RESUMO
Recombinant expression and purification of proteins have become a staple of modern drug discovery as it enables more precise in vitro analyses of drug targets, which may help obtain biochemical and biophysical parameters of a known enzyme and even uncover unknown characteristics indicative of novel enzymatic functions. Such information is often necessary to prepare adequate screening assays and drug-discovery experiments in general. Toxoplasma gondii is an obligate protozoan parasite that is a member of the phylum Apicomplexa, can develop several neuro-degenerative symptoms and, in specific cases, certain death for human hosts. Its relict non-photosynthetic plastid, the apicoplast, harbours a unique de novo long-chain fatty acid synthesis pathway of a prokaryotic character, FASII. The FASII pathway shows plasticity and, is essential for many intracellular and membranal components, along with fatty acid uptake via salvaging from the host, therefore, its disruption causes parasite death. TgFabG, a FASII enzyme responsible for a single reduction step in the pathway, was recombinantly expressed, purified and biochemically and biophysically characterised in this study. The bioengineering hurdle of expressing the recombinant gene of a eukaryotic, signal peptide-containing protein in a prokaryotic system was overcome for the apicomplexan enzyme TgFabG, by truncating the N-terminal signal peptide. TgFabG was ultimately recombinantly produced in a plasmid expression vector from its 1131 base pair gene, purified as 260 and 272 amino acid proteins using a hexahistidine (6 × Histag) affinity chromatography and its biochemical (enzyme activity and kinetics) and biophysical characteristics were analysed in vitro.
Assuntos
Apicoplastos , Toxoplasma , Humanos , Apicoplastos/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Proteína de Transporte de Acila/metabolismo , Oxirredutases/metabolismo , Ácidos Graxos/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Theileria and Babesia species are eukaryotic protozoan parasites classified under the order Piroplasmida of the phylum Apicomplexa. Tick vectors transmit these microorganisms in tropical and subtropical regions to a wide range of animals, including ruminants, causing fatal and life-threatening diseases such as bovine babesiosis and theileriosis. Resistance to commercially available drugs requires the search for new drug candidates. Histone deacetylase (HDAC) has a potential to be utilized as a drug target; therefore, it may be considered as an effective alternative. Previous studies revealed that HDAC inhibitors, identified for human use, show promising anti-parasitic effects. We have herein focused on the class I HDAC enzyme, HDAC1, of the Babesia and Theileria species to discover potential benzamide inhibitors by following a streamlined workflow of computer-aided drug design methodology. Molecular docking and molecular dynamics simulations revealed that benzamide derivatives stably interacted with the HDAC1 active site in both parasites as hypothesized. Furthermore, specific residue insertions at the entry point of the active site cleft of parasitic HDAC1 could enable ways to design parasite-specific drugs without adversely affecting host enzymes.
Assuntos
Antiprotozoários/farmacologia , Babesia/enzimologia , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Histona Desacetilase 1/antagonistas & inibidores , Simulação de Dinâmica Molecular , Theileria/enzimologia , Animais , Bovinos , Desenho de Fármacos , Humanos , Simulação de Acoplamento MolecularRESUMO
In this study, the inhibition potential of 3- and 4-arylcoumarin derivatives on Theileria annulata enolase (TaENO) was assessed for the first time in the literature. Firstly, protein stabilization analyses of TaENO were performed and it was found that the enzyme remains stable with the addition of 6 M ethylene glycol at + 4 °C. Inhibitor screening analyses were carried out using 25 coumarin derivatives on highly purified TaENO (> 95%), and four coumarin derivatives [4-(3,4-dimethoxyphenyl)-6,7-dihydroxy-2H-chromen-2-one (C8); 4-(3,4-dihydroxyphenyl)-7,8 dihydroxy-2H-chromen-2-one (C9); 4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2H-chromen-2 one (C21); and 3-(3,4-dihydroxyphenyl)-7,8-dihydroxy-2H-chromen-2-one (C23)] showed the highest inhibitory effects with the IC50 values of 10.450, 13.170, 8.871 and 10.863 µM, respectively. The kinetic results indicated that these compounds inhibited the enzyme by uncompetitive inhibition. In addition, the successful binding of the most potent inhibitor (C21) into TaENO was confirmed by using MALDI-TOF mass spectrophotometry. Molecular docking analyses have predicted that C8 and C21 coumarin derivatives which showed high inhibitory effects on TaENO were interacted with high affinity to the potential regions out of the active site. Taken together, these coumarin derivatives (C8, C9, C21 and C23) are first known potent, nonsubstrate, uncompetitive inhibitors of TaENO and these results will facilitate further in vitro and in vivo analysis toward structure-based drug design studies.
