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1.
Methods Mol Biol ; 2836: 219-233, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38995543

RESUMO

Channels, tunnels, and pores serve as pathways for the transport of molecules and ions through protein structures, thus participating to their functions. MOLEonline ( https://mole.upol.cz ) is an interactive web-based tool with enhanced capabilities for detecting and characterizing channels, tunnels, and pores within protein structures. MOLEonline has two distinct calculation modes for analysis of channel and tunnels or transmembrane pores. This application gives researchers rich analytical insights into channel detection, structural characterization, and physicochemical properties. ChannelsDB 2.0 ( https://channelsdb2.biodata.ceitec.cz/ ) is a comprehensive database that offers information on the location, geometry, and physicochemical characteristics of tunnels and pores within macromolecular structures deposited in Protein Data Bank and AlphaFill databases. These tunnels are sourced from manual deposition from literature and automatic detection using software tools MOLE and CAVER. MOLEonline and ChannelsDB visualization is powered by the LiteMol Viewer and Mol* viewer, ensuring a user-friendly workspace. This chapter provides an overview of user applications and usage.


Assuntos
Bases de Dados de Proteínas , Software , Conformação Proteica , Interface Usuário-Computador , Modelos Moleculares , Canais Iônicos/metabolismo , Canais Iônicos/química , Biologia Computacional/métodos , Proteínas/química , Proteínas/metabolismo , Navegador
2.
Nucleic Acids Res ; 52(D1): D413-D418, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-37956324

RESUMO

ChannelsDB 2.0 is an updated database providing structural information about the position, geometry and physicochemical properties of protein channels-tunnels and pores-within deposited biomacromolecular structures from PDB and AlphaFoldDB databases. The newly deposited information originated from several sources. Firstly, we included data calculated using a popular CAVER tool to complement the data obtained using original MOLE tool for detection and analysis of protein tunnels and pores. Secondly, we added tunnels starting from cofactors within the AlphaFill database to enlarge the scope of the database to protein models based on Uniprot. This has enlarged available channel annotations ∼4.6 times as of 1 September 2023. The database stores information about geometrical features, e.g. length and radius, and physico-chemical properties based on channel-lining amino acids. The stored data are interlinked with the available UniProt mutation annotation data. ChannelsDB 2.0 provides an excellent resource for deep analysis of the role of biomacromolecular tunnels and pores. The database is available free of charge: https://channelsdb2.biodata.ceitec.cz.


Assuntos
Bases de Dados de Proteínas , Proteínas , Software , Aminoácidos , Proteínas/química , Conformação Proteica
3.
Bioorg Med Chem ; 95: 117504, 2023 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-37871508

RESUMO

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Trifosfato de Adenosina/metabolismo , Antituberculosos/química , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo
4.
J Steroid Biochem Mol Biol ; 233: 106365, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37468002

RESUMO

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.


Assuntos
Neoplasias da Mama , Estrona , Humanos , Feminino , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Estrona/farmacologia , Receptores de Estrogênio/metabolismo , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Tamoxifeno/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estradiol/farmacologia , Estradiol/uso terapêutico
5.
Br J Pharmacol ; 180(7): 829-842, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-34855983

RESUMO

BACKGROUND AND PURPOSE: Opioids and benzodiazepines are frequently combined in medical as well as in non-medical contexts. At high doses, such combinations often result in serious health complications attributed to pharmacodynamics interactions. Here, we investigate the contribution of the metabolic interactions between oxycodone, diazepam and diclazepam (a designer benzodiazepine) in abuse/overdose conditions through ex vivo, in vivo and in silico approaches. EXPERIMENTAL APPROACH: A preparation of pooled human liver microsomes was used to study oxycodone metabolism in the presence or absence of diazepam or diclazepam. In mice, diazepam or diclazepam was concomitantly administered with oxycodone to mimic acute intoxication. Diclazepam was introduced on Day 10 in mice continuously infused with oxycodone for 15 days to mimic chronic intoxication. In silico modelling was used to study the molecular interactions of the three drugs with CYP3A4 and 2D6. KEY RESULTS: In mice, in acute conditions, both diazepam and diclazepam inhibited the metabolism of oxycodone. In chronic conditions and at pharmacologically equivalent doses, diclazepam drastically enhanced the production of oxymorphone. In silico, the affinity of benzodiazepines was higher than oxycodone for CYP3A4, inhibiting oxycodone metabolism through CYP3A4. Oxycodone metabolism is likely to be diverted towards CYP2D6. CONCLUSION AND IMPLICATIONS: Acute doses of diazepam or diclazepam result in the accumulation of oxycodone, whereas chronic administration induces the accumulation of oxymorphone, the toxic metabolite. This suggests that overdoses of opioids in the presence of benzodiazepines are partly due to metabolic interactions, which in turn explain the patterns of toxicity dependent on usage. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.


