RESUMO
Helicobacter pylori (H. pylori) is a pathogen that can remain in the stomach of an infected person for their entire life. As a result, this leads to the development of severe gastric diseases such as gastric cancer. In addition, current therapies have several problems including antibiotics resistance. Therefore, new practical options to eliminate this bacterium, and its induced affections, are required to avoid morbidity and mortality worldwide. One strategy in the search for new drugs is to detect compounds that inhibit a limiting step in a central metabolic pathway of the pathogen of interest. In this work, we tested 55 compounds to gain insights into their possible use as new inhibitory drugs of H. pylori glucose-6-phosphate dehydrogenase (HpG6PD) activity. The compounds YGC-1; MGD-1, MGD-2; TDA-1; and JMM-3 with their respective scaffold 1,3-thiazolidine-2,4-dione; 1H-benzimidazole; 1,3-benzoxazole, morpholine, and biphenylcarbonitrile showed the best inhibitory activity (IC50 = 310, 465, 340, 204 and 304 µM, respectively). We then modeled the HpG6PD protein by homology modeling to conduct an in silico study of the chemical compounds and discovers its possible interactions with the HpG6PD enzyme. We found that compounds can be internalized at the NADP+ catalytic binding site. Hence, they probably exert a competitive inhibitory effect with NADP+ and a non-competitive or uncompetitive effect with G6P, that of the compounds binding far from the enzyme's active site. Based on these findings, the tested compounds inhibiting HpG6PD represent promising novel drug candidates against H. pylori.
Assuntos
Simulação por Computador , Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Helicobacter pylori/enzimologia , Vetores Genéticos/metabolismo , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Helicobacter pylori/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Recombinantes/isolamento & purificação , Homologia Estrutural de ProteínaRESUMO
Among the diseases transmitted by vectors, there are those caused by viruses named arboviruses (arthropod-borne viruses). In past years, viruses transmitted by mosquitoes have been of relevance in global health, such as Chikungunya (CHIKV), Dengue (DENV), and Zika (ZIKV), which have Aedes aegypti as a common vector, thus raising the possibility of multi-infection. Previous reports have described the general structure of RNA-dependent RNA polymerases termed right-hand fold, which is conserved in positive single-stranded RNA viruses. Here, we report a comparison between sequences and the computational structure of RNA-dependent RNA polymerases from CHIKV, DENV, and ZIKV and the conserved sites to be considered for the design of an antiviral drug against the three viruses. We show that the sequential identity between consensus sequences from CHIKV and DENV is 8.1% and the similarity is 15.1%; the identity between CHIKV and ZIKV is 9.3%, and the similarity is 16.6%; and the identity between DENV and ZIKV is 68.6%, and the similarity is 79.2%. Nevertheless, the structural alignment shows that the root-mean-square deviation (RMSD) measurement value in general structure comparison between CHIKV RdRp and ZIKV RdRp was 1.248 Å, RMSD between CHIKV RdRp and DENV RdRp was 1.070 Å, and RMSD between ZIKV RdRp and DENV RdRp was 1.106 Å. Despite the low identity and similarity of CHIKV sequence with DENV and ZIKV, we show that A, B, C, and E motifs are structurally well conserved. These structural similarities offer a window into drug design against these arboviruses giving clues about critical target sites.
Assuntos
Vírus Chikungunya/química , Vírus da Dengue/enzimologia , RNA Polimerase Dependente de RNA/química , Proteínas não Estruturais Virais/química , Zika virus/enzimologia , Motivos de Aminoácidos , Vírus Chikungunya/genética , Vírus da Dengue/genética , Humanos , Infecções por Vírus de RNA/genética , Infecções por Vírus de RNA/terapia , RNA Polimerase Dependente de RNA/genética , Homologia Estrutural de Proteína , Proteínas não Estruturais Virais/genética , Zika virus/genéticaRESUMO
Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.
Assuntos
Giardia lamblia/enzimologia , Gluconatos/química , NADP/química , Fosfogluconato Desidrogenase/química , Proteínas de Protozoários/química , Ribulosefosfatos/química , Motivos de Aminoácidos , Sítios de Ligação , Clonagem Molecular/métodos , Expressão Gênica , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimologia , Giardia lamblia/genética , Gluconatos/metabolismo , Humanos , Cinética , Modelos Moleculares , NADP/metabolismo , Via de Pentose Fosfato/genética , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosefosfatos/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato , TermodinâmicaRESUMO
Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.