Assuntos
Cumarínicos/química , Fosfopiruvato Hidratase/antagonistas & inibidores , Theileria annulata/efeitos dos fármacos , Domínio Catalítico , Desenho de Fármacos , Cinética , Simulação de Acoplamento Molecular/métodos , Relação Estrutura-AtividadeRESUMO
Among the thermophilic Bacillaceae family members, α-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus α-amylase genes displayed ≥ 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (≤ 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (≤ 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 α-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli α-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 α-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around α-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around α-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3.
Assuntos
Anoxybacillus/enzimologia , Bacillaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Glicosídeo Hidrolases/metabolismo , alfa-Amilases/química , alfa-Amilases/classificação , alfa-Amilases/metabolismo , Sequência de Aminoácidos , Anoxybacillus/classificação , Anoxybacillus/genética , Bacillaceae/genética , Bacillus/classificação , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Ensaios Enzimáticos , Estabilidade Enzimática , Evolução Molecular , Geobacillus/metabolismo , Glicosídeo Hidrolases/genética , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , alfa-Amilases/genéticaRESUMO
BACKGROUND & OBJECTIVES: Trypanothione reductase (TR) is a member of FAD-dependent NADPH oxidoreductase protein family and it is a key enzyme which connects the NADPH and the thiol-based redox system. Inhibition studies indicate that TR is an essential enzyme for parasite survival. Therefore, it is an attractive target enzyme for novel drug candidates. There is no structural model for TR of Leishmania mexicana (LmTR) in the protein databases. In this work, 3D structure of TR from L. mexicana was identified by template-based in silico homology modeling method, resultant model was validated, structurally analyzed and possible ligand binding pockets were identified. METHODS: For computational molecular modeling study, firstly, template was identified by BLAST search against PDB database. Multiple alignments were achieved by ClustalW2. Molecular modeling of LmTR was done and possible drug targeting sites were identified. Refinement of the model was done by performing local energy minimization for backbone, hydrogen and side chains. Model was validated by web-based servers. RESULTS: A reliable 3D model for TR from L. mexicana was modeled by using L. infantum trypanothione reductase (LiTR) as a template. RMSD results according to C-alpha, visible atoms and backbone were 0.809 Å, 0.732 Å and 0.728 Å respectively. Ramachandran plot indicates that model shows an acceptable stereochemistry. CONCLUSION: Modeled structure of LmTR shows high similarity with LiTR based on overall structural features like domains and folding patterns. Predicted structure will provide a source for the further docking studies of various peptide-based inhibitors.
Assuntos
Leishmania mexicana/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biologia Computacional/métodos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de SequênciaRESUMO
Acinetobacter baumannii is one of the most serious opportunistic pathogens according to WHO. The difference between bacterial and mammalian fatty acid biosynthesis pathways makes FASII enzymes attractive targets in drug discovery. 3-oxoacyl-[acyl-carrier-protein] synthase I (FabB) from the FAS II pathway catalyze the condensation of malonyl ACP with acyl-ACP, and elongates the fatty acid chain by two carbons. To investigate potential inhibitors of the A. baumannii FabB, we used computational approaches including homology modeling, high-throughput virtual screening, molecular docking, molecular dynamics simulations, and MM-GBSA free energy calculations. After the high-throughput virtual screening, the resulting ligands were further screened using the QM-polarized ligand docking (QPLD) and induced fit docking (IFD) approaches. Molecular dynamics simulations were performed for 100 ns. And according to binding free energy calculations, we have identified nine compounds with the best binding affinities. Three of these compounds were selected for an additional 1 µs MD simulation to assess ligand stability. Two of them named L6 and L7 showed promised stability and affinity to the target. Here, we present novel compounds against A. baumannii FabB via structure-based computational approaches. These compounds might pave the way for the design of new lead structures and inhibitors for multidrug-resistant A. baumannii.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase , Acinetobacter baumannii , Simulação de Acoplamento Molecular , Proteína de Transporte de Acila , Glicogênio Sintase , Ligantes , Simulação de Dinâmica Molecular , Ácidos Graxos , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/químicaRESUMO
