Mayo, FL, United States
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Roberts E.R.,University of Massachusetts Amherst | Srinivasan J.,Rockford College | Andrzejewski Jr. C.,Baystate Medical Center | Andrzejewski Jr. C.,Tufts University | And 4 more authors.
Journal of Experimental Medicine | Year: 2013

Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defned. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by 7-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1IC and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infltrating T cells during active disease. Conditionally deleting Notch1 or administering 7-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH 1IC was signifcantly elevated and bound to the TBX21 promoter, showing NOTCH 1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH 1IC levels, decreased NOTCH 1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH 1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention. ©2013 Roderick et al.


Maharvi G.M.,Chemical Synthesis Core Facility | Edwards A.O.,University of Oregon | Fauq A.H.,Chemical Synthesis Core Facility
Tetrahedron Letters | Year: 2010

First syntheses of a deuterium-labeled very long C34-containing polyunsaturated fatty acid, 34:5n5, and three other unlabeled very long chain C30-32 containing polyunsaturated fatty acids are reported. These syntheses were achieved by coupling chemically modified C22- and C20-containing polyunsaturated fatty acids with carbanions derived from arylalkyl sulfones, followed by sodium amalgam-mediated desulfonylation. © 2010 Elsevier Ltd. All rights reserved.


Shin H.M.,University of Massachusetts Amherst | Shin H.M.,University of Massachusetts Medical School | Tilahun M.E.,University of Massachusetts Amherst | Tilahun M.E.,National Institute of Allergy and Infectious Diseases | And 14 more authors.
Frontiers in Immunology | Year: 2014

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCΘ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCΘ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling. © 2014 Shin, Tilahun, Cho, Chandiran, Kuksin, Keerthivasan, Fauq, Golde, Miele, Thome, Osborne and Minter.


Bourassa P.,University of Québec | Dubeau S.,University of Québec | Maharvi G.M.,Chemical Synthesis Core Facility | Fauq A.H.,Chemical Synthesis Core Facility | And 2 more authors.
European Journal of Medicinal Chemistry | Year: 2011

The breast anticancer drug tamoxifen and its metabolites bind serum albumins. We located the binding sites of tamoxifen, 4-hydroxytamoxifen and endoxifen on bovine serum albumin (BSA). FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to characterize the drug binding mode, binding constant and the effect of drug binding on BSA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bind BSA via hydrophobic and hydrophilic interactions with overall binding constants of K tam-BSA = 1.96 (±0.2) × 10 4 M -1, K 4-hydroxytam-BSA = 1.80 (±0.4) × 10 4 M -1 and K endox-BSA = 8.01 (±0.8) × 10 3 M -1. The number of bound drug molecules per protein is 1.7 (tamoxifen), 1.4 (4-hydroxitamoxifen) and 1.13 (endoxifen). The participation of several amino acid residues in drug-protein complexes is stabilized by extended hydrogen bonding network with the free binding energy of -13.47 (tamoxifen), -13.79 (4-hydroxtamoxifen) and -12.72 kcal/mol (endoxifen). The order of binding is 4-hydroxy-tamoxen > tamoxifen > endoxifen. BSA conformation was altered by a major reduction of α-helix from 63% (free BSA) to 41% with tamoxifen, to 39% with 4-hydroxytamoxifen, and to 47% with endoxifen. In addition, an increase in turn and random coil structures was found, suggesting partial protein unfolding. These results suggest that serum albumins might act as carrier proteins for tamoxifen and its metabolites in delivering them to target tissues. © 2011 Elsevier Masson SAS. All rights reserved.


Bourassa P.,University of Quebec at Trois - Rivieres | Dubeau S.,University of Quebec at Trois - Rivieres | Maharvi G.M.,Chemical Synthesis Core Facility | Fauq A.H.,Chemical Synthesis Core Facility | And 2 more authors.
Biochimie | Year: 2011

Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. The aim of this study was to examine the interaction of human serum albumin (HSA) with tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen at physiological conditions, using constant protein concentration and various drug contents. FTIR, UV-Visible, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding mode, the binding constant and the effects of drug complexation on HSA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bound HSA via both hydrophobic and hydrophilic interactions with overall binding constants of Ktam = 1.8 (±0.2) × 104 M -1, K4-hydroxytam = 1.8 (±0.4) × 10 4 M-1 and Kendox = 2.0 (±0.5) × 104 M-1. The number of bound drugs per protein is 1.2 (tamoxifen), 1.7 (4-hydroxitamoxifen) and 1.0 (endoxifen). Structural modeling showed the participation of several amino acid residues in drug-HSA complexation, with extended H-bonding network. HSA conformation was altered by tamoxifen and its metabolites with a major reduction of α-helix and an increase in β-sheet, random coil and turn structures, indicating a partial protein unfolding. Our results suggest that serum albumins can act as carrier proteins for tamoxifen and its metabolites in delivering them to target tissues. © 2011 Elsevier Masson SAS. All rights reserved.