Assuntos
Overdose de Drogas , Oxicodona , Humanos , Animais , Camundongos , Oximorfona , Citocromo P-450 CYP3A , Benzodiazepinas/toxicidade , Diazepam/farmacologia , Analgésicos Opioides/toxicidade , Modelos Animais
6.
Eur J Med Chem ; 244: 114831, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36242986

RESUMO

Mycobacterial zinc metalloprotease-1 (Zmp1) is an essential enzyme for intracellular survival and pathogenicity of Mycobacterium tuberculosis. However, the exact mechanism of function of this enzyme remains unclear. This paper examines the effect of novel organic molecules on the inhibition of Zmp1. We followed our previous results and synthesised three libraries of new hydroxamates. All compounds were studied for their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis by MALDI-TOF MS. Furthermore, a macrophage infection assay was performed to evaluate intracellular antimycobacterial activity. In the whole-cell assay, no direct activity of synthesised heterocyclic hydroxamates was observed against Mycobacterium tuberculosis and Mycobacterium bovis. No acute cellular toxicity was observed against the murine RAW 264.7 macrophage cell line and human MRC-5 lung fibroblast cell line. However, thiazolidinediones 2 showed the dose-dependent inhibition of intracellular survival of Mycobacterium tuberculosis H37Ra. The inhibition was structure-dependent, with the most active derivative 2f inducing an 83.2% reduction of bacterial survival within the macrophage host cell. The promising biological activity confirmed thiazolidinediones 2 as Zmp1 inhibitors that can be used as tool compounds for further exploration of the role of Zmp1 for in vivo pathogenicity. In the long run, thiazolidinediones 2 show the potential to act as a scaffold for Zmp1 inhibitors to target intracellular Mtb as a novel tuberculosis treatment strategy.


Assuntos
Mycobacterium tuberculosis , Tiazolidinedionas , Humanos , Camundongos , Animais , Zinco/metabolismo , Metaloproteases/metabolismo , Proteínas de Bactérias , Ácidos Hidroxâmicos/farmacologia , Tiazolidinedionas/farmacologia
7.
Nucleic Acids Res ; 49(W1): W431-W437, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33956157

RESUMO

Large biomolecular structures are being determined experimentally on a daily basis using established techniques such as crystallography and electron microscopy. In addition, emerging integrative or hybrid methods (I/HM) are producing structural models of huge macromolecular machines and assemblies, sometimes containing 100s of millions of non-hydrogen atoms. The performance requirements for visualization and analysis tools delivering these data are increasing rapidly. Significant progress in developing online, web-native three-dimensional (3D) visualization tools was previously accomplished with the introduction of the LiteMol suite and NGL Viewers. Thereafter, Mol* development was jointly initiated by PDBe and RCSB PDB to combine and build on the strengths of LiteMol (developed by PDBe) and NGL (developed by RCSB PDB). The web-native Mol* Viewer enables 3D visualization and streaming of macromolecular coordinate and experimental data, together with capabilities for displaying structure quality, functional, or biological context annotations. High-performance graphics and data management allows users to simultaneously visualise up to hundreds of (superimposed) protein structures, stream molecular dynamics simulation trajectories, render cell-level models, or display huge I/HM structures. It is the primary 3D structure viewer used by PDBe and RCSB PDB. It can be easily integrated into third-party services. Mol* Viewer is open source and freely available at https://molstar.org/.


Assuntos
Substâncias Macromoleculares/química , Modelos Moleculares , Software , Internet , Conformação Proteica
8.
J Steroid Biochem Mol Biol ; 211: 105904, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33933576