Toxoplasma gondii is an opportunistic obligate parasite, ubiquitous around the globe with seropositivity rates that range from 10% to 90% and infection by the parasite of pregnant women causes pre-natal death of the foetus in most cases and severe neurodegenerative syndromes in some. No vaccine is currently available, and since drug-resistance is common among T. gondii strains, discovering lead compounds for drug design using diverse tactics is necessary. In this study, the sole constituent isoform of an enzymatic 3-oxoacyl-[acyl-carrier-protein] reduction step in an apicoplast-located fatty acid biosynthesis pathway was chosen as a possible drug target. FASII is prokaryotic therefore, targeting it would pose fewer side-effects to human hosts. After a homology 3D modelling of TgFabG, a high-throughput virtual screening of 9867 compounds, the elimination of ligands was carried out by a flexible ligand molecular docking and 200 ns molecular dynamics simulations, with additional DCCM and PC plot analyses. Molecular Dynamics and related post-MD analyses of the top 3 TgFabG binders selected for optimal free binding energies, showed that L2 maintained strong H-bonds with TgFabG and facilitated structural reorientation expected of FabGs, namely an expansion of the Rossmann Fold and a flexible lid capping. The most flexible TgFabG sites were the α7 helix (the flexible lid region) and the ß4-α4 and ß5-α6 loops. For TgFabG-L2, the movements of these regions toward the active site enabled greater ligand stability. Thus, L2 ("Skimmine"; PubChem ID: 320361), was ultimately selected as the optimal candidate for the discovery of lead compounds for rational drug design.Communicated by Ramaswamy H. Sarma.
Assuntos
3-Oxoacil-(Proteína Carreadora de Acil) Redutase , Proteínas de Protozoários , Toxoplasma , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase/genética , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase/metabolismo , Feminino , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Gravidez , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Toxoplasma/genéticaRESUMO
In this study, the Nsp12-Nsp8 complex of SARS-CoV-2 was targeted with structure-based and computer-aided drug design approach because of its vital role in viral replication. Sequence analysis of RNA-dependent RNA polymerase (Nsp12) sequences from 30,366 different isolates were analysed for possible mutations. FDA-approved and investigational drugs were screened for interaction with both mutant and wild-type Nsp12-Nsp8 interfaces. Sequence analysis revealed that 70.42% of Nsp12 sequences showed conserved P323L mutation, located in the Nsp8 binding cleft. Compounds were screened for interface interaction, any with XP GScores lower than -7.0 kcal/mol were considered as possible interface inhibitors. RX-3117 (fluorocyclopentenyl cytosine) and Nebivolol had the highest binding affinities in both mutant and wild-type enzymes, therefore they were selected and resultant protein-ligand complexes were simulated for analysis of stability over 100 ns. Although the selected ligands had partial mobility in the binding cavity, they were not removed from the binding pocket after 100 ns. The ligand RX-3117 remained in the same position in the binding pocket of the mutant and wild-type enzyme after 100 ns MD simulation. However, the ligand Nebivolol folded and embedded in the binding pocket of mutant Nsp12 protein. Overall, FDA-approved and investigational drugs are able to bind to the Nsp12-Nsp8 interaction interface and prevent the formation of the Nsp12-Nsp8 complex. Interruption of viral replication by drugs proposed in this study should be further tested to pave the way for in vivo studies towards the treatment of COVID-19.Communicated by Ramaswamy H. Sarma.
Assuntos
COVID-19 , SARS-CoV-2 , Drogas em Investigação , Humanos , Proteínas não Estruturais Virais , Replicação ViralRESUMO
Bacteriodes fragilis is an anaerobic bacterium found in the human intestinal flora. In this study, BfEno was targeted with a structure-based drug design approach because inhibition of this enzyme may prevent both the aerobic and anaerobic pathways due to its role in the glycolytic pathway. First, the gene encoding BfEno was cloned, expressed and the protein produced over 95% purity. The Km and Vmax values of BfEno were determined as 314.9 µM and 256.2 µmol/min.mg, respectively. Drug-like chemicals were retrieved from the ZINC database for high-throughput virtual screening analyses. As a result of screening study, the ZINC91441604 has been proposed to bind to the active site of the enzyme and remain stable. The same compound exhibited weak binding to the human enolases than the bacterial enolase. Hence, ZINC91441604 may be proposed as a novel candidate for further in vitro and in vivo drug analysis towards the treatment of B. fragilis infections.
