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 fármacosRESUMO
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/químicaRESUMO
In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens. The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.
Assuntos
Corynebacterium pseudotuberculosis/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Polímeros/química , Polímeros/farmacologia , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/química , Domínio Catalítico , Corynebacterium pseudotuberculosis/genética , Ligantes , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxirredução , Polieletrólitos , Ligação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificaçãoRESUMO
We propose and analyze a model, based on signal-tuned Gabor functions, for the receptive fields and responses of V1 cells. Signal-tuned Gabor functions are gaussian-modulated sinusoids whose parameters are obtained from a given, spatial, or spectral "tuning" signal. These functions can be proven to yield exact representations of their tuning signals and have recently been proposed as the kernels of a variant Gabor transform-the signal-tuned Gabor transform (STGT)-which allows the accurate detection of spatial and spectral events. Here we show that by modeling the receptive fields of simple and complex cells as signal-tuned Gabor functions and expressing their responses as STGTs, we are able to replicate the properties of these cells when tested with standard grating and slit inputs, at the same time emulating their stimulus-dependent character as revealed by recent neurophysiological studies.
Assuntos
Neurônios/fisiologia , Córtex Visual/fisiologia , Percepção Visual/fisiologia , Algoritmos , Modelos Neurológicos , Distribuição Normal , Campos Visuais/fisiologiaRESUMO
Studies examining various spacer groups that link the two aromatic rings of combretastatin A-4 (CA4) have shown that the biological activity of analogs does not require the cis-stilbene configuration of CA4. Oxygen or nitrogen, carbonyl, methylene and ethylene spacers, for example, are present in CA4 analogs that show good activity. Up to now sulfur was not tested for this purpose. In this article we describe the synthesis of sulfide, sulfoxide and sulfone spacers between two aromatic rings comparable to those of CA4. We also compared them with CA4 for inhibitory effects on cell growth, tubulin polymerization, and the binding of [(3)H]colchicine to tubulin. We found that the sulfide is highly active and may be a lead compound for the preparation of antitumor compounds.