RESUMO

One of the main directions of steroid research is the preparation of modified derivatives in which, in addition to changes in physicochemical properties, receptor binding is significantly altered, thus a bioactivity different from that of the parent compound predominates. In the frame of this work, 2-arylidene derivatives were first synthesized by regioselective modification of the A-ring of natural sex hormone, 5α-dihydrotestosterone (DHT). After Claisen-Schmidt condensations of DHT with (hetero)aromatic aldehydes in alkaline EtOH, heterocyclizations of the α,ß-enones were performed with 3-amino-1,2,4-triazole, 3-aminopyrazole and 3-amino-5-methylpyrazole in the presence of t-BuOK in DMF to afford 7'-epimeric mixtures of A-ring-fused azolo-dihydropyrimidines, respectively. Depending on the electronic demand of the substituents of the arylidene moiety, spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the heteroring led to triazolo[1,5-a]pyrimidines and pyrazolo[1,5-a]pyrimidines in good yields, while, using the Jones reagent as a strong oxidant, 17-oxidation also occurred. The crystal structures of an arylidene and a triazolopyrimidine product have been determined by single crystal X-ray diffraction and both were found to crystallize in the monoclinic crystal system at P21 space group. Most derivatives were found to diminish the transcriptional activity of androgen receptor (AR) in reporter cell line. The candidate compound (17ß-hydroxy-2-(4-chloro)benzylidene-5α-androstan-3-one, 2f) showed to suppress androgen-mediated AR transactivation in a dose-dependent manner. We confirmed the cellular interaction of 2f with AR, described the binding in AR-binding cavity by the flexible docking and showed the ability of the compound to suppress the expression of AR-regulated genes in two prostate cancer cell lines.


Assuntos
Antagonistas de Receptores de Andrógenos/síntese química , Antagonistas de Receptores de Andrógenos/farmacologia , Di-Hidrotestosterona/química , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Sobrevivência Celular , Humanos , Masculino , Conformação Molecular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas
9.
Int J Mol Sci ; 22(1)2020 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-33375728

RESUMO

Brassinosteroids are a class of plant hormones that regulate a broad range of physiological processes such as plant growth, development and immunity, including the suppression of biotic and abiotic stresses. In this paper, we report the synthesis of new brassinosteroid analogues with a nitrogen-containing side chain and their biological activity on Arabidopis thaliana. Based on molecular docking experiments, two groups of brassinosteroid analogues were prepared with short and long side chains in order to study the impact of side chain length on plants. The derivatives with a short side chain were prepared with amide, amine and ammonium functional groups. The derivatives with a long side chain were synthesized using amide and ammonium functional groups. A total of 25 new brassinosteroid analogues were prepared. All 25 compounds were tested in an Arabidopsis root sensitivity bioassay and cytotoxicity screening. The synthesized substances showed no significant inhibitory activity compared to natural 24-epibrassinolide. In contrast, in low concentration, several compounds (8a, 8b, 8e, 16e, 22a and 22e) showed interesting growth-promoting activity. The cytotoxicity assay showed no toxicity of the prepared compounds on cancer and normal cell lines.


Assuntos
Brassinosteroides/síntese química , Brassinosteroides/farmacologia , Técnicas de Química Sintética , Nitrogênio/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Brassinosteroides/química , Estrutura Molecular , Desenvolvimento Vegetal/efeitos dos fármacos
10.
Eur J Med Chem ; 185: 111812, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31703818

RESUMO

Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the intracellular growth of Mycobacterium tuberculosis at a concentration of 100 and 10 µM. The thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated library.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Metaloproteases/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Humanos , Ácidos Hidroxâmicos/química , Metaloproteases/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Relação Estrutura-Atividade , Tiazolidinedionas/química
11.
Eur J Med Chem ; 179: 483-492, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31271960

RESUMO

Prostate cancer is one of the main causes of male cancer-related deaths worldwide and the suppression of androgen receptor signalling is established as an effective strategy for the treatment. A series of galeterone analogues including several steroid-fused azacycles, as well as 17-(benzimidazol-1-ylimino), 16α-(benzimidazol-2-ylamino), and 16α-(benzothiazol-2-ylamino) steroid derivatives, were synthesized and tested against prostate cancer cell lines. Candidate compound 3f was shown to reduce AR-regulated transcription in a dose-dependent manner in nanomolar ranges and suppress expression of AR-regulated proteins Nkx3.1 and PSA in 22Rv1-ARE14 and VCaP cancer cell lines. Flexible docking study revealed similar position of 3f within AR binding site in comparison of galeterone even with stronger binding energy.