Assuntos
Infecções Bacterianas , Bacteroides fragilis , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Composição de Bases , Humanos , Fosfopiruvato Hidratase/química , Filogenia , RNA Ribossômico 16S/metabolismo , Análise de Sequência de DNARESUMO
Theileria annulata secretes peptidyl prolyl isomerase enzyme (TaPIN1) to manipulate the host cell oncogenic signaling pathway by disrupting the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) protein level leading to an increased level of c-Jun proto-oncogene. Buparvaquone is a hydroxynaphthoquinone anti-theilerial drug and has been used to treat theileriosis. However, TaPIN1 contains the A53â¯P mutation that causes drug resistance. In this study, potential TaPIN1 inhibitors were investigated using a library of naphthoquinone derivatives. Comparative models of mutant (m) and wild type (wt) TaPIN1 were predicted and energy minimization was followed by structure validation. A naphthoquinone (hydroxynaphthalene-1,2-dione, hydroxynaphthalene-1,4-dione) and hydroxynaphthalene-2,3-dione library was screened by Schrödinger Glide HTVS, SP and XP docking methodologies and the docked compounds were ranked by the Glide XP scoring function. The two highest ranked docked compounds Compound 1 (4-hydroxy-3-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxynaphthalene-1,2-dione) and Compound 2 (6-acetyl-1,4,5,7,8-pentahydroxynaphthalene-2,3-dione) were used for further molecular dynamics (MD) simulation studies. The MD results showed that ligand Compound 1 was located in the active site of both mTaPIN1 and wtTaPIN1 and could be proposed as a potential inhibitor by acting as a substrate antagonist. However, ligand Compound 2 was displaced away from the binding pocket of wtTaPIN1 but was located near the active site binding pocket of mTaPIN1 suggesting that could be selectively evaluated as a potential inhibitor against the mTaPIN1. Compound 1 and Compound 2 ligands are potential inhibitors but Compound 2 is suggested as a better inhibitor for mTaPIN1. These ligands could also further evaluated as potential inhibitors against human peptidyl prolyl isomerase which causes cancer in humans by using the same mechanism as TaPIN1.
Assuntos
Inibidores Enzimáticos/química , Naftoquinonas/química , Peptidilprolil Isomerase/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Theileria annulata/enzimologia , Domínio Catalítico , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Inibidores Enzimáticos/metabolismo , Ensaios de Triagem em Larga Escala , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Naftoquinonas/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Proto-Oncogene Mas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
SARS-CoV-2 has caused COVID-19 outbreak with nearly 2 M infected people and over 100K death worldwide, until middle of April 2020. There is no confirmed drug for the treatment of COVID-19 yet. As the disease spread fast and threaten human life, repositioning of FDA approved drugs may provide fast options for treatment. In this aspect, structure-based drug design could be applied as a powerful approach in distinguishing the viral drug target regions from the host. Evaluation of variations in SARS-CoV-2 genome may ease finding specific drug targets in the viral genome. In this study, 3458 SARS-CoV-2 genome sequences isolated from all around the world were analyzed. Incidence of C17747T and A17858G mutations were observed to be much higher than others and they were on Nsp13, a vital enzyme of SARS-CoV-2. Effect of these mutations was evaluated on protein-drug interactions using in silico methods. The most potent drugs were found to interact with the key and neighbor residues of the active site responsible from ATP hydrolysis. As result, cangrelor, fludarabine, folic acid and polydatin were determined to be the most potent drugs which have potency to inhibit both the wild type and mutant SARS-CoV-2 helicase. Clinical data supporting these findings would be important towards overcoming COVID-19.
Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , RNA Helicases/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Betacoronavirus/enzimologia , Betacoronavirus/genética , Sítios de Ligação , COVID-19 , Simulação por Computador , Infecções por Coronavirus/virologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Ácido Fólico/farmacologia , Genoma Viral , Glucosídeos/farmacologia , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Mutação , Pandemias , Pneumonia Viral/virologia , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , SARS-CoV-2 , Estilbenos/farmacologia , Vidarabina/análogos & derivados , Vidarabina/farmacologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide (103EWGYC107) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENOΔEWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENOΔEK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENOΔEK exhibited a small decrease of substrate binding affinity compared to the active site of TaENOΔEWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.
