km of 6.726 1/[ACh] mM-1, these results showed a more effective inhibition by fucosterol respect to neostigmine. Structural analysis revealed that neostigmine reaches the AChE binding site reported elsewhere, whereas fucosterol can act as a no-competitive and competitive acetylcholinesterase inhibitor, in agree with kinetic enzymatic experiments. Binding free energy calculations revealed that fucosterol reaches the acetylcholinesterase binding site with higher affinity than neostigmine, which is according to experimental results. Whereas the per-residue decomposition free energy analysis let us identify crucial residues involved in the molecular recognition of ligands by AChE. Results corroborate the ability of theoretical methods to provide crucial information at the atomic level about energetic and structural differences in the binding interaction and affinity from fucosterol with AChE. Communicated by Ramaswamy H. Sarma.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Sargassum/química , Estigmasterol/análogos & derivados , Sítios de Ligação/efeitos dos fármacos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Estigmasterol/farmacologiaRESUMO
BACKGROUND: In order to develop new larvicidal agents derived from phytochemicals, the larvicidal activity of fifty molecules that are constituent of essential oils was evaluated against Culex quinquefasciatus Say. Terpenes, terpenoids and phenylpropanoids molecules were included in the in vitro evaluation, and QSAR models using genetic algorithms were built to identify molecular and structural properties of biological interest. Further, to obtain structural details on the possible mechanism of action, selected compounds were submitted to docking studies on sterol carrier protein-2 (SCP-2) as possible target. RESULTS: Results showed high larvicidal activity of carvacrol and thymol on the third and fourth larval stage with a median lethal concentration (LC50) of 5.5 and 11.1 µg/mL respectively. Myrcene and carvacrol were highly toxic for pupae, with LC50 values of 31.8 and 53.2 µg/mL. Structure-activity models showed that the structural property π-bonds is the largest contributor of larvicidal activity while ketone groups should be avoided. Similarly, property-activity models attributed to the molecular descriptor LogP the most contribution to larvicidal activity, followed by the absolute total charge (Qtot) and molar refractivity (AMR). The models were statistically significant; thus the information contributes to the design of new larvicidal agents. Docking studies show that all molecules tested have the ability to interact with the SCP-2 protein, wherein α-humulene and ß-caryophyllene were the compounds with higher binding energy. CONCLUSIONS: The description of the molecular properties and the structural characteristics responsible for larvicidal activity of the tested compounds were used for the development of mathematical models of structure-activity relationship. The identification of molecular and structural descriptors, as well as studies of molecular docking on the SCP-2 protein, provide insight on the mechanism of action of the active molecules, and the information can be used for the design of new structures for synthesis as potential new larvicidal agents.
RESUMO
Estrogen (17ß-estradiol) is essential for normal growth and differentiation in the mammary gland. In the last three decades, previous investigations have revealed that Estrogen Receptor Alpha (ERα) plays a critical role in breast cancer. More recently, observations regarding the widespread expression of ERß-like proteins in normal and neoplastic mammary tissues have suggested that ERß is also involved in the mentioned pathology. Design of new drugs both steroidal and nonsteroidal that target any of these receptors represents a promise to treat breast cancer although it remains a challenge due to the sequence similarity between their catalytic domains. In this work, we propose a new set of compounds that could effectively target the estrogen receptors ERα and ERß. These ligands were designed based on the chemical structure of the ERß-selective agonist Diarylpropionitrile (DPN). The designed ligands were submitted to in silico ADMET studies, yielding in a filtered list of ligands that showed better drug-like properties. Molecular dynamics simulations of both estrogen receptors and docking analysis were carried-out employing the designed compounds, from which two were chosen due to their promising characteristics retrieved from theoretical results (docking analysis or targeting receptor predictions). They were chemically synthetized and during the process, two precursor ligands were also obtained. These four ligands were subjected to biological studies from which it could be detected that compound mol60b dislplayed inhibitory activity and its ability to activate the transcription via an estrogenic mechanism of action was also determined. Interestinly, this observation can be related to theoretical binding free energy calculations, where the complex: ERß-mol60b showed the highest energy ΔGbind value in comparison to others.