Assuntos
Androstadienos/farmacologia , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Androstadienos/síntese química , Androstadienos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Estrutura Molecular , Células PC-3 , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Relação Estrutura-Atividade
12.
Database (Oxford) ; 20192019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31250015

RESUMO

Biological membranes act as barriers or reservoirs for many compounds within the human body. As such, they play an important role in pharmacokinetics and pharmacodynamics of drugs and other molecular species. Until now, most membrane/drug interactions have been inferred from simple partitioning between octanol and water phases. However, the observed variability in membrane composition and among compounds themselves stretches beyond such simplification as there are multiple drug-membrane interactions. Numerous experimental and theoretical approaches are used to determine the molecule-membrane interactions with variable accuracy, but there is no open resource for their critical comparison. For this reason, we have built Molecules on Membranes Database (MolMeDB), which gathers data about over 3600 compound-membrane interactions including partitioning, penetration and positioning. The data have been collected from scientific articles published in peer-reviewed journals and complemented by in-house calculations from high-throughput COSMOmic approach to set up a baseline for further comparison. The data in MolMeDB are fully searchable and browsable by means of name, SMILES, membrane, method or dataset and we offer the collected data openly for further reuse and we are open to further additions. MolMeDB can be a powerful tool that could help researchers better understand the role of membranes and to compare individual approaches used for the study of molecule/membrane interactions.


Assuntos
Bases de Dados de Compostos Químicos , Humanos , Membranas
13.
Nucleic Acids Res ; 46(W1): W368-W373, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29718451

RESUMO

MOLEonline is an interactive, web-based application for the detection and characterization of channels (pores and tunnels) within biomacromolecular structures. The updated version of MOLEonline overcomes limitations of the previous version by incorporating the recently developed LiteMol Viewer visualization engine and providing a simple, fully interactive user experience. The application enables two modes of calculation: one is dedicated to the analysis of channels while the other was specifically designed for transmembrane pores. As the application can use both PDB and mmCIF formats, it can be leveraged to analyze a wide spectrum of biomacromolecular structures, e.g. stemming from NMR, X-ray and cryo-EM techniques. The tool is interconnected with other bioinformatics tools (e.g., PDBe, CSA, ChannelsDB, OPM, UniProt) to help both setup and the analysis of acquired results. MOLEonline provides unprecedented analytics for the detection and structural characterization of channels, as well as information about their numerous physicochemical features. Here we present the application of MOLEonline for structural analyses of α-hemolysin and transient receptor potential mucolipin 1 (TRMP1) pores. The MOLEonline application is freely available via the Internet at https://mole.upol.cz.


Assuntos
Biologia Computacional , Internet , Conformação Proteica , Software , Modelos Moleculares
14.
J Med Chem ; 61(9): 3855-3869, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29672049

RESUMO

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.


Assuntos
Antineoplásicos/farmacologia , Diaminas/farmacologia , Descoberta de Drogas , Leucemia Mieloide Aguda/patologia , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Diaminas/química , Diaminas/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismo
15.
J Inorg Biochem ; 183: 117-136, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29653695

RESUMO

Mammalian cytochromes P450 are an important class of enzymes involved in the biotransformation of many endo- and exogenous compounds. Cytochrome P450 isoforms are attached to the membrane of the endoplasmic reticulum or mitochondria, and their catalytic domains move along the membrane surface while being partially immersed in the membrane environment. Their active sites are connected to both the membrane and cytosolic environments via a complex network of access channels. Consequently, they can accept substrates from both environments. The membrane also supports the interactions of cytochromes P450 with their redox partners. In this review, we provide an overview of current knowledge of the structure, flexibility, and interactions with substrates and redox partners of cytochrome P450 on membranes, amalgamating information derived from both experiments and simulations.


Assuntos
Membrana Celular/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Humanos , Oxirredução , Ligação Proteica
16.
Steroids ; 127: 46-55, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28837783

RESUMO

A series of phenyl analogues of brassinosteroids was prepared via alkene cross-metathesis using commercially available styrenes and 24-nor-5α-chola-2,22-dien-6-one. All derivatives were successfully docked into the active site of BRI1 using AutoDock Vina. Plant growth promoting activity was measured using the pea inhibition biotest and Arabidopsis root sensitivity assay and then was compared with naturally occuring brassinosteroids. Differences in the production of plant hormone ethylene were also observed in etiolated pea seedlings after treatment with the new and also five known brassinosteroid phenyl analogues. Antiproliferative activity was also studied using normal human fibroblast and human cancer cell lines.


Assuntos
Alcenos/química , Brassinosteroides/síntese química , Brassinosteroides/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/química , Brassinosteroides/metabolismo , Domínio Catalítico , Técnicas de Química Sintética , Simulação de Acoplamento Molecular , Pisum sativum/efeitos dos fármacos , Pisum sativum/crescimento & desenvolvimento , Proteínas Quinases/química , Proteínas Quinases/metabolismo
17.
Biochim Biophys Acta Biomembr ; 1859(10): 2113-2122, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28774819

RESUMO

The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1µM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.


