RESUMO

The potential outcome of flavivirus and alphavirus co-infections is worrisome due to the development of severe diseases. Hundreds of millions of people worldwide live under the risk of infections caused by viruses like chikungunya virus (CHIKV, genus Alphavirus), dengue virus (DENV, genus Flavivirus), and zika virus (ZIKV, genus Flavivirus). So far, neither any drug exists against the infection by a single virus, nor against co-infection. The results described in our study demonstrate the inhibitory potential of two flavonoids derived from citrus plants: Hesperetin (HST) against NS2B/NS3pro of ZIKV and nsP2pro of CHIKV and, Hesperidin (HSD) against nsP2pro of CHIKV. The flavonoids are noncompetitive inhibitors and the determined IC50 values are in low µM range for HST against ZIKV NS2B/NS3pro (12.6 ± 1.3 µM) and against CHIKV nsP2pro (2.5 ± 0.4 µM). The IC50 for HSD against CHIKV nsP2pro was 7.1 ± 1.1 µM. The calculated ligand efficiencies for HST were > 0.3, which reflect its potential to be used as a lead compound. Docking and molecular dynamics simulations display the effect of HST and HSD on the protease 3D models of CHIKV and ZIKV. Conformational changes after ligand binding and their effect on the substrate-binding pocket of the proteases were investigated. Additionally, MTT assays demonstrated a very low cytotoxicity of both the molecules. Based on our results, we assume that HST comprise a chemical structure that serves as a starting point molecule to develop a potent inhibitor to combat CHIKV and ZIKV co-infections by inhibiting the virus proteases.

Assuntos

Vírus Chikungunya/enzimologia , Citrus/química , Hesperidina/farmacologia , Peptídeo Hidrolases/metabolismo , Zika virus/enzimologia , Animais , Vírus Chikungunya/efeitos dos fármacos , Chlorocebus aethiops , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeo Hidrolases/química , Extratos Vegetais/química , Conformação Proteica , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismo , Zika virus/efeitos dos fármacos

RESUMO

Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.

Assuntos

Corynebacterium pseudotuberculosis/metabolismo , Vitamina B 12/metabolismo , Adenina/química , Adenina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinética , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometria de Fluorescência , Homologia Estrutural de Proteína , Suramina/química , Suramina/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/química

RESUMO

Carbendazim (MBC) is a fungicide widely used in agriculture which allows the high productivity of several cultures, a necessary condition considering the growing of the world population. Moreover, MBC has environmental impact mainly on the soil and water sources, and consequently, on animal and human lives. However, even though the toxicity of fungicides is well established, their action mechanism in cell membranes are not completely understood. Herein, we investigate the interaction of different polar headgroups: dimethyldioctadecylammonium bromide (DODAB), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG); and different chain unsaturation degrees DPPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with MBC. Lipid monolayers at the air/water interface were applied as mimetic systems of cell membranes to investigate the interaction with MBC dissolved in the ultrapure water subphase. It was found that the interaction is driven preferably by electrostatic forces of the headgroups, with higher affinity for DODAB (cationic), intermediate for DPPC (zwitterionic), and absent for DPPG (anionic), considering the monolayer in the condensed phase. DODAB-MBC electrostatic interaction was consistent with FTIR (cast films). We also investigated giant unilamellar vesicles (GUVs) of zwitterionic lipids (DPPC, POPC, and DOPC) with distinct chain unsaturations in the presence of MBC by confocal microscopy and molecular dynamic (MD) simulations. The results indicate that, unlike the chain unsaturation, the polar headgroups play key role on the lipid-MBC interaction.