RESUMEN
H2O2-oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalytic cysteine residues (Cc(SH) undergo rapid S-glutathionylation. Restoration of the enzyme activity is accomplished by thiol/disulfide SN2 displacement (directly or enzymatically) forming glutathione disulfide (G(SS)G) and active enzyme, a process that should be facile as Cc(SH) reside on the subunit surface. As S-glutathionylated GAPDH accumulates following ischemic and/or oxidative stress, in vitro/silico approaches have been employed to address this paradox. Cc(SH) residues were selectively oxidized and S-glutathionylated. Kinetics of GAPDH dehydrogenase recovery demonstrated that glutathione is an ineffective reactivator of S-glutathionylated GAPDH compared to dithiothreitol. Molecular dynamic simulations (MDS) demonstrated strong binding interactions between local residues and S-glutathione. A second glutathione was accommodated for thiol/disulfide exchange forming a tightly bound glutathione disulfide G(SS)G. The proximal sulfur centers of G(SS)G and Cc(SH) remained within covalent bonding distance for thiol/disulfide exchange resonance. Both these factors predict inhibition of dissociation of G(SS)G, which was verified by biochemical analysis. MDS also revealed that both S-glutathionylation and bound G(SS)G significantly perturbed subunit secondary structure particularly within the S-loop, region which interacts with other cellular proteins and mediates NAD(P)+ binding specificity. Our data provides a molecular rationale for how oxidative stress elevates S-glutathionylated GAPDH in neurodegenerative diseases and implicates novel targets for therapeutic intervention.
Asunto(s)
Peróxido de Hidrógeno , Enfermedades Neurodegenerativas , Humanos , Disulfuro de Glutatión/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glutatión/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Disulfuros/químicaRESUMEN
Oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by reactive oxygen species such as H2O2 activate pleiotropic signaling pathways is associated with pathophysiological cell fate decisions. Oxidized GAPDH binds chaperone proteins with translocation of the complex to the nucleus and mitochondria initiating autophagy and cellular apoptosis. In this study, we establish the mechanism by which H2O2-oxidized GAPDH subunits undergo a subunit conformational rearrangement. H2O2 oxidizes both the catalytic cysteine and a vicinal cysteine (four residues downstream) to their respective sulfenic acids. A 'two-cysteine switch' is activated, whereby the sulfenic acids irreversibly condense to an intrachain thiosulfinic ester resulting in a major metastable subunit conformational rearrangement. All four subunits of the homotetramer are uniformly and independently oxidized by H2O2, and the oxidized homotetramer is stabilized at low temperatures. Over time, subunits unfold forming disulfide-linked aggregates with the catalytic cysteine oxidized to a sulfinic acid, resulting from thiosulfinic ester hydrolysis via the highly reactive thiosulfonic ester intermediate. Molecular Dynamic Simulations provide additional mechanistic insights linking GAPDH subunit oxidation with generating a putative signaling conformer. The low-temperature stability of the H2O2-oxidized subunit conformer provides an operable framework to study mechanisms associated with gain-of-function activities of oxidized GAPDH to identify novel targets for the treatment of neurodegenerative diseases.
Asunto(s)
Cisteína , Ácidos Sulfénicos , Cisteína/metabolismo , Ésteres , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Peróxido de Hidrógeno , Oxidación-Reducción , Ácidos Sulfénicos/metabolismoRESUMEN
Acetylcholinesterase (AChE) is a main drug target, and its inhibitors have demonstrated functionality in the symptomatic treatment of Alzheimer's disease (AD). In this study, a series of novel AChE inhibitors were designed and their inhibitory activity was evaluated with 2D quantitative structure-activity relationship (QSAR) studies using a training set of 20 known compounds for which IC50 values had previously been determined. The QSAR model was calculated based on seven unique descriptors. Model validation was determined by predicting IC50 values for a test set of 20 independent compounds with measured IC50 values. A correlation analysis was carried out comparing the statistics of the measured IC50 values with predicted ones. These selectivity-determining descriptors were interpreted graphically in terms of principal component analyses (PCA). A 3D pharmacophore model was also created based on the activity of the training set. In addition, absorption, distribution, metabolism, and excretion (ADME) descriptors were also determined to evaluate their pharmacokinetic properties. Finally, molecular docking of these novel molecules into the AChE binding domain indicated that three molecules (6c, 7c, and 7h) should have significantly higher affinities and solvation energies than the known standard drug donepezil. The docking studies of 2H-thiazolo[3,2-a]pyrimidines (6a-6j) and 5H-thiazolo[3,2-a] pyrimidines (7a-7j) with human AChE have demonstrated that these ligands bind to the dual sites of the enzyme. Simple and ecofriendly syntheses and diastereomeric crystallizations of 2H-thiazolo [3,2-a]pyrimidines and 5H-thiazolo[3,2-a] pyrimidines are described. The solid-state structures for the HBr salts of compounds 6a, 6e, 7a, and 7i have been determined using single-crystal X-ray diffraction techniques, and X-ray powder patterns were measured for the bulk solid remaining after solvent was removed from solutions containing 6a and 7a. These studies provide valuable insight for designing more potent and selective inhibitors for the treatment of AD.
Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/farmacología , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Interfaz Usuario-Computador , Acetilcolinesterasa/química , Técnicas de Química Sintética , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacocinética , Donepezilo , Evaluación Preclínica de Medicamentos , Humanos , Indanos/síntesis química , Indanos/metabolismo , Indanos/farmacocinética , Indanos/farmacología , Piperidinas/síntesis química , Piperidinas/metabolismo , Piperidinas/farmacocinética , Piperidinas/farmacología , Conformación Proteica , Relación Estructura-Actividad CuantitativaRESUMEN
Amyloid binding alcohol dehydrogenase, a mitochondrial protein, is a cofactor facilitating amyloid-ß peptide (Aß) induced cell stress. Antagonizing Aß-ABAD interaction protects against aberrant mitochondrial and neuronal function and improves learning memory in the Alzheimer's disease mouse model. Therefore, it offers a potential target for Alzheimer's drug design, by identifying potential inhibitors of Aß-ABAD interaction. 2D QSAR methods were applied to novel compounds with known IC(50) values, which formed a training set. A correlation analysis was carried out comparing the statistics of the measured IC(50) with predicted values. These selectivity-determining descriptors were interpreted graphically in terms of principle component analyses, which are highly informative for the lead optimization process with respect to activity enhancement. A 3D pharmacophore model also was created. The 2D QSAR and 3D pharmacophore models will assist in high-throughput screening. In addition, ADME descriptors were also determined to study their pharmacokinetic properties. Finally, amyloid binding alcohol dehydrogenase molecular docking study of these novel molecules was undertaken to determine whether these compounds exhibit significant binding affinity with the binding site. We have synthesized only the compounds that have shown the best drug-like properties as candidates for further studies.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/antagonistas & inhibidores , Enfermedad de Alzheimer/tratamiento farmacológico , Organofosfonatos/síntesis química , Tiazoles/síntesis química , 3-Hidroxiacil-CoA Deshidrogenasas/química , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Animales , Sitios de Unión , Células CACO-2 , Diseño de Fármacos , Humanos , Ligandos , Ratones , Simulación del Acoplamiento Molecular , Organofosfonatos/química , Organofosfonatos/farmacología , Relación Estructura-Actividad Cuantitativa , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC50 45 nM at the human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing hormone receptor. Structure-activity relationships for the series were explored to optimize receptor affinity and pharmacokinetic properties, resulting in compounds with Ki values <1nM and brain levels after oral dosing approximately 100-fold higher than receptor affinities.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Azetidinas/farmacología , Animales , Azetidinas/sangre , Azetidinas/farmacocinética , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Perros , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Ratas , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
In the AD brain, there are elevated amounts of soluble and insoluble Abeta peptides which enhance the expression of membrane bound and soluble receptor for advanced glycation end products (RAGE). The binding of soluble Abeta to soluble RAGE inhibits further aggregation of Abeta peptides, while membrane bound RAGE-Abeta interactions elicit activation of the NF-kappaB transcription factor promoting sustained chronic neuroinflammation. Atomic force microscopy observations demonstrated that the N-terminal domain of RAGE, by interacting with Abeta, is a powerful inhibitor of Abeta polymerization even at prolonged periods of incubation. Hence, the potential RAGE-Abeta structural interactions were further explored utilizing a series of computational chemistry algorithms. Our modeling suggests that a soluble dimeric RAGE assembly creates a positively charged well into which the negative charges of the N-terminal domain of dimeric Abeta dock.
Asunto(s)
Microscopía de Fuerza Atómica , Receptores Inmunológicos/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Dimerización , Disulfuros/química , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Inmunoglobulina G/inmunología , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , SolubilidadRESUMEN
In this paper we explore the potential functional role of the A beta peptides in the context of Alzheimer's disease (AD). We begin by defining the morphology of the amyloid deposits in relation to surrounding glial cells and, more importantly, in relation to the brain vasculature. Amyloid accumulation in the brain's microvasculature causes disturbances in the blood-brain barrier (BBB), and in larger arteries, impairment in control of regional cerebral blood flow due to myocyte degeneration. We postulate that the deposition of vascular amyloid may represent a hydrophobic protein plaster to seal leaks in the BBB, occasionally observed in aging and catastrophically common in AD. The vasoconstrictive activity of A beta may also be related to leaky vessels whereby decreasing the arterial diameter may also help to control breaches in the BBB. The admission of plasma neurotoxic proteins into the brain may be controlled by activation of microglia elicited by soluble A beta peptides creating a subtle, but permanent brain inflammatory reaction. We also delve into the influence that cholesterol metabolism may have in membrane topology and A beta production, and the close correlations that exist between cardiovascular disease and AD. Finally, we speculate about the possibility of a peripheral source of A beta that may, by crossing the BBB, contribute to the vascular and parenchymal deposits of A beta in the AD brain.