Gene/Protein Disease Symptom Drug Enzyme Compound
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The exponential increase in the number of new nanomaterials that are being produced increases the likelihood of adverse biological effects in humans and the environment. In this study we compared the effects of cationic nanoparticles in five different cell lines that represent portal-of-entry or systemic cellular targets for engineered nanoparticles. Although 60 nm NH(2)-labeled polystyrene (PS) nanospheres were highly toxic in macrophage (RAW 264.7) and epithelial (BEAS-2B) cells, human microvascular endothelial (HMEC), hepatoma (HEPA-1), and pheochromocytoma (PC-12) cells were relatively resistant to particle injury. While the death pathway in RAW 264.7 cells involves caspase activation, the cytotoxic response in BEAS-2B cells is more necrotic in nature. Using fluorescent-labeled NH(2)-PS, we followed the routes of particle uptake. Confocal microscopy showed that the cationic particles entered a LAMP-1 positive lysosomal compartment in RAW 264.7 cells from where the particles could escape by lysosomal rupture. A proton pump inhibitor interfered in this pathway. Subsequent deposition of the particles in the cytosol induced an increase in mitochondrial Ca(2+) uptake and cell death that could be suppressed by cyclosporin A (CsA). In contrast, NH(2)-PS toxicity in BEAS-2B cells did not involve the LAMP-1 endosomal compartment, stimulation of proton pump activity, or an increase in mitochondrial Ca(2+). Particles were taken up by caveolae, and their toxicity could be disrupted by cholesterol extraction from the surface membrane. Although the particles induced mitochondrial damage and ATP depletion, CsA did not affect cytotoxicity. Cationic particles were taken up into HEPA-1, HMEC, and PC-12 cells, but this did not lead to lysosomal permeabilization, increased Ca(2+) flux, or mitochondrial damage. Taken together, the results of this study demonstrate the importance of cell-specific uptake mechanisms and pathways that could lead to sensitivity or resistance to cationic particle toxicity.
ACS Nano 2008 Jan
PMID:Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. 1920 51

Heat shock protein 90 (Hsp90) is a promising cancer drug target, as multiple oncogenic proteins are destabilized simultaneously when it loses its activity in tumor cells. Highly selective Hsp90 inhibitors, including the natural antibiotics geldanamycin (GdA) and radicicol (RAD), inactivate this essential molecular chaperone by occupying its nucleotide binding site. Often cancer drug therapy is compromised by the development of resistance, but a resistance to these Hsp90 inhibitors should not arise readily by mutation of those amino acids within Hsp90 that facilitate inhibitor binding, as these are required for the essential ATP binding/ATPase steps of the chaperone cycle and are tightly conserved. Despite this, the Hsp90 of a RAD-producing fungus is shown to possess an unusually low binding affinity for RAD but not GdA. Within its nucleotide binding site a normally conserved leucine is replaced by isoleucine, though the chaperone ATPase activity is not severely affected. Inserted into the Hsp90 of yeast, this conservative leucine to isoleucine substitution recreated this lowered affinity for RAD in vitro. It also generated a substantially enhanced resistance to RAD in vivo. Co-crystal structures reveal that the change to isoleucine is associated with a localized increase in the hydration of an Hsp90-bound RAD but not GdA. To the best of our knowledge, this is the first demonstration that it is possible for Hsp90 inhibitor resistance to arise by subtle alteration to the structure of Hsp90 itself.
ACS Chem Biol 2009 Apr 17
PMID:Structural basis of the radicicol resistance displayed by a fungal hsp90. 1923 53

Silver nanoparticles (Ag-np) are being used increasingly in wound dressings, catheters, and various household products due to their antimicrobial activity. The toxicity of starch-coated silver nanoparticles was studied using normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). The toxicity was evaluated using changes in cell morphology, cell viability, metabolic activity, and oxidative stress. Ag-np reduced ATP content of the cell caused damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. DNA damage, as measured by single cell gel electrophoresis (SCGE) and cytokinesis blocked micronucleus assay (CBMN), was also dose-dependent and more prominent in the cancer cells. The nanoparticle treatment caused cell cycle arrest in G(2)/M phase possibly due to repair of damaged DNA. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. The transmission electron microscopic (TEM) analysis indicated the presence of Ag-np inside the mitochondria and nucleus, implicating their direct involvement in the mitochondrial toxicity and DNA damage. A possible mechanism of toxicity is proposed which involves disruption of the mitochondrial respiratory chain by Ag-np leading to production of ROS and interruption of ATP synthesis, which in turn cause DNA damage. It is anticipated that DNA damage is augmented by deposition, followed by interactions of Ag-np to the DNA leading to cell cycle arrest in the G(2)/M phase. The higher sensitivity of U251 cells and their arrest in G(2)/M phase could be explored further for evaluating the potential use of Ag-np in cancer therapy.
ACS Nano 2009 Feb 24
PMID:Cytotoxicity and genotoxicity of silver nanoparticles in human cells. 1923 62

Resistance to Hsp90 inhibition has become an important concern as several clinical trials are currently in progress for the treatment of cancer. A summary of known mechanisms of resistance to Hsp90 inhibitors is provided, including the recent solution of the Humicola fuscoatra Hsp90 structure, the organism responsible for the biosynthesis of radicicol. Through careful analyses of Hsp90 structures, a plausible mechanism for resistance to Hsp90 inhibitors has been obtained by single mutations about the N-terminal ATP-binding site.
ACS Chem Biol 2009 Apr 17
PMID:Hydrating for resistance to radicicol. 1937 Nov 33

