|Cell-penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases.|
|Jump to Full Text|
|PMID: 21095575 Owner: NLM Status: MEDLINE|
|Posttranslational modification of metazoan nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) is essential, dynamic, and inducible and can compete with protein phosphorylation in signal transduction. Inhibitors of O-GlcNAcase, the enzyme removing O-GlcNAc, are useful tools for studying the role of O-GlcNAc in a range of cellular processes. We report the discovery of nanomolar OGA inhibitors that are up to 900,000-fold selective over the related lysosomal hexosaminidases. When applied at nanomolar concentrations on live cells, these cell-penetrant molecules shift the O-GlcNAc equilibrium toward hyper-O-GlcNAcylation with EC₅₀ values down to 3 nM and are thus invaluable tools for the study of O-GlcNAc cell biology.|
|Helge C Dorfmueller; Vladimir S Borodkin; Marianne Schimpl; Xiaowei Zheng; Robert Kime; Kevin D Read; Daan M F van Aalten|
Related Documents :
|16822055 - Investigation of living pancreas tumor cells by digital holographic microscopy.
1961365 - From here to eternity: brain aging in an evolutionary perspective.
20419255 - Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery ...
394365 - Cryptococcus neoformans: gastronomic delight of a soil ameba.
23107495 - Monitoring of bacterial load in terms of culturable and non-culturable cells on new mat...
16832065 - Temporally restricted substrate interactions direct fate and specification of neural pr...
|Type: Journal Article; Research Support, Non-U.S. Gov't|
|Title: Chemistry & biology Volume: 17 ISSN: 1879-1301 ISO Abbreviation: Chem. Biol. Publication Date: 2010 Nov|
|Created Date: 2010-11-24 Completed Date: 2011-03-14 Revised Date: 2014-02-20|
Medline Journal Info:
|Nlm Unique ID: 9500160 Medline TA: Chem Biol Country: United States|
|Languages: eng Pagination: 1250-5 Citation Subset: IM|
|Copyright © 2010 Elsevier Ltd. All rights reserved.|
|APA/MLA Format Download EndNote Download BibTex|
Amino Acid Substitution
Enzyme Inhibitors / chemistry*, pharmacology
Hexosaminidases / antagonists & inhibitors*, metabolism
Protein Structure, Tertiary
beta-N-Acetylhexosaminidases / antagonists & inhibitors*, genetics, metabolism
|087590//Wellcome Trust; G0900138//Medical Research Council; //Wellcome Trust|
|0/Enzyme Inhibitors; EC 3.2.1.-/Hexosaminidases; EC 184.108.40.206/hexosaminidase C; EC 220.127.116.11/beta-N-Acetylhexosaminidases; V956696549/Acetylglucosamine|
Journal ID (nlm-ta): Chem Biol
Publisher: Cell Press
Â© 2010 Elsevier Ltd.
Received Day: 21 Month: 6 Year: 2010
Revision Received Day: 24 Month: 8 Year: 2010
Accepted Day: 1 Month: 9 Year: 2010
pmc-release publication date: Day: 24 Month: 11 Year: 2010
Print publication date: Day: 24 Month: 11 Year: 2010
Volume: 17 Issue: 11
First Page: 1250 Last Page: 1255
PubMed Id: 21095575
Publisher Id: CHBIOL1789
|Cell-Penetrant, Nanomolar O-GlcNAcase Inhibitors Selective against Lysosomal Hexosaminidases|
|Helge C. Dorfmueller1|
|Vladimir S. Borodkin1|
|Kevin D. Read2|
|Daan M.F. van Aalten1∗||Email: email@example.com|
1Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland
2Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland
|∗Corresponding author firstname.lastname@example.org
The O-GlcNAc modification was discovered more than two decades ago by Torres and Hart (1984). This modification consists of a single GlcNAc sugar that is dynamically and reversibly transferred onto/hydrolyzed from serine/threonine residues on proteins in the nucleocytoplasm (Hart et al., 2007; Hanover et al., 2010). Similar to protein phosphorylation, the O-GlcNAc modification has been shown to be involved in signaling processes and to occupy serine/threonine residues identical, or adjacent, to known protein phosphorylation sites (Roquemore et al., 1996; Chou et al., 1992; Zachara and Hart, 2006; Hart et al., 2007). O-GlcNAc transfer is performed by a single enzyme, the O-GlcNAc transferase (OGT) (Kreppel et al., 1997; Lubas et al., 1997). The O-GlcNAc modification is removed by a single enzyme termed O-GlcNAcase (OGA) (Gao et al., 2001).