Fauq A.H.,Chemical Synthesis Core Facility | Maharvi G.M.,Chemical Synthesis Core Facility | Sinha D.,Chemical Synthesis Core Facility
Bioorganic and Medicinal Chemistry Letters | Year: 2010

A mixture of the (Z)- and (E)-isomers of 4-hydroxy-N-desmethyltamoxifen was conveniently prepared in four steps. These geometrical isomers were then neatly separated by semi-preparative Reverse Phase High Performance Liquid Chromatography (RP-HPLC) using specified conditions. Additionally, the isolated E-isomer could be equilibrated in aqueous strong acid in acetonitrile or trifluoroacetic acid/dichloromethane to give a clean 1:1 mixture of Z/E isomers that was re-subjected to HPLC separation. In this way, most of the undesired (E)-isomer could be readily converted to the desired (Z)-isomer providing quick access to over 200 mg quantities of pure endoxifen (Z-isomer), a potent antiestrogenic metabolite of tamoxifen traditionally used in breast cancer treatment. © 2010 Elsevier Ltd. All rights reserved.


Maharvi G.M.,Chemical Synthesis Core Facility | Fauq A.H.,Chemical Synthesis Core Facility
Tetrahedron Letters | Year: 2010

A concise, convergent racemic synthesis of BMS-708163 is reported. Two fragments consisting of N-4chlorophenylsulfonyl-3,3,3-trifluorpropylglycine and a 1,2,4-oxadiazole derivative of 2-fluorobenzyl alcohol were prepared in separate pots and then coupled together via a Mitsunobu reaction. Since a convenient chiral synthesis of optically pure (D)-3,3,3-trifluoropropyl glycine methyl ester was developed using Schällkopf reagent alkylation, this methodology can also be adopted for the enantioselective synthesis of BMS-708163. © 2010 Elsevier Ltd. All rights reserved.


Seegmiller J.C.,Mayo Medical School | Burns B.E.,Mayo Medical School | Fauq A.H.,Chemical Synthesis Core Facility | Mukhtar N.,Chemical Synthesis Core Facility | And 2 more authors.
Clinical Chemistry | Year: 2010

BACKGROUND: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radio-contrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. METHODS: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8-487.0, 614.8-456.0, 614.8 -361.1, and 614.8 -177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. RESULTS: Mean recovery was 90%-110% in both urine and plasma matrices. Imprecision was ≤15% for the m/z 614.8-487.0 and 614.8-456.0 transitions over a 10-day period at 1 mg/L. Method comparison for 159 patient samples (53 clearances) provided the following Passing-Bablok regressions: plasma iothalamate LC-MS/MS (y) vs CE-UV (x), y = 0.99x + 0.36; urine iothalamate LC-MS/MS vs CE-UV, y = 1.01x + 0.31; corrected GFR LC-MS/MS vs CE-UV, y = 1.00x + 0.00. Interfering substances prevented accurate iothalamate quantification by CE-UV in 2 patients, whereas these samples could be analyzed by LC-MS/MS. CONCLUSIONS: Iothalamate can be quantified by LCMS/MS for GFR measurement. This method circumvents potential problems with interfering substances that occasionally confound accurate GFR determinations. © 2010 American Association for Clinical Chemistry.


Maharvi G.M.,Chemical Synthesis Core Facility | Bharucha A.E.,Mayo Medical School | Fauq A.H.,Chemical Synthesis Core Facility
Bioorganic and Medicinal Chemistry Letters | Year: 2013

Magnetic resonance imaging (MRI) is used to evaluate gastrointestinal (GI) structure and functions in humans. Despite filling the viscus lumen with a contrast agent, visualization of the viscus wall is limited. To overcome this limitation, we de novo synthesized a conjugate that covalently combines a Gd-based MRI contrast agent, encaged with a chelating agent (DOTA), with pantoprazole, which is a widely used proton pump inhibitor that binds to proton pumps in the stomach and colon. The DOTA linkage was installed at a mechanism-based strategic location in the pantoprazole molecule to minimize a possible negative effect of the structural modification on the drug. It is anticipated that by defining the wall of the stomach and colon, this compound will facilitate functional MRI of the GI tract in humans. © 2013 Elsevier Ltd. All rights reserved.


PubMed | Chemical Synthesis Core Facility
Type: Journal Article | Journal: Bioorganic & medicinal chemistry letters | Year: 2013

Magnetic resonance imaging (MRI) is used to evaluate gastrointestinal (GI) structure and functions in humans. Despite filling the viscus lumen with a contrast agent, visualization of the viscus wall is limited. To overcome this limitation, we de novo synthesized a conjugate that covalently combines a Gd-based MRI contrast agent, encaged with a chelating agent (DOTA), with pantoprazole, which is a widely used proton pump inhibitor that binds to proton pumps in the stomach and colon. The DOTA linkage was installed at a mechanism-based strategic location in the pantoprazole molecule to minimize a possible negative effect of the structural modification on the drug. It is anticipated that by defining the wall of the stomach and colon, this compound will facilitate functional MRI of the GI tract in humans.

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