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.
ACS Chem Biol 2009 Jun 19
PMID:Discovery of antibacterial biotin carboxylase inhibitors by virtual screening and fragment-based approaches. 1953 54

How primitive enzymes emerged from a primordial pool remains a fundamental unanswered question with important practical implications in synthetic biology. Here we show that a de novo evolved ATP binding protein, selected solely on the basis of its ability to bind ATP, mediates the regiospecific hydrolysis of ATP to ADP when crystallized with 1 equiv of ATP. Structural insights into this reaction were obtained by growing protein crystals under saturating ATP conditions. The resulting crystal structure refined to 1.8 A resolution reveals that this man-made protein binds ATP in an unusual bent conformation that is metal-independent and held in place by a key bridging water molecule. Removal of this interaction using a null mutant results in a variant that binds ATP in a normal linear geometry and is incapable of ATP hydrolysis. Biochemical analysis, including high-resolution mass spectrometry performed on dissolved protein crystals, confirms that the reaction is accelerated in the crystalline environment. This observation suggests that proteins with weak chemical reactivity can emerge from high affinity ligand binding sites and that constrained ligand-binding geometries could have helped to facilitate the emergence of early protein enzymes.
ACS Chem Biol 2009 Aug 21
PMID:A synthetic protein selected for ligand binding affinity mediates ATP hydrolysis. 1952 80

We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca(2+)) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC(50)) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC(50)) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyltransferase, reactive oxygen species formation, long-chain acetyl-CoA synthetase and ATP-sensitive K(+) channel activity or activation of uncoupling protein 2 (UCP2) accounted for only approximately 20% of OA's excitatory effects and approximately 40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose dependent. This glucose-OA interaction provides a potential mechanism whereby such "metabolic sensing" neurons can respond to differences in the metabolic states associated with fasting and feeding.
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PMID:Characteristics and mechanisms of hypothalamic neuronal fatty acid sensing. 1953 76

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140 degrees to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl-binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl-binding pocket.
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PMID:The 2.1 A crystal structure of an acyl-CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl-binding pocket for small branched acyl substrates. 1954 69

ABCG2 is a membrane-localized, human transporter protein that has been demonstrated to reduce the intracellular accumulation of substrates through ATP-dependent efflux. Highly expressed in placental syncytiotrophoblasts, brain microvasculature, and the gastrointestinal tract, ABCG2 has been shown to mediate normal tissue protection as well as limit oral bioavailability of substrate compounds. Development of ABCG2 inhibitors for clinical use may allow increased penetration of therapeutic agents into sanctuary sites and increased gastrointestinal absorption. Previously identified inhibitors have lacked potency or specificity or were toxic at concentrations needed to inhibit ABCG2; none are in clinical development. A previously developed high-throughput assay measuring inhibition of ABCG2-mediated pheophorbide a transport was applied to natural product extract libraries. Among the active samples were extracts from the marine ascidian Botryllus tyreus. Bioassay-guided fractionation resulted in purification of a series of botryllamides. Ten botryllamides were obtained, two of which (designated I and J) were novel. Activity against ABCG2 was confirmed by assessing the ability of the compounds to inhibit ABCG2-mediated BODIPY-prazosin transport in ABCG2-transfected HEK293 cells, compete with [(125)I]-iodoarylazidoprazosin (IAAP) labeling of ABCG2, stimulate ABCG2-associated ATPase activity, and reverse ABCG2-mediated resistance.
ACS Chem Biol 2009 Aug 21
PMID:Botryllamides: natural product inhibitors of ABCG2. 1955 20

DNA synthesis is catalyzed by an ensemble of proteins designated the replicase. The efficient assembly of this multiprotein complex is essential for the continuity of DNA replication and is mediated by clamp-loading accessory proteins that use ATP binding and hydrolysis to coordinate these events. As a consequence, the ability to selectively inhibit the activity of these accessory proteins provides a rational approach to regulate DNA synthesis. Toward this goal, we tested the ability of several non-natural nucleotides to inhibit ATP-dependent enzymes associated with DNA replicase assembly. Kinetic and biophysical studies identified 5-nitro-indolyl-2'-deoxyribose-5'-triphosphate as a unique non-natural nucleotide capable of selectively inhibiting the bacteriophage T4 clamp loader versus the homologous enzyme from Escherichia coli. Modeling studies highlight the structural diversity between the ATP-binding site of each enzyme and provide a mechanism accounting for the differences in potencies for various substituted indolyl-2'-deoxyribose-5'-triphosphates. An in vivo assay measuring plaque formation demonstrates the efficacy and selectivity of 5-nitro-indolyl-2'-deoxyribose as a cytostatic agent against T4 bacteriophage while leaving viability of the E. coli host unaffected. This strategy provides a novel approach to develop agents that selectively inhibit ATP-dependent enzymes that are required for efficient DNA replication.
ACS Chem Biol 2010 Feb 19
PMID:Selective inhibition of DNA replicase assembly by a non-natural nucleotide: exploiting the structural diversity of ATP-binding sites. 1999 7


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