A range of approaches has been used to gain insight into the function of O-GlcNAc and its possible regulation of signaling pathways, including genetic manipulation (Bowe et al., 2006; Ngoh et al., 2009) and blocking O-GlcNAc hydrolysis with small molecules that inhibit hOGA. A number of hOGA inhibitors have been described and used to demonstrate the involvement of O-GlcNAc in the insulin response (Akimoto et al., 2007), regulation of Tau phosphorylation (Yuzwa et al., 2008), Akt activation (Vosseller et al., 2002), and, recently, in regulation of cellular volume (Nagy et al., 2009). However, many of these inhibitors possess limited selectivity over the structurally related lysosomal hexosaminidases A/B (HexA/B) and/or are only active on cells in the high micromolar range, increasing the risk of off-target effects, as suggested recently by Macauley et al. (2008), Yuzwa et al. (2008), and Dorfmueller et al. (2006, 2009).
Here we have attempted to target a conserved hOGA active site cysteine with suicide inhibitors, based on the GlcNAcstatin scaffold. These novel compounds are up to 900000-fold selective over HexA/B, penetrating the OGA active site as revealed by X-ray crystallography. The compounds penetrate live cells, inducing hyper-O-GlcNAcylation when used at low nanomolar concentrations. The new GlcNAcstatins will be useful tools for studying the role of O-GlcNAc in cellular signaling pathways.
GlcNAcstatins (Figure 1A), a novel family of potent human OGA inhibitors, have recently been reported to possess 150-fold selectivity over hHexA/B (Dorfmueller et al., 2006, 2009). Based on the structural data of GlcNAcstatins in complex with a bacterial OGA from C. perfringens (CpOGA) (Figure 2A), we assumed that increased selectivity for hOGA over the HexA/B could be achieved by extending the size of the N-acyl derivative. However, similar elaboration of the NAG-thiazoline/PUGNAc scaffolds reduced inhibition from nanomolar to micromolar range (Macauley et al., 2005; Stubbs et al., 2006). A structural comparison of the active site pockets from CpOGA (Dorfmueller et al., 2009) and hHexA (Lemieux et al., 2006) and hHexB (Mark et al., 2003) reveals obvious differences in the N-acetyl binding pocket (Figure 2A). The β-hexosaminidases have a narrower (difference of approximately 1.4 Å) and shallower (difference of approximately 5.0 Å) pocket than the OGA enzymes (Figure 2A). Interestingly, a cysteine residue is located at the bottom of the hOGA active site (Cys215) (Figure 2B). This cysteine is conserved in metazoan OGAs, and hOGA is potently inhibited by a thiol-reactive compound (Dong and Hart, 1994).
In an attempt to generate a potent, selective hOGA “suicide” inhibitor, the N-acyl group of GlcNAcstatin D was extended and modified to contain thiol-reactive groups that could irreversibly react with the cysteine located in a pocket at the bottom of the active site. GlcNAcstatin F carries a 3-mercaptopropanamide side chain (Figure 1A) and GlcNAcstatin G a penta-2,4-dienamide derivative, both potentially able to react with the hOGA Cys215. GlcNAcstatin H, a saturated derivative of GlcNAcstatin G, was synthesized as a control (Figure 1A). The synthesis will be reported elsewhere.
