scholarly journals Achieving a Good Crystal System for Crystallographic X-ray Fragment Screening

2018 ◽  
Author(s):  
Patrick M. Collins ◽  
Alice Douangamath ◽  
Romain Talon ◽  
Alexandre Dias ◽  
José Brandão-Neto ◽  
...  
Keyword(s):  
User Program ◽  
X Ray ◽  
Crystal System ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Surface Sites ◽  
Screening Experiment ◽  
Active User ◽  
Density Maps

The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program.  The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites:  this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user programme that is generating large amounts of data:  in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals.  It has also generated a large number of learnings concerning the main remaining bottleneck, namely obtaining a suitable crystal system that will support a successful fragment screen.  Here we discuss the practicalities of generating screen-ready crystals that have useful electron density maps, and how to ensure they will be successfully reproduced and usable at a facility outside the home lab.

scholarly journals Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

2018 ◽  
Vol 19 (11) ◽  
pp. 3664 ◽  
Author(s):  
Ali Munawar ◽  
Steven Beelen ◽  
Ahmad Munawar ◽  
Eveline Lescrinier ◽  
Sergei Strelkov
Keyword(s):  
Zika Virus ◽  
Dissociation Constants ◽  
Global Public Health ◽  
Human Pathogens ◽  
Allosteric Site ◽  
Replication Complex ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Atomic Precision

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015–2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

Predicting the Success of Fragment Screening by X-Ray Crystallography

2011 ◽  
pp. 91-114 ◽  
Author(s):  
Douglas R. Davies ◽  
Darren W. Begley ◽  
Robert C. Hartley ◽  
Bart L. Staker ◽  
Lance J. Stewart
Keyword(s):  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening

Application of Fragment Screening by X-ray Crystallography to the Discovery of Aminopyridines as Inhibitors of β-Secretase§

2007 ◽  
Vol 50 (6) ◽  
pp. 1124-1132 ◽  
Author(s):  
Miles Congreve ◽  
David Aharony ◽  
Jeffrey Albert ◽  
Owen Callaghan ◽  
James Campbell ◽  
...  
Keyword(s):  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening

scholarly journals Parallel Screening of Low Molecular Weight Fragment Libraries

2012 ◽  
Vol 18 (2) ◽  
pp. 147-159 ◽  
Author(s):  
Jerome Wielens ◽  
Stephen J. Headey ◽  
David I. Rhodes ◽  
Roger J. Mulder ◽  
Olan Dolezal ◽  
...  
Keyword(s):  
Screening Methods ◽  
Resonance Spectroscopy ◽  
Lead Discovery ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Lead Compounds ◽  
Preliminary Identification ◽  
Std Nmr ◽  
Fragment Libraries

Fragment screening is becoming widely accepted as a technique to identify hit compounds for the development of novel lead compounds. In neighboring laboratories, we have recently, and independently, performed a fragment screening campaign on the HIV-1 integrase core domain (IN) using similar commercially purchased fragment libraries. The two campaigns used different screening methods for the preliminary identification of fragment hits; one used saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR), and the other used surface plasmon resonance (SPR) spectroscopy. Both initial screens were followed by X-ray crystallography. Using the STD-NMR/X-ray approach, 15 IN/fragment complexes were identified, whereas the SPR/X-ray approach found 6 complexes. In this article, we compare the approaches that were taken by each group and the results obtained, and we look at what factors could potentially influence the final results. We find that despite using different approaches with little overlap of initial hits, both approaches identified binding sites on IN that provided a basis for fragment-based lead discovery and further lead development. Comparison of hits identified in the two studies highlights a key role for both the conditions under which fragment binding is measured and the criteria selected to classify hits.

scholarly journals PANDDAs: multi-dataset methods for finding hits from fragment screening by X-ray crystallography

2015 ◽  
Vol 71 (a1) ◽  
pp. s258-s258
Author(s):  
Nicholas M. Pearce ◽  
Sebastian Kelm ◽  
Jiye Shi ◽  
Charlotte M. Deane ◽  
Frank von Delft
Keyword(s):  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening

scholarly journals Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2020 ◽  
Vol 76 (1) ◽  
pp. 40-46
Author(s):  
James H. Thorpe ◽  
Ian D. Wall ◽  
Robert H. Sinnamon ◽  
Amy N. Taylor ◽  
Robert A. Stavenger
Keyword(s):  
Drug Discovery ◽  
Acinetobacter Baumannii ◽  
Structural Data ◽  
X Ray ◽  
Traditional Medicines ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Binding Pockets ◽  
Antibacterial Target ◽  
Robustness Check

Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.

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.

Unambiguous determination of H-atom positions: comparing results from neutron and high-resolution X-ray crystallography

2010 ◽  
Vol 66 (5) ◽  
pp. 558-567 ◽  
Author(s):  
Anna S. Gardberg ◽  
Alexis Rae Del Castillo ◽  
Kevin L. Weiss ◽  
Flora Meilleur ◽  
Matthew P. Blakeley ◽  
...  
Keyword(s):  
Neutron Diffraction ◽  
Electron Density ◽  
Pyrococcus Furiosus ◽  
X Ray Diffraction ◽  
Neutron Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Density Maps ◽  
Improved Model

The locations of H atoms in biological structures can be difficult to determine using X-ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 Å resolution neutron diffraction studies of fully perdeuterated and selectively CH3-protonated perdeuterated crystals ofPyrococcus furiosusrubredoxin (D-rubredoxin and HD-rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 Å resolution X-ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the σ level at the D-atom coordinate in simulated-annealing composite D-OMIT maps. It is shown that 1.65 Å resolution RT neutron data for perdeuterated rubredoxin are ∼8 times more likely overall to provide high-confidence positions for D atoms than 1.1 Å resolution X-ray data at 100 K or RT. At or above the 1.0σ level, the joint X-ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D-atom positions for D-rubredoxin and HD-rubredoxin, respectively. The X-ray-only 1.1 Å resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D-atom positions above the 1.0σ level for D-rubredoxin and HD-rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron-density maps, permitting the location of more D atoms than electron-density maps from models refined against X-ray data only.

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