RESUMO
With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.
Assuntos
Antiprotozoários/uso terapêutico , Chalcona/metabolismo , Chalcona/farmacologia , Citosol/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/farmacologia , Células Cultivadas , Chalcona/administração & dosagem , Chalcona/análogos & derivados , Citosol/enzimologia , Citosol/parasitologia , Descoberta de Drogas , Humanos , Leishmania/classificação , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: In the field of protein engineering and biotechnology, the discovery and characterization of structural patterns is highly relevant as these patterns can give fundamental insights into protein-ligand interaction and protein function. This paper presents GSP4PDB, a bioinformatics web tool that enables the user to visualize, search and explore protein-ligand structural patterns within the entire Protein Data Bank. RESULTS: We introduce the notion of graph-based structural pattern (GSP) as an abstract model for representing protein-ligand interactions. A GSP is a graph where the nodes represent entities of the protein-ligand complex (amino acids and ligands) and the edges represent structural relationships (e.g. distances ligand - amino acid). The novel feature of GSP4PDB is a simple and intuitive graphical interface where the user can "draw" a GSP and execute its search in a relational database containing the structural data of each PDB entry. The results of the search are displayed using the same graph-based representation of the pattern. The user can further explore and analyse the results using a wide range of filters, or download their related information for external post-processing and analysis. CONCLUSIONS: GSP4PDB is a user-friendly and efficient application to search and discover new patterns of protein-ligand interaction.
Assuntos
Ligantes , Proteínas/metabolismo , Interface Usuário-Computador , Animais , Bases de Dados de Proteínas , Humanos , Ligação de Hidrogênio , Mapas de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/química , Dedos de ZincoRESUMO
The electrostatic potential plays a key role in many biological processes like determining the affinity of a ligand to a given protein target, and they are responsible for the catalytic activity of many enzymes. Understanding the effect that amino acid mutations will have on the electrostatic potential of a protein, will allow a thorough understanding of which residues are the most important in a protein. MutantElec, is a friendly web application for in silico generation of site-directed mutagenesis of proteins and the comparison of electrostatic potential between the wild type protein and the mutant(s), based on the three-dimensional structure of the protein. The effect of the mutation is evaluated using different approach to the traditional surface map. MutantElec provides a graphical display of the results that allows the visualization of changes occurring at close distance from the mutation and thus uncovers the local and global impact of a specific change. © 2017 Wiley Periodicals, Inc.
Assuntos
Simulação por Computador , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação , Eletricidade Estática , Aminoácidos/química , Aminoácidos/genética , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Interface Usuário-ComputadorRESUMO
With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html ). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate.
Assuntos
Aminoácidos/química , Proteínas/química , Software , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Sítios de Ligação , Bases de Dados de Proteínas , Internet , LigantesRESUMO
Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3 Å; corresponding to a diferric µ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.