The new GlcNAcstatin derivatives were evaluated in kinetic studies for their ability to inhibit recombinant hOGA. The pH optimum of hOGA is 7.3 (Figure 1D), whereas the first GlcNAcstatin inhibitor reported (GlcNAcstatin C) inhibits with maximum potency at pH 6.6 (Ki = 2.9 nM) (Figure 1D). At pH 7.3, GlcNAcstatins F–H show time-independent inhibition in the 2.6–11.2 nM range (Table 1 and Figures 1A and 1B). To assess selectivity, inhibition of hHexA/B was also investigated (Figure 1C). The extension of the N-propionyl side chain of GlcNAcstatin D with an additional thiol group (GlcNAcstatin F) increases selectivity for hOGA to 1000-fold (Figure 1C and Table 1), showing that the elongated N-acyl substitution abolishes the binding of the compound to hHexA/B (Table 1). Strikingly, the more extended GlcNAcstatin G inhibits hHexA/B with an approximate IC50 of only 7 mM (Figure 1C and Table 1), thus resulting in a >900,000-fold selectivity for GlcNAcstatin G toward hOGA, representing the most selective hOGA inhibitor reported to date.
The molecular basis of the increased selectivity of the new GlcNAcstatin derivatives was investigated by X-ray crystallography using the CpOGA V331C mutant that perfectly mimics the hOGA active site (Dorfmueller et al., 2009) (Figure 2B). The CpOGA-V331C GlcNAcstatin F complex (2.5 Å resolution) reveals that the inhibitor-binding mode is similar to the previously reported GlcNAcstatin C/D complexes (Dorfmueller et al., 2006, 2009) (Figure 2B). The 3-mercaptopropanamide moiety points straight into the pocket of the active site, whereas all hydrogen bonds seen in the GlcNAcstatin C/D complexes are conserved. Interestingly, the sulfhydryl group of GlcNAcstatin F approaches the active site cysteine (Cys215 in hOGA, equivalent to Cys331 in the V331C-CpOGA mutant) to within 3.5 Å (Figure 2B), although the side chain thiol points away from the inhibitor, and no disulfide bond is formed, in agreement with the absence of time-dependent inhibition. A structural superposition of the GlcNAcstatin F-V331C-CpOGA complex onto the structure of hHexA (Figure 2C) reveals that the shallower N-acetyl binding pocket in the HexA structure would produce steric clashes with the 3-mercaptopropanamide moiety, also explaining the increased selectivity for hOGA of >900,000-fold for the further elongated penta-2,4-dienamide substituent (GlcNAcstatin G).
No direct evidence has so far been provided for any known hOGA inhibitor penetrating the cell and inducing cellular hyper-O-GlcNAcylation by direct inhibition of intracellular hOGA. We have used a mass-spectrometric approach to measure intracellular GlcNAcstatin G concentrations in HEK293 cells. HEK293 cells were treated in triplicate with two inhibitor concentrations (1 and 10 μM) for 1.5 and 6 hr. An equal number of cultured cells were lysed, and the intracellular volume was investigated by UPLC-MS/MS. On the basis of the average cell size (13.1–15.7 μm) and the number of cultured cells (1.4 × 106 to 2.2 × 106), the maximal cellular volume and, thus, the minimal dilution factor of the intracellular volume upon the addition of lysis buffer were calculated (280-fold). Treatment of HEK293 cells with 10 μM GlcNAcstatin G resulted in a minimal inhibitor concentration of 270 nM after 1.5 hr and 380 nM after 6 hr. Application of a 1 μM extracellular concentration resulted in a 10-fold lower intracellular concentration of at least 30 and 40 nM, after 1.5 and 6 hr treatment, respectively. To our knowledge, these data provide the first direct evidence that GlcNAcstatins are cell-penetrant compounds.