Assuntos
Dipeptídeos/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fosfopiruvato Hidratase/química , Proteínas de Protozoários/química , Theileria annulata/enzimologia , Animais , Domínio Catalítico/genética , Bovinos , Dipeptídeos/genética , Fosfopiruvato Hidratase/genética , Proteínas de Protozoários/genética , Especificidade por Substrato/genética , Theileria annulata/genéticaRESUMO
Bacteroides fragilis is an anaerobic bacterium naturally hosted in the human colon flora. B. fragilisdlactate dehydrogenase (BfdLDH) is an important enzyme which catalyzes the conversion of dlactate to pyruvate and regulates anaerobic glycolysis. In this study BfdLDH has been targeted for structure based drug design. B. fragilisdlactate dehydrogenase has been expressed, purified and inhibitory activities of 25 coumarin derivatives previously synthetize for their antioxidant activity were evaluated. Among the 25 coumarin derivatives, compound 6a, 5l, and 6b exhibited the highest inhibitory activity with IC50 values of 0,47⯵M, 0,57⯵M ve 0,057⯵M, respectively. The results indicate that the mechanism by which 6a, 5l and 6b coumarin derivatives inhibit BfdLDH by reversible non-competitive inhibition. Docking experiments were carried out to further explain the results and compare the theoretical and experimental affinity of these compounds to the BfdLDH protein. According to docking results, all coumarins bind to the site occupied by pyruvate and the nicotinamide ring of NADH.
Assuntos
Proteínas de Bactérias/química , Bacteroides fragilis/enzimologia , Cumarínicos/química , L-Lactato Desidrogenase/química , Simulação de Acoplamento Molecular , Relação Estrutura-AtividadeRESUMO
A novel implant coating material containing graphene oxide (GO) and collagen (COL), and hydroxyapatite (HA) was fabricated with the aid of tannic acid by electrodeposition. The surface of Ti16Nb alloy was subjected to anodic oxidation, and then HA-GO coating was applied to Ti16Nb surface by cathodic method. Then, COL was deposited on the surface of the HA-GO coating by the biomimetic method. HA, HA-GO, HA-GO-COL coatings on the surface of the Ti16Nb alloy have increased the corrosion resistance by the formation of a barrier layer on the surface. For HA-GO-COL coating, the highest corrosion resistance is obtained due to the compactness and homogeneity of the coating structure. The contact angle of the bare Ti16Nb is approximately 65°, while the contact angle of the coated samples is close to 0°. Herein, the increased surface wettability is important for cell adhesion. The surface roughness of the uncoated Ti16Nb alloy was between 1 and 3⯵m, while the surface roughness of the coated surfaces was measured between 20 and 110⯵m. The contact between the bone and the implant has been improved. Graphene oxide-containing coatings have improved the antibacterial properties compared to the GO-free coating using S. aureus. The hardness and elastic modulus of the coatings were measured by the nanoindentation test, and the addition of GO and collagen to the HA coating resulted in an increase in strength. The addition of GO to the HA coating reduced the viability of 3â¯T3 fibroblast cells, whereas the addition of collagen to HA-GO coat increased the cell adhesion and viability.
Assuntos
Ligas/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Colágeno/farmacologia , Durapatita/farmacologia , Galvanoplastia/métodos , Grafite/farmacologia , Compostos de Estanho/química , Células 3T3 , Animais , Corrosão , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Camundongos , Testes de Sensibilidade Microbiana , Nanotubos/química , Nanotubos/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Compostos de Estanho/farmacologia , Difração de Raios XRESUMO
Phthalocyanines are considered as good DNA binders, which makes them promising anti-tumor drug leads. The purpose of this study is to investigate the interactions between DNA and quaternary metallophthalocyanine derivatives (Q-MPc) possessing varying metals (M = Zn, Ni, Cu, Fe, Mg and Ca) by molecular docking since there seems to be a lack of information in the literature regarding this issue. In this direction, Autodock Vina and Molegro Virtual Docker programs were employed. Autodock Vina results reveal that each Q-MPc derivative binds to DNA strongly with similar binding energies and almost identical binding modes. They bind to the grooves of DNA by constituting favorable interactions between phosphate groups of DNA and Q-MPcs. Although changing the metal has no significant effect on binding, presence of quaternary amine substituents increases the binding constant Kb by about 2-fold comparing to the core Pc (ZnPc). Contrary to Autodock Vina, the calculated Molegro Virtual Docker binding scores have been more diverse indicating that the scoring function of Molegro is better in differentiating these metals. Despite the fact that Molegro is superior to Autodock Vina in terms of metal characterization, Autodock Vina and Molegro exhibit similar binding sites for the studied metallophthalocyanines. We propose that Q-MPc derivatives designed in this study are promising anti-tumor lead compounds since they tightly bind to DNA with considerably high Kb values. Cationic substituents and presence of metal have both positive effects on DNA binding which is critical for designing DNA-active drugs. Additional calculations employing molecular dynamics (MD) simulations verified the stability of Q-MPc-DNA complexes which remained in contact after 20 ns via attractive interactions mainly between DNA backbone and the Pc metal center.