Assuntos
Antineoplásicos/farmacologia , Nitrilas/farmacologia , Propionatos/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Nitrilas/síntese química , Nitrilas/química , Propionatos/síntese química , Propionatos/química , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Relação Estrutura-AtividadeRESUMO
Human immunodeficiency virus type-1 (HIV-1) has infected more than 40 million people around the world. HIV-1 treatment still has several side effects, and the development of a vaccine, which is another potential option for decreasing human infections, has faced challenges. This work presents a computational study that includes a quantitative structure activity relationship(QSAR) using density functional theory(DFT) for reported peptides to identify the principal quantum mechanics descriptors related to peptide activity. In addition, the molecular recognition properties of these peptides are explored on major histocompatibility complex I (MHC-I) through docking and molecular dynamics (MD) simulations accompanied by the Molecular Mechanics Generalized Born Surface Area (MMGBSA) approach for correlating peptide activity reported elsewhere vs. theoretical peptide affinity. The results show that the carboxylic acid and hydroxyl groups are chemical moieties that have an inverse relationship with biological activity. The number of sulfides, pyrroles and imidazoles from the peptide structure are directly related to biological activity. In addition, the HOMO orbital energy values of the total absolute charge and the Ghose-Crippen molar refractivity of peptides are descriptors directly related to the activity and affinity on MHC-I. Docking and MD simulation studies accompanied by an MMGBSA analysis show that the binding free energy without considering the entropic contribution is energetically favorable for all the complexes. Furthermore, good peptide interaction with the most affinity is evaluated experimentally for three proteins. Overall, this study shows that the combination of quantum mechanics descriptors and molecular modeling studies could help describe the immunogenic properties of peptides from HIV-1.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Sítios de Ligação , HIV-1 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peptídeos/metabolismo , Ligação ProteicaRESUMO
The goal of this study was to identify neuraminidase (NA) residue mutants from human influenza AH1N1 using sequences from 1918 to 2012. Multiple alignment studies of complete NA sequences (5732) were performed. Subsequently, the crystallographic structure of the 1918 influenza (PDB ID: 3BEQ-A) was used as a wild-type structure and three-dimensional (3-D) template for homology modeling of the mutated selected NA sequences. The 3-D mutated NAs were refined using molecular dynamics (MD) simulations (50 ns). The refined 3-D models were used to perform docking studies using oseltamivir. Multiple sequence alignment studies showed seven representative mutations (A232V, K262R, V263I, T264V, S367L, S369N, and S369K). MD simulations applied to 3-D NAs showed that each NA had different active-site shapes according to structural surface visualization and docking results. Moreover, Cartesian principal component analyses (cPCA) show structural differences among these NA structures caused by mutations. These theoretical results suggest that the selected mutations that are located outside of the active site of NA could affect oseltamivir recognition and could be associated with resistance to oseltamivir.
Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Neuraminidase/química , Proteínas Virais/química , Sequência de Aminoácidos , Sítios de Ligação , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação Puntual , Análise de Componente Principal , Estrutura Terciária de ProteínaRESUMO
The recent finding of a specific receptor for prorrenin/renin (PRR) has brought new insights into the physiology of the renin-angiotensin-aldosterone system. No undoubtable role has been described for this receptor so far. Its role seems to be important in chronic illnesses such as hypertension, possibly participating in the cardiovascular remodeling process, and diabetes where participation in inflammation development has been described. It is not possible, however, to explore the PRR function using classical pharmacological approaches due to the lack of specific agonists or antagonists. Two synthetic peptides have been described to accomplish these roles, but no conclusive data have been provided. There are no X-ray crystallography studies available to describe the structure and potential sites for drug development. So, the aim of this work was to model and theoretically describe the PRR. We describe and characterize the whole receptor protein, its spatial conformation and the potential interactions of PRR with the synthetic peptides available, describing the amino acid residues responsible for these interactions. This information provides the basis for directed development of drugs, seeking to agonize or antagonize PRR activity and study its function in health and ill stages.