The novel GlcNAcstatin derivatives were investigated for their activity on endogenous hOGA in a cell-based assay with HEK293 cells. Cells were exposed for 6 hr to a range of inhibitor concentrations and investigated for an increase of cellular O-GlcNAcylation levels by western blotting using an anti-O-GlcNAc antibody (CTD110.6), followed by the fitting of densitometric data to a standard dose-response curve (Figure 3A), yielding EC50s of 20 nM for both GlcNAcstatins C and G, and an EC50 of 290 nM for GlcNAcstatin F. Treatment with concentrations as low as 100 nM of GlcNAcstatin G in the culture media appears to already induce maximum levels of O-GlcNAcylation (Figure 3A). Furthermore, we determined the EC50 of GlcNAcstatins G and H by O-GlcNAc microscopy assay (Figure 3B; see Figure S1 available online). Elevated total O-GlcNAc levels in HEK293 cells were quantified and normalized over a reference DAPI signal. Dose-response curves of these analyses yielded EC50 values of 3 and 42 nM for GlcNAcstatins G and H, respectively, in good agreement with the immunoblotting-derived data (Figures 3A and 3B).
In conclusion these new compounds and, in particular, GlcNAcstatin G provide potent and selective chemical biological dissection tools as an attractive alternative to genetic approaches for modulating intracellular O-GlcNAc levels in cells to study the role of O-GlcNAc in signal transduction.
Selective inhibition of human O-GlcNAcase is a useful strategy to study the role of the O-GlcNAc modification in living cells. Previously reported compounds showed undesirable inhibition of the human lysosomal hexosaminidases, and there has been no direct evidence of the cell penetration of these compounds. This work reports derivatives of the GlcNAcstatin scaffold, showing potent inhibition of human O-GlcNAcase with up to 900,000-fold weaker inhibition of the lysosomal hexosaminidases. These are the most selective human O-GlcNAcase inhibitors known to date and penetrate live cells to induce cellular hyper-O-GlcNAcylation levels with EC50s in the low nanomolar range.
Cloning, expression, purification, and crystallization of hOGA and/or CpOGA have been described previously (Dorfmueller et al., 2009). Diffraction data for a CpOGA-V331C GlcNAcstatin F complex were collected on BM-14 (ESRF, Grenoble) to 2.4 Å (Table 2). Refinement to 2.5 Å resolution was initiated from the protein model in the CpOGA-GlcNAcstatin C complex (PDB entry 2J62) (Dorfmueller et al. ), and completed by iterative model building using COOT (Emsley and Cowtan, 2004) and refinement with REFMAC (Murshudov et al., 1997) (Final R, Rfree at 2.5 Å resolution: 0.197, 0.236).
Steady-state kinetics (measurements were performed in triplicate) and inhibition constants (Ki) for GlcNAcstatin derivatives were determined using the fluorogenic assay as described previously (Dorfmueller et al., 2009).
The mode of inhibition was visually verified by a Lineweaver-Burk plot (Figure 1B) and the Ki determined by fitting all fluorescence intensity data to the standard equation for competitive inhibition in GraFit (Leatherbarrow, 2001) (Table 1). IC50 measurements with hHexA/B (Sigma A6152) activities against GlcNAcstatins F, G, and H were performed using the fluorogenic 4MU-NAG substrate and standard reaction mixtures as described previously (Dorfmueller et al., 2009) (Figure 1C).
For the O-GlcNAc western blots, HEK293 cells were cultured and treated with GlcNAcstatin derivatives as described previously (Dorfmueller et al., 2009). Cell lysates were separated by SDS PAGE, and O-GlcNAcylation was detected by western blotting with the anti-O-GlcNAc CTD110.6 antibody and quantified (Dorfmueller et al., 2009). To calculate the EC50 values of GlcNAcstatins C, F, and G, the data, background corrected with the untreated cells, were plotted against the inhibitor concentrations using the program GraphPad Prism (http://www.graphpad.com).