Assuntos
DNA/química , Indóis/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Sítios de Ligação , Cristalografia por Raios X , Isoindóis , Ligantes , Estrutura MolecularRESUMO
Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases.
Assuntos
L-Lactato Desidrogenase/química , Oligopeptídeos/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Sequência de Bases , Domínio Catalítico , Escherichia coli , Ligação de Hidrogênio , L-Lactato Desidrogenase/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Proteínas de Protozoários/genética , Ácido Pirúvico/química , Theileria annulata/enzimologiaRESUMO
Theileria annulata is a parasite that causes theileriosis in cattle. Reports about drug resistance made essential to develop new drug. LDH of Theileria schizonts is the vital enzyme for its anaerobic metabolism. TaLDH gene was first cloned into pGEM-T cloning vector with two introns in our previous study. Here we report cloning of TaLDH without introns into pLATE 31 vector in E. coli BL21(DE3). Protein was in an inactive form. Two mutations were fixed to express the active protein. Protein was purified by affinity chromatography and evaluated by SDS-PAGE and size exclusion chromatography. Optimum pH of enzyme was performed in pH 7.5, and enzyme was stabilized at 20-40 °C. Enzyme kinetics of recombinant TaLDH were found to be in the direction of pyruvate to lactate K m 0.1324 and K i 4.295 mM, k cat, 44.55/s and k cat /K m, 3.3693 × 10(5)/M/s. 3D structure of TaLDH was predicted, and possible drug binding sites were determined by homology modelling.
Assuntos
L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Theileria annulata/enzimologia , Sítios de Ligação , Simulação por Computador , Estabilidade Enzimática , Modelos Moleculares , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Theileria annulata/genéticaRESUMO
Theileria annulata is an apicomplexan parasite which is responsible for tropical theileriosis in cattle. Due to resistance of T. annulata against commonly used antitheilerial drug, new drug candidates should be identified urgently. Enolase might be a druggable protein candidate which has an important role in glycolysis, and could also be related to several cellular functions as a moonlight protein. In this study; we have described three-dimensional models of open and closed conformations of T. annulata enolase by homology modeling method for the first time with the comprehensive domain, active site and docking analyses. Our results show that the enolase has similar folding patterns within enolase superfamily with conserved catalytic loops and active site residues. We have described specific insertions, possible plasminogen binding sites, electrostatic potential surfaces and positively charged pockets as druggable regions in T. annulata enolase.
Assuntos
Fosfopiruvato Hidratase/química , Theileria annulata/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Propriedades de SuperfícieRESUMO
The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish.
RESUMO
The research investigated the effect of Zr, Nb and Ti additions on mechanical, electrochemical properties and biocompatibility of injection molded 316L stainless steel. Addition of elemental powder is promoted to get high performance of sintered 316L stainless steels. The amount of additive powder plays a role in determining the sintered microstructure and all properties. In this study, 316L stainless steel powders used with the elemental Zr, Nb and Ti powders. A feedstock containing 62.5 wt% powders loading was molded at different injection molded temperature. The binders were completely removed from molded components by solvent and thermal debinding at different temperatures. The debinded samples were sintered at 1350°C for 60 min. Mechanical, electrochemical property and biocompatibility of the sintered samples were performed mechanical, electrochemical, SBF immersion tests and cell culture experiments. Results of study showed that sintered 316L and 316L with additives samples exhibited high corrosion properties and biocompatibility in a physiological environment.