Assuntos
Simulação de Dinâmica Molecular , Conformação Proteica , Receptores de Superfície Celular/química , Renina/química , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/metabolismo , Renina/metabolismo , Alinhamento de Sequência , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Following the influenza A (H1N1) pandemic in Mexico and around the world in 2009, the numbers of oseltamivir-resistant clinical cases have increased through a mechanism that remains unclear. In this work, we focus on studying the mutated NA structures ADA71175 (GenBank) and 3CKZ (PDB ID). Recently crystallized NA (PDB ID: 3NSS) was used as a wild-type structure and template to construct the three-dimensional (3D) structure of ADA71175. Then, the NA mutants and 3NSS natives as well as their refined monomer structures as determined through MD simulations (snapshots at 50 ns) were used as models to perform a docking study using a set of aryl-oseltamivir derivatives. These aryl-oseltamivir derivatives have better recognition properties than oseltamivir because of cation-π interactions with a cluster of Arg residues (118, 292, and 371) at the binding site. This cluster of Arg residues represents a potential binding site for aryl-oseltamivir derivatives that are potentially new NA inhibitors.
Assuntos
Antivirais/química , Arginina/química , Farmacorresistência Viral , Vírus da Influenza A Subtipo H1N1 , Modelos Moleculares , Neuraminidase/química , Oseltamivir/química , Sequência de Aminoácidos , Antivirais/farmacologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Dados de Sequência Molecular , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Oseltamivir/farmacologia , Ligação Proteica , Conformação Proteica , Alinhamento de SequênciaRESUMO
The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.
Assuntos
Benzodioxóis/química , Estradiol/análogos & derivados , Estradiol/química , Quinolinas/química , Receptores de Estrogênio/química , Receptores Acoplados a Proteínas G/química , Tamoxifeno/química , Motivos de Aminoácidos , Sítios de Ligação , Fulvestranto , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , TermodinâmicaRESUMO
MCM-41 and FSM-16 were used for enzyme immobilization on account of their good physical and chemical properties. In this work, the catalytic activity of acetylcholinesterase (AChE) immobilized on these materials was investigated, using neostigmina as AChE inhibitor. The results show that AChE was adsorbed on MCM-41 and on FSM-16-TIPB. AChE immobilized on the latter material maintained 70% of its activity and the material did not hydrolyze ACh (as MCM-41) by itself. Therefore, FSM-16-TIPB was the best material, considering also that when neostigmine was applied to AChE immobilized on FSM-16-TIPB, the activity of AChE decreased as occurs in its free from. Hence, this model could be useful in the evaluation of different kinds of AChE inhibitors, allowing the recycling of enzymes and making possible several assays and thereby, lowering cost.
Assuntos
Acetilcolinesterase/metabolismo , Enzimas Imobilizadas/metabolismo , Dióxido de Silício/metabolismo , Acetilcolinesterase/química , Adsorção , Animais , Catálise , Bovinos , Enzimas Imobilizadas/química , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Porosidade , Soluções , Espectrofotometria Ultravioleta , Difração de Raios XRESUMO
In the present study the acetylcholinesterase (AChE) inhibition and acute toxicity of two succinic acid derivatives were compared with tacrine. Administration of a single dose of each of two succinic acid derivatives produced a time and dose-dependent inhibition of brain AChE activity. Although the magnitude of the cholinergic effects observed with the two succinic acid derivatives was similar to that seen with tacrine and other AChE inhibitors, the toxicity study showed that the new inhibitors have less adverse side effects.