For the microscopy studies, HEK293 cells were seeded in clear flat-bottom 96 well assay plates (Corning 3340) and grown for 12 hr at 37°C under 5% CO2. Cells were treated with concentrations of GlcNAcstatins G and H (1% final DMSO concentration) for 6 hr. Cells were washed twice with 50 μl PBS and fixed in 3.7% formaldehyde for 10 min at 37°C. After washing with TBST (0.1% Triton X-100) and permeabilized for further 10 min, the cells were blocked with 2% BSA and 0.1% normal donkey serum in TBST for 1 hr at RT, followed by washing twice with TBST. Cells were immunostained with the RL2 O-GlcNAc antibody (1/500) for 12 hr at 4°C and fluorescent secondary (1/500) for 1 hr at RT. Cells were further washed twice with TBS before incubation with DAPI (5 μg/ml) and CellMask (1/25000). Cells were washed twice with TBS and stored in 50 μl PBS. Images were taken and analyzed with the IN Cell Analyzer 1000 system (GE Healthcare).
HEK293 cells were treated as described above with two concentrations (1 and 10 μM) of GlcNAcstatin G for 1.5 and 6 hr. Cells were washed twice in ice-cold PBS and harvested in lysis buffer. The soluble fraction was separated from cell debris by centrifugation and direct analysis performed by UPLC-MS/MS on a Quattro Premier XE mass spectrometer using positive electrospray ionization (Waters, UK) in multiple reaction-monitoring mode. A calibration curve was constructed in lysis buffer to cover approximately 3 orders of magnitude for GlcNAcstatin G (i.e., 0.25–500 nM). To calculate the minimal intracellular concentration of GlcNAcstatin G, the number and average size (diameter in μm) of cultured HEK293 cells that were used in this study were analyzed using a Cellometer Auto T4 from Nexcelom Bioscience. Subsequently, the maximal intracellular volume was approximated as V = (4/3) × π × r3, assuming a spherical cell and ignoring effects of intracellular compartments and proteins. To calculate the total maximal volume of the intracellular volume in a 2 ml culture, the total number of HEK293 cells was calculated and multiplied by the average single-cell volume. This volume was used to determine the minimal dilution factor of the intracellular volume upon cell lysis. To determine the cellular uptake and, thus, the minimal concentration of freely available GlcNAcstatin G, the concentration of the compound determined by UPLC-MS/MS was corrected using the dilution factor.
|Akimoto Y.,Hart G.W.,Wells L.,Vosseller K.,Yamamoto K.,Munetomo E.,Ohara-Imaizumi M.,Nishiwaki C.,Nagamatsu S.,Hirano H.,Kawakami H.. Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki ratsGlycobiology17Year: 200712714017095531|
|Bowe D.B.,Sadlonova A.,Toleman C.A.,Novak Z.,Hu Y.,Huang P.,Mukherjee S.,Whitsett T.,Frost A.R.,Paterson A.J.,Kudlow J.E.. O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptorsMol. Cell. Biol.26Year: 20068539855016966374|
|Chou C.F.,Smith A.J.,Omary M.B.. Characterization and dynamics of O-linked glycosylation of human cytokeratin 8 and 18J. Biol. Chem.267Year: 1992390139061371281|
|Dong D.L.,,Hart G.W.. Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosolJ. Biol. Chem.269Year: 199419321193308034696|
|Dorfmueller H.C.,Borodkin V.S.,Schimpl M.,Shepherd S.M.,Shpiro N.A.,van Aalten D.M.F.. GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-GlcNAcylation levelsJ. Am. Chem. Soc.128Year: 2006164841648517177381|
|Dorfmueller H.C.,Borodkin V.S.,Schimpl M.,van Aalten D.M.F.. GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylationBiochem. J.420Year: 200922122719275764|
|Emsley P.,Cowtan K.. Coot: model-building tools for molecular graphicsActa Crystallogr. D Biol. Crystallogr.60Year: 20042126213215572765|
