Publications by authors named "Heather E Murrey"

10 Publications

  • Page 1 of 1

Development of Sulfonamide Photoaffinity Inhibitors for Probing Cellular γ-Secretase.

ACS Chem Neurosci 2016 08 17;7(8):1166-73. Epub 2016 Jun 17.

Chemical Biology Program, Memorial Sloan Kettering Cancer Center , 1275 York Avenue, New York, New York 10065, United States.

γ-Secretase is a multiprotein complex that catalyzes intramembrane proteolysis associated with Alzheimer's disease and cancer. Here, we have developed potent sulfonamide clickable photoaffinity probes that target γ-secretase in vitro and in cells by incorporating various photoreactive groups and walking the clickable alkyne handle to different positions around the molecule. We found that benzophenone is preferred over diazirine as a photoreactive group within the sulfonamide scaffold for labeling γ-secretase. Intriguingly, the placement of the alkyne at different positions has little effect on probe potency but has a significant impact on the efficiency of labeling of γ-secretase. Moreover, the optimized clickable photoprobe, 163-BP3, was utilized as a cellular probe to effectively assess the target engagement of inhibitors with γ-secretase in primary neuronal cells. In addition, biotinylated 163-BP3 probes were developed and used to capture the native γ-secretase complex in the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) solubilized state. Taken together, these next generation clickable and biotinylated sulfonamide probes offer new tools to study γ-secretase in biochemical and cellular systems. Finally, the data provide insights into structural features of the sulfonamide inhibitor binding site in relation to the active site and into the design of clickable photoaffinity probes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschemneuro.6b00127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576034PMC
August 2016

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.

J Am Chem Soc 2015 Sep 31;137(35):11461-75. Epub 2015 Aug 31.

Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.5b06847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572613PMC
September 2015

Discovery of indole-derived pyridopyrazine-1,6-dione γ-secretase modulators that target presenilin.

Bioorg Med Chem Lett 2015 Feb 25;25(4):908-13. Epub 2014 Dec 25.

Pfizer Worldwide Research & Development, 610 Main Street, Cambridge, MA 02139, USA.

Herein we describe design strategies that led to the discovery of novel pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) incorporating an indole motif as a heterocyclic replacement for a naphthyl moiety that was present in the original lead 9. Tactics involving parallel medicinal chemistry and in situ monomer synthesis to prepare focused libraries are discussed. Optimized indole GSM 29 exhibited good alignment of in vitro potency and physicochemical properties, and moderate reduction of brain Aβ42 was achieved in a rat efficacy model when dosed orally at 30mg/kg. Labeling experiments using a clickable, indole-derived GSM photoaffinity probe demonstrated that this series binds to the presenilin N-terminal fragment (PS1-NTF) of the γ-secretase complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2014.12.059DOI Listing
February 2015

γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

J Biol Chem 2013 Apr 8;288(14):9710-20. Epub 2013 Feb 8.

Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139, USA.

γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic β-amyloid peptides (i.e. Aβ42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.398602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617273PMC
April 2013

Identification of the plasticity-relevant fucose-alpha(1-2)-galactose proteome from the mouse olfactory bulb.

Biochemistry 2009 Aug;48(30):7261-70

Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucalpha(1-2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucalpha(1-2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucalpha(1-2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a "glycocode" to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucalpha(1-2)Gal sugars in the OB is regulated by the alpha(1-2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucalpha(1-2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi900640xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717711PMC
August 2009

The chemical neurobiology of carbohydrates.

Chem Rev 2008 May 2;108(5):1708-31. Epub 2008 May 2.

Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cr078215fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004190PMC
May 2008

Protein fucosylation regulates synapsin Ia/Ib expression and neuronal morphology in primary hippocampal neurons.

Proc Natl Acad Sci U S A 2006 Jan 22;103(1):21-6. Epub 2005 Dec 22.

Howard Hughes Medical Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

Although fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] carbohydrates have been implicated in cognitive processes such as long-term memory, the molecular mechanisms by which these sugars influence neuronal communication are not well understood. Here, we present molecular insights into the functions of Fucalpha(1-2)Gal sugars, demonstrating that they play a role in the regulation of synaptic proteins and neuronal morphology. We show that synapsins Ia and Ib, synapse-specific proteins involved in neurotransmitter release and synaptogenesis, are the major Fucalpha(1-2)Gal glycoproteins in mature cultured neurons and the adult rat hippocampus. Fucosylation has profound effects on the expression and turnover of synapsin in cells and protects synapsin from degradation by the calcium-activated protease calpain. Our studies suggest that defucosylation of synapsin has critical consequences for neuronal growth and morphology, leading to stunted neurite outgrowth and delayed synapse formation. We also demonstrate that Fucalpha(1-2)Gal carbohydrates are not limited to synapsin but are found on additional glycoproteins involved in modulating neuronal architecture. Together, our studies identify important roles for Fucalpha(1-2)Gal sugars in the regulation of neuronal proteins and morphological changes that may underlie synaptic plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0503381102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1324972PMC
January 2006

Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways.

J Biol Chem 2003 Dec 30;278(50):50810-7. Epub 2003 Sep 30.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M309469200DOI Listing
December 2003

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.

Proc Natl Acad Sci U S A 2002 Dec 27;99(25):16309-13. Epub 2002 Nov 27.

Diabetes Unit, Section of Endocrinology, Boston Medical Center and Departments of Medicine and Physiology, Boston University School of Medicine, Boston, MA 02118, USA.

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 micro g/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 micro g/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 micro g/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 micro g) to C57 BL6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15-30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.222657499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC138607PMC
December 2002

Oligomerization state-dependent activation of NF-kappa B signaling pathway by adipocyte complement-related protein of 30 kDa (Acrp30).

J Biol Chem 2002 Aug 26;277(33):29359-62. Epub 2002 Jun 26.

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.

Adipocyte complement-related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived hormone that affects lipid and glucose metabolism in muscle and liver, but its physical and biochemical properties are poorly characterized. Here we have used several approaches to show that Acrp30 expressed in and purified from Escherichia coli and human embryonic kidney 293T cells forms trimers and hexamers; 293T cells also produce a higher molecular weight species. Similar Acrp30 oligomers were found in mouse serum as well as in 3T3-L1 adipocyte-conditioned medium, although in different proportions. In parallel, we assessed whether Acrp30 is a signaling molecule by searching for promoter or enhancer elements that respond to Acrp30 or its isolated trimeric globular C-terminal domain, gAcrp30. Acrp30 addition to C2C12 myocytes or myotubes led to activation of NF-kappa B transcription factor in a manner dependent upon phosphorylation and degradation of I kappa B-alpha. Importantly, only hexameric and larger isoforms of Acrp30 activated NF-kappa B; trimeric Acrp30 or gAcrp30 could not activate NF-kappa B. Our data indicate that oligomerization of Acrp30 is important for at least some of its biological activities, and changes in the relative abundance of each oligomeric isoform in plasma may regulate Acrp30 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.C200312200DOI Listing
August 2002