Assuntos
Trifosfato de Adenosina/metabolismo , Corantes Fluorescentes/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Citoplasma/metabolismo , Cinética , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida/métodos , Triptofano/metabolismo
18.
Toxicol Lett ; 265: 86-96, 2017 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-27871908

RESUMO

The Pregnane X (PXR), Vitamin D (VDR) and Farnesoid X (FXR) nuclear receptors have been shown to be receptors of bile acids controlling their detoxification or synthesis. Chenodeoxycholic (CDCA) and lithocholic (LCA) acids are ligands of FXR and VDR, respectively, whereas 3-keto and acetylated derivates of LCA have been described as ligands for all three receptors. In this study, we hypothesized that oxidation or acetylation at position 3, 7 and 12 of bile acids DCA (deoxycholic acid), LCA, CA (cholic acid), and CDCA by detoxification enzymes or microbiome may have an effect on the interactions with bile acid nuclear receptors. We employed reporter gene assays in HepG2 cells, the TR-FRET assay with recombinant PXR and RT-PCR to study the effects of acetylated and keto bile acids on the nuclear receptors activation and their target gene expression in differentiated hepatic HepaRG cells. We demonstrate that the DCA 3,12-diacetate and CA 3,7,12-triacetate derivatives are ligands of PXR and DCA 3,12-diacetate induces PXR target genes such as CYP3A4, CYP2B6 and ABCB1/MDR1. In conclusion, we found that acetylated DCA and CA are potent ligands of PXR. Whether the acetylated bile acid derivatives are novel endogenous ligands of PXR with detoxification or physiological functions should be further studied in ongoing experiments.


Assuntos
Ácido Cólico/química , Ácido Desoxicólico/química , Receptores de Esteroides/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Acetilação , Animais , Técnicas de Cultura de Células , Ácido Cólico/metabolismo , Ácido Cólico/farmacologia , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP3A/genética , Ácido Desoxicólico/metabolismo , Ácido Desoxicólico/farmacologia , Relação Dose-Resposta a Droga , Genes Reporter , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Oxirredução , Plasmídeos , Receptor de Pregnano X , Ligação Proteica , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido
19.
J Comput Chem ; 37(28): 2485-94, 2016 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-27620738

RESUMO

Molecular docking is a powerful tool for theoretical prediction of the preferred conformation and orientation of small molecules within protein active sites. The obtained poses can be used for estimation of binding energies, which indicate the inhibition effect of designed inhibitors, and therefore might be used for in silico drug design. However, the evaluation of ligand binding affinity critically depends on successful prediction of the native binding mode. Contemporary docking methods are often based on scoring functions derived from molecular mechanical potentials. In such potentials, nonbonded interactions are typically represented by electrostatic interactions between atom-centered partial charges and standard 6-12 Lennard-Jones potential. Here, we present implementation and testing of a scoring function based on more physically justified exponential repulsion instead of the standard Lennard-Jones potential. We found that this scoring function significantly improved prediction of the native binding modes in proteins bearing narrow active sites such as serine proteases and kinases. © 2016 Wiley Periodicals, Inc.

20.
Eur J Med Chem ; 123: 803-813, 2016 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-27541263

RESUMO

Two new series of N-substituted indole derivatives 4a-l and 5a-h were synthesized. Their chemical structures were confirmed using spectroscopic tools including IR, (1)H NMR, (13)C NMR mass spectroscopy and elemental analyses. The results showed no significant cytotoxic activity on either cancer or normal human cells. Anti-inflammatory activity for all target compounds was evaluated in vitro. Compounds 5a-h were found to have better anti-inflammatory activity than 4a-l. The inhibitory activity of COX-2 and 5-LOX were tested for 5a-h. Three compounds, 5c, 5d and 5f showed excellent COX-2 inhibitory activity with IC50 ranging from 0.98 to 1.23 µM compared to the reference celecoxib (1.54 µM). These compounds had a reasonable selectivity index between 7.03 and 8.05. Additionally, p-methylbenzoyl derivative 5g (IC50 = 5.78 µM) had superior 5-LOX inhibitory activity, higher than quercetin. 5e was close to quercetin in its LOX inhibitory activity. Compounds 5a-h were docked inside the active site of COX-2 and 5-LOX enzymes.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ciclo-Oxigenase 2/metabolismo , Desenho de Fármacos , Indóis/síntese química , Indóis/farmacologia , Simulação de Acoplamento Molecular , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Araquidonato 5-Lipoxigenase/química , Domínio Catalítico , Linhagem Celular Tumoral , Técnicas de Química Sintética , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase 2/síntese química , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Indóis/química , Indóis/metabolismo , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Bases de Schiff/química
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