|Gao Y.,Wells L.,Comer F.I.,Parker G.J.,Hart G.W.. Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brainJ. Biol. Chem.276Year: 20019838984511148210|
|Hanover J.A.,Krause M.W.,Love D.C.. The hexosamine signaling pathway: O-GlcNAc cycling in feast or famineBiochim. Biophys. Acta1800Year: 2010809519647043|
|Hart G.W.,Housley M.P.,Slawson C.. Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteinsNature446Year: 20071017102217460662|
|Kreppel L.K.,Blomberg M.A.,Hart G.W.. Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeatsJ. Biol. Chem.272Year: 1997930893159083067|
|Leatherbarrow, R.J. (2001). Version 5, Erithacus Software Ltd., Horley, UK.|
|Lemieux M.J.,Mark B.L.,Cherney M.M.,Withers S.G.,Mahuran D.J.,James M.N.G.. Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysisJ. Mol. Biol.359Year: 200691392916698036|
|Lubas W.A.,Frank D.W.,Krause M.,Hanover J.A.. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeatsJ. Biol. Chem.272Year: 1997931693249083068|
|Macauley M.S.,Whitworth G.E.,Debowski A.W.,Chin D.,Vocadlo D.J.. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitorsJ. Biol. Chem.280Year: 2005253132532215795231|
|Macauley M.S.,Bubb A.K.,Martinez-Fleites C.,Davies G.J.,Vocadlo D.J.. Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistanceJ. Biol. Chem.283Year: 2008346873469518842583|
|Mark B.L.,Mahuran D.J.,Cherney M.M.,Zhao D.,Knapp S.,James M.N.. Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs diseaseJ. Mol. Biol.327Year: 20031093110912662933|
|Murshudov G.N.,Vagin A.A.,Dodson E.J.. Refinement of macromolecular structures by the maximum-likelihood methodActa Crystallogr. D Biol. Crystallogr.53Year: 199724025515299926|
|Nagy T.,Balasa A.,Frank D.,Rab A.,Rideg O.,Kotek G.,Magyarlaki T.,Bogner P.,Kovács G.L.,Miseta A.. O-GlcNAc modification of proteins affects volume regulation in Jurkat cellsEur. Biophys. J.39Year: 20091207121720043149|
|Ngoh G.A.,Facundo H.T.,Hamid T.,Dillmann W.,Zachara N.E.,Jones S.P.. Unique hexosaminidase reduces metabolic survival signal and sensitizes cardiac myocytes to hypoxia/reoxygenation injuryCirc. Res.104Year: 2009414919023128|
|Roquemore E.P.,Chevrier M.R.,Cotter R.J.,Hart G.W.. Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallinBiochemistry35Year: 1996357835868639509|
|Stubbs K.A.,Zhang N.,Vocadlo D.J.. A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective inhibitors of O-GlcNAcaseOrg. Biomol. Chem.4Year: 200683984516493467|
|Torres C.R.,Hart G.W.. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAcJ. Biol. Chem.259Year: 1984330833176421821|
|Vosseller K.,Wells L.,Lane M.D.,Hart G.W.. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytesProc. Natl. Acad. Sci. USA99Year: 20025313531811959983|
|Yuzwa S.,Macauley M.,Heinonen J.,Shan X.,Dennis R.,He Y.,Whitworth G.,Stubbs K.,McEachern E.,Davies G.,Vocadlo D.. A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivoNat. Chem. Biol.4Year: 200848349018587388|
|Zachara N.E.,Hart G.W.. Cell signaling, the essential role of O-GlcNAc!Biochim. Biophys. Acta1761Year: 200659961716781888|
Coordinates and structure factors are deposited in the RCSB protein data bank (PDB entry 2XPK).
Document S1. Supplemental Experimental Procedures, Six Figures, and One Table
Click here for additional data file (mmc1.pdf)
We thank the ESRF, Grenoble, for beam time on BM-14. This work was supported by a Wellcome Trust Senior Fellowship and a Lister Institute for Preventive Medicine Research Prize to D.M.F.v. H.C.D. is supported by the College of Life Sciences Alumni Studentship.
Previous Document: Activation state-dependent binding of small molecule kinase inhibitors: structural insights from bio...
Next Document: Inhibitor of fatty acid amide hydrolase normalizes cardiovascular function in hypertension without a...