The differences in binding 12-carbon aliphatic ligands by bovine β-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography

2013 ◽  
Vol 26 (8) ◽  
pp. 357-367 ◽  
Author(s):  
Joanna I. Loch ◽  
Piotr Bonarek ◽  
Agnieszka Polit ◽  
Sylwia Świątek ◽  
Marta Dziedzicka-Wasylewska ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
Β Lactoglobulin

scholarly journals X-Ray Crystallography and Isothermal Titration Calorimetry Studies of the Salmonella Zinc Transporter ZntB

Structure ◽  
2011 ◽  
Vol 19 (5) ◽  
pp. 700-710 ◽  
Author(s):  
Qun Wan ◽  
Md Faiz Ahmad ◽  
James Fairman ◽  
Bonnie Gorzelle ◽  
María de la Fuente ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Zinc Transporter ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography

scholarly journals Crystal Structure of a Virus-Encoded Putative Glycosyltransferase

2010 ◽  
Vol 84 (23) ◽  
pp. 12265-12273 ◽  
Author(s):  
Ye Xiang ◽  
Ulrich Baxa ◽  
Ying Zhang ◽  
Alasdair C. Steven ◽  
Gentry L. Lewis ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Gene Product ◽  
Isothermal Titration Calorimetry ◽  
Active Form ◽  
Structural Proteins ◽  
Titration Calorimetry ◽  
Type B ◽  
Active Centers ◽  
X Ray ◽  
X Ray Crystallography

ABSTRACT The chloroviruses (family Phycodnaviridae), unlike most viruses, encode some, if not most, of the enzymes involved in the glycosylation of their structural proteins. Annotation of the gene product B736L from chlorovirus NY-2A suggests that it is a glycosyltransferase. The structure of the recombinantly expressed B736L protein was determined by X-ray crystallography to 2.3-Å resolution, and the protein was shown to have two nucleotide-binding folds like other glycosyltransferase type B enzymes. This is the second structure of a chlorovirus-encoded glycosyltransferase and the first structure of a chlorovirus type B enzyme to be determined. B736L is a retaining enzyme and belongs to glycosyltransferase family 4. The donor substrate was identified as GDP-mannose by isothermal titration calorimetry and was shown to bind into the cleft between the two domains in the protein. The active form of the enzyme is probably a dimer in which the active centers are separated by about 40 Å.

Investigation of the inclusion of the herbicide metobromuron in native cyclodextrins by powder X-ray diffraction and isothermal titration calorimetry

2009 ◽  
Vol 344 (17) ◽  
pp. 2388-2393 ◽  
Author(s):  
Vincent J. Smith ◽  
Natalia M. Rougier ◽  
Rita H. de Rossi ◽  
Mino R. Caira ◽  
Elba I. Buján ◽  
...  
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
X Ray Diffraction ◽  
X Ray

scholarly journals Fragment-Based Screening for Inhibitors of PDE4A Using Enthalpy Arrays and X-ray Crystallography

2012 ◽  
Vol 17 (4) ◽  
pp. 469-480 ◽  
Author(s):  
Michael I. Recht ◽  
Vandana Sridhar ◽  
John Badger ◽  
Leslie Hernandez ◽  
Barbara Chie-Leon ◽  
...  
Keyword(s):  
Spectroscopic Properties ◽  
Titration Calorimetry ◽  
Label Free ◽  
Lead Discovery ◽  
High Resolution Data ◽  
Affinity Ligands ◽  
X Ray ◽  
X Ray Crystallography ◽  
Chemical Structures ◽  
Fragment Library

Fragment-based screening has typically relied on X-ray or nuclear magnetic resonance methods to identify low-affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher throughput method to reliably prescreen a fragment library to identify a subset of compounds for structural analysis. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with K I <2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed, and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and provides a list of candidate fragments for inhibition of PDE4A.

scholarly journals A High-Throughput Screening Triage Workflow to Authenticate a Novel Series of PFKFB3 Inhibitors

2017 ◽  
Vol 23 (1) ◽  
pp. 11-22
Author(s):  
Stephen A. St-Gallay ◽  
Neil Bennett ◽  
Susan E. Critchlow ◽  
Nicola Curtis ◽  
Gareth Davies ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Isothermal Calorimetry ◽  
Binding Mode ◽  
Carbonyl Oxygen ◽  
Enzyme Concentration ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Hill

A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.

scholarly journals Mass spectrometry for fragment screening

2017 ◽  
Vol 61 (5) ◽  
pp. 465-473 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Andrew J. Whitehouse ◽  
Anthony G. Coyne ◽  
Chris Abell
Keyword(s):  
Drug Discovery ◽  
High Sensitivity ◽  
Titration Calorimetry ◽  
Label Free ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Differential Scanning Fluorimetry ◽  
Biophysical Techniques

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

scholarly journals Structure of a highly acidic β-lactamase from the moderate halophileChromohalobactersp. 560 and the discovery of a Cs+-selective binding site

2015 ◽  
Vol 71 (3) ◽  
pp. 541-554 ◽  
Author(s):  
Shigeki Arai ◽  
Yasushi Yonezawa ◽  
Nobuo Okazaki ◽  
Fumiko Matsumoto ◽  
Chie Shibazaki ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Nuclear Power ◽  
Specific Binding ◽  
Radioactive Isotopes ◽  
Titration Calorimetry ◽  
X Ray ◽  
X Ray Crystallography ◽  
Specific Binding Sites ◽  
Enzymatic Function

Environmentally friendly absorbents are needed for Sr2+and Cs+, as the removal of the radioactive Sr2+and Cs+that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+or Sr2+. The crystal structure of a halophilic β-lactamase fromChromohalobactersp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space groupP31using X-ray crystallography. Moreover, the locations of bound Sr2+and Cs+ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+(90 mMNa+/10 mMCs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+and Cs+ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes.

scholarly journals Identification and Optimization of PDE10A Inhibitors Using Fragment-Based Screening by Nanocalorimetry and X-ray Crystallography

2013 ◽  
Vol 19 (4) ◽  
pp. 497-507 ◽  
Author(s):  
Michael I. Recht ◽  
Vandana Sridhar ◽  
John Badger ◽  
Pierre-Yves Bounaud ◽  
Cheyenne Logan ◽  
...  
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Spectroscopic Properties ◽  
Low Complexity ◽  
Titration Calorimetry ◽  
Label Free ◽  
Lead Discovery ◽  
X Ray ◽  
X Ray Crystallography ◽  
Heavy Atoms

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

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