Novel sulfonylurea derivatives as H3 receptor antagonists. Preliminary SAR studies

The combination of antagonism at histamine H3 receptor and the stimulation of insulin secretion have been proposed as an approach to new dual therapeutic agents for the treatment of type 2 diabetes mellitus associated with obesity. We have designed and synthesized a new series of non-imidazole derivatives, based on a basic amine ring connected through an alkyl spacer of variable length to a phenoxysulfonylurea moiety. These compounds were initially evaluated for histamine H3 receptor binding affinities, suggesting that a propoxy chain linker between the amine and the core ring could be essential for optimal binding affinity. Compound 56, 1-(naphthalen-1-yl)-3-[(p-(3-pyrrolidin-1-ylpropoxy)benzene)]sulfonylurea exhibited the best H3 antagonism affinity. However, since all these derivatives failed to block KATP channels, the link of these two related moieties should not be considered a good pharmacophore for obtaining new dual H3 antagonists with insulinotropic activity, suggesting the necessity to propose a new chemical hybrid prototype

In vitro and in vivo anti-Trypanosoma cruzi activity of new arylamine Mannich base-type derivatives

Chagas disease is a neglected tropical disease with 6-7 million people infected worldwide and there is no effective treatment. Therefore, there is an urgent need to continue researching in order to discover novel therapeutic alternatives. We present a series of arylaminoketone derivatives as means of identifying new drugs to treat Chagas disease in the acute phase with greater activity, less toxicity and with a larger spectrum of action than that corresponding to the reference drug benznidazole. Indexes of high selectivity found in vitro formed the basis for later in vivo assays in BALB/c mice. Murine model results show that compounds 3, 4, 7 and 10 induced a remarkable decrease in parasitemia levels in acute phase and the parasitemia reactivation following immunosuppression, and curative rates were higher than with benznidazole. These high anti-parasitic activities encourage us to propose these compounds as promising molecules for developing an easy to synthesize anti-Chagas agent

Using normal mode analysis on protein structural models. How far can we go on our predictions?

International audienceNormal Mode Analysis is a fast and inexpensive approach that is largely used to gain insight into functional protein motions, and more recently to create conformations for further computational studies. However, when the protein structure is unknown, the use of computational models is necessary. Here, we analyze the capacity of normal mode analysis in internal coordinate space to predict protein motion, its intrinsic flexibility and atomic displacements, using protein models instead of native structures, and the possibility to use it for model refinement. Our results show that normal mode analysis is quite insensitive to modelling errors, but that calculations are strictly reliable only for very accurate models. Our study also suggests that internal normal mode analysis is a more suitable tool for the improvement of structural models, and for integrating them with experimental data or in other computational techniques, such as protein docking or more refined molecular dynamics simulations

Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores

Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double beta-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices

Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function

Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized. Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, this classification guideline may be useful to aid future studies on these abundant toxins

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry

Aerolysin nanopores decode digital information stored in tailored macromolecular analytes

International audienceDigital data storage is a growing need for our society and finding alternative solutions than those based on silicon or magnetic tapes is a challenge in the era of “big data.” The recent development of polymers that can store information at the molecular level has opened up new opportunities for ultrahigh density data storage, long-term archival, anticounterfeiting systems, and molecular cryptography. However, synthetic informational polymers are so far only deciphered by tandem mass spectrometry. In comparison, nanopore technology can be faster, cheaper, nondestructive and provide detection at the single-molecule level; moreover, it can be massively parallelized and miniaturized in portable devices. Here, we demonstrate the ability of engineered aerolysin nanopores to accurately read, with single-bit resolution, the digital information encoded in tailored informational polymers alone and in mixed samples, without compromising information density. These findings open promising possibilities to develop writing-reading technologies to process digital data using a biological-inspired platform

Structural insights into the allosteric site of Arabidopsis NADP-malic enzyme 2: role of the second sphere residues in the regulatory signal transmission

Structure–function studies contribute to deciphering how small modifcations in the primary structure could introduce desirable characteristics into enzymes without afecting its overall functioning. Malic enzymes (ME) are ubiquitous and responsible for a wide variety of functions. The availability of a high number of ME crystal structures from diferent species facilitates comparisons between sequence and structure. Specifcally, the structural determinants necessary for fumarate allosteric regulation of ME has been of particular interest. NADP-ME2 from Arabidopsis thaliana exhibits a distinctive and complex regulation by fumarate, acting as an activator or an inhibitor according to substrate and efector concentrations. However, the 3D structure for this enzyme is not yet reported. In this work, we characterized the NADP-ME2 allosteric site by structural modeling, molecular docking, normal mode analysis and mutagenesis. The regulatory site model and its docking analysis suggested that other C4 acids including malate, NADP-ME2 substrate, could also ft into fumarate’s pocket. Besides, a non-conserved cluster of hydrophobic residues in the second sphere of the allosteric site was identifed. The substitution of one of those residues, L62, by a less fexible residue as tryptophan, resulted in a complete loss of fumarate activation and a reduction of substrate afnities for the active site. In addition, normal mode analysis indicated that conformational changes leading to the activation could originate in the region surrounding L62, extending through the allosteric site till the active site. Finally, the results in this work contribute to the understanding of structural determinants necessary for allosteric regulation providing new insights for enzyme optimization.Fil: Gerrard Wheeler, Mariel Claudia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Arias, Cintia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Da Fonseca Rezende e Mello, Juliana. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Cirauqui Diaz, Nuria. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Rodrigues, Carlos Rangel. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Drincovich, María Fabiana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina.Fil: Mendonça Teles de Souza, Alessandra. Universidade Federal do Rio de Janeiro. Faculdade de Farmácia. Laboratório de Modelagem Molecular & QSAR (ModMolQSAR); Brazil.Fil: Alvarez, Clarisa Ester. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET); Argentina