Assuntos
Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Mutantes/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzotiazóis/química , Benzotiazóis/metabolismo , Sítios de Ligação , AMP Cíclico/química , AMP Cíclico/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Ligantes , Domínios Proteicos , Homologia Estrutural de Proteína , Termodinâmica , Triazóis/química , Triazóis/metabolismoRESUMO
Several antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat α7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, ICh, which activates α7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat α7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited ICh with the order: norfluoxetine ~ mirtazapine ~ imipramine < bupropion ~ fluoxetine ~ venlafaxine ~ escitalopram. The constructed homology model of the rat α7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the α7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of ICh and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the α7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.
Assuntos
Antidepressivos/química , Antidepressivos/farmacologia , Canais Iônicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Antidepressivos/classificação , Colina/farmacologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ratos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.
Assuntos
Doença de Chagas/imunologia , Defensinas/administração & dosagem , Microbioma Gastrointestinal/genética , Proteínas de Insetos/genética , Lectinas/metabolismo , Rhodnius/fisiologia , Trypanosoma cruzi/fisiologia , Animais , Defensinas/metabolismo , Vetores de Doenças , Proteínas de Peixes/genética , Inativação Gênica , Imunidade Inata , Proteínas de Insetos/metabolismo , Lectinas/genética , Simulação de Acoplamento Molecular , RNA Ribossômico 16S/genética , Homologia Estrutural de ProteínaRESUMO
Internal structure similarity in proteins can be observed at the domain and subdomain levels. From an evolutionary perspective, structurally similar elements may arise divergently by gene duplication and fusion events but may also be the product of convergent evolution under physicochemical constraints. The characterization of proteins that contain repeated structural elements has implications for many fields of protein science including protein domain evolution, structure classification, structure prediction, and protein engineering. FiRES (Find Repeated Elements in Structure) is an algorithm that relies on a topology-independent structure alignment method to identify repeating elements in protein structure. FiRES was tested against two hand curated databases of protein repeats: MALIDUP, for very divergent duplicated domains; and RepeatsDB for short tandem repeats. The performance of FiRES was compared to that of lalign, RADAR, HHrepID, CE-symm, ReUPred, and Swelfe. FiRES was the method that most accurately detected proteins either with duplicated domains (accuracy = 0.86) or with multiple repeated units (accuracy = 0.92). FiRES is a new methodology for the discovery of proteins containing structurally similar elements. The FiRES web server is publicly available at http://fires.ifc.unam.mx. The scripts, results, and benchmarks from this study can be downloaded from https://github.com/Claualvarez/fires.
Assuntos
Algoritmos , Proteínas/química , Software , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Benchmarking , Bases de Dados de Proteínas , Evolução Molecular , Duplicação Gênica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.
Assuntos
Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Idoso , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Neoplasias Esofágicas/diagnóstico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC , Homologia Estrutural de Proteína , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Bacterial chemoreceptors control the activity of the associated CheA kinase in response to chemical gradients and, consequently, regulate the swimming behavior of the cell. However, such control is not direct but requires the participation of the essential coupling protein CheW, which is structurally homologous to the carboxy-terminal domain of the kinase. The actual role of this small coupling protein is somehow intriguing. It has been demonstrated that it is absolutely essential for chemoreceptor control of the kinase, in spite of the occurrence of direct contacts between chemoreceptors and CheA. In addition, CheW plays an essential role in the assembly of the large macromolecular arrays that combine chemoreceptors of different specificities, and it is therefore responsible for molecular interactions that provide such arrays with remarkable signaling properties. In this work, we analyze truncated CheW derivatives that are still able to control the kinase but have lost the ability to connect signaling units. We demonstrate that these two activities can work separately and speculate about the significance of the roles of these two different activities in the context of the chemoreceptor cluster.
Assuntos
Proteínas de Bactérias/ultraestrutura , Quimiotaxia , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/ultraestrutura , Histidina Quinase/ultraestrutura , Proteínas Quimiotáticas Aceptoras de Metil/ultraestrutura , Proteínas de Bactérias/genética , Sítios de Ligação , Quimiotaxia/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Histidina Quinase/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transdução de Sinais/genética , Homologia Estrutural de ProteínaRESUMO
Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.