Entity

Time filter

Source Type

Cambridge, MA, United States

Hudson R.P.,University of Toronto | Chong P.A.,University of Toronto | Protasevich I.I.,University of Alabama at Birmingham | Vernon R.,University of Toronto | And 10 more authors.
Journal of Biological Chemistry | Year: 2012

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Thibodeau P.H.,University of Texas Southwestern Medical Center | Thibodeau P.H.,University of Pittsburgh | Richardson III J.M.,University of Texas Southwestern Medical Center | Wang W.,University of Alabama at Birmingham | And 10 more authors.
Journal of Biological Chemistry | Year: 2010

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Zhang Z.,Harvard University | Kolodziej A.F.,Epix Pharmaceuticals | Greenfield M.T.,Epix Pharmaceuticals | Caravan P.,Harvard University
Angewandte Chemie - International Edition | Year: 2011

Kept within bounds: For peptide-targeted contrast agents, internal motion limits the relaxivity gain. A focused library approach identified an N-terminal thymine peptide nucleic acid (PNA) that reaches an additional binding pocket on the protein fibrin. The heteroditopic binding rigidifies the molecule upon binding, resulting in increased protein-bound relaxivity (see picture). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Porvasnik S.L.,University of Florida | Germain S.,University of Florida | Embury J.,University of Florida | Gannon K.S.,Eolas Biosciences United States | And 5 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2010

Pulmonary arterial hypertension (PAH) is a life-threatening disease that results in right ventricular failure. 5-((4-(6-Chlorothieno[2,3-d]pyrimidin-4- ylamino)piperidin-1-yl)methyl)-2-fluorobenzonitrile monofumarate (PRX-08066) is a selective 5-hydroxytryptamine receptor 2B (5-HT2BR) antagonist that causes selective vasodilation of pulmonary arteries. In the current study, the effects of PRX-08066 were assessed by using the monocrotaline (MCT)-induced PAH rat model. Male rats received 40 mg/kg MCT or phosphate-buffered saline and were treated orally twice a day with vehicle or 50 or 100 mg/kg PRX-08066 for 5 weeks. Pulmonary and cardiac functions were evaluated by hemodynamics, heart weight, magnetic resonance imaging (MRI), pulmonary artery (PA) morphology, and histology. Cardiac MRI demonstrated that PRX-08066 (100 mg/kg) significantly (P < 0.05) improved right ventricular ejection fraction. PRX-08066 significantly reduced peak PA pressure at 50 and 100 mg/kg (P < 0.05 and < 0.01, respectively) compared with MCT control animals. PRX-08066 therapy also significantly reduced right ventricle (RV)/body weight and RV/left ventricle + septum (P < 0.01 and < 0.001, respectively) compared with MCT-treated animals. Morphometric assessment of pulmonary arterioles revealed a significant reduction in medial wall thickening and lumen occlusion associated with both doses of PRX-08066 (P < 0.01). The 5-HT2BR antagonist PRX-08066 significantly attenuated the elevation in PA pressure and RV hypertrophy and maintained cardiac function. Pulmonary vascular remodeling was also diminished compared with MCT control rats. PRX-08066 prevents the severity of PAH in the MCT rat model. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics. Source


Dumas S.,Epix Pharmaceuticals | Jacques V.,Epix Pharmaceuticals | Sun W.-C.,Epix Pharmaceuticals | Troughton J.S.,Epix Pharmaceuticals | And 5 more authors.
Investigative Radiology | Year: 2010

Rationale and Objectives: The donor atoms that bind to gadolinium in contrast agents influence inner-sphere water exchange and electronic relaxation, both of which determine observed relaxivity. The effect of these molecular parameters on relaxivity is greatest when the contrast agent is protein bound. We sought to determine an optimal donor atom set to yield high relaxivity compounds. Methods: A total of 38 gadolinium-1,4,7,10-tetraazacyclo-dodecane-N, N′,N′′,N′′′-tetraacetato derivatives were prepared and relaxivity was determined in the presence and absence of human serum albumin as a function of temperature and magnetic field. Each compound had a common albumin-binding group and differed only by substitution of different donor groups at one of the macrocycle nitrogens. Oxygen-17 isotope relaxometry at 7.05 T was performed to estimate water exchange rates. Results: Changing a single donor atom resulted in changes in water exchange rates ranging across 3 orders of magnitude. Donor groups increased water exchange rate in the order: phosphonate ∼ phenolate > α-substituted acetate > acetate > hydroxamate ∼ sulfonamide > amide ∼ pyridyl ∼ imidazole. Relaxivites at 0.47 and 1.4 T, 37°C, ranged from 12.3 to 55.6 mMs and from 8.3 to 32.6 mMs respectively. Optimal relaxivities were observed when the donor group was an α-substituted acetate. Electronic relaxation was slowest for the acetate derivatives as well. Conclusions: Water exchange dynamics and relaxivity can be predictably tuned by choice of donor atoms. © 2010 by Lippincott Williams & Wilkins. Source

Discover hidden collaborations