I04-1-Macromolecular Crystallography (fixed wavelength)
|
Open Access
Abstract: Fragment based methods are now widely used to identify starting points in drug discovery and generation of tools for chemical biology. A significant challenge is optimization of these weak binding fragments to hit and lead compounds. We have developed an approach where individual reaction mixtures of analogues of hits can be evaluated without purification of the product. Here, we describe experiments to optimise the processes and then assess such mixtures in the high throughput crystal structure determination facility, XChem. Diffraction data for crystals of the proteins Hsp90 and PDHK2 soaked individually with 83 crude reaction mixtures are analysed manually or with the automated XChem procedures. The results of structural analysis are compared with binding measurements from other biophysical techniques. This approach can transform early hit to lead optimisation and the lessons learnt from this study provide a protocol that can be used by the community.
|
Sep 2020
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
|
May 2020
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Storm
Hassell-hart
,
Andrew
Runcie
,
Tobias
Krojer
,
Jordan
Doyle
,
Ella
Lineham
,
Cory A.
Ocasio
,
Brenno A. D.
Neto
,
Oleg
Fedorov
,
Graham
Marsh
,
Hannah
Maple
,
Robert
Felix
,
Rebecca
Banks
,
Alessio
Ciulli
,
Sarah
Picaud
,
Panagis
Filippakopoulos
,
Frank
Von Delft
,
Paul
Brennan
,
Helen J. S.
Stewart
,
Timothy J.
Chevassut
,
Martin
Walker
,
Carol
Austin
,
Simon
Morley
,
John
Spencer
Diamond Proposal Number(s):
[19301]
Abstract: (+)-JD1, a rationally designed ferrocene analogue of the BET bromodomain (BRD) probe molecule (+)-JQ1, has been synthesized and evaluated in biophysical, cell-based assays as well as in pharmacokinetic studies. It displays nanomolar activity against BRD isoforms, and its cocrystal structure was determined in complex with the first bromodomain of BRD4 and compared with that of (+)-JQ1, a known BRD4 small-molecule probe. At 1 μM concentration, (+)-JD1 was able to inhibit c-Myc, a key driver in cancer and an indirect target of BRD4.
|
Dec 2019
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Inga
Pfeffer
,
Lennart
Brewitz
,
Tobias
Krojer
,
Sacha A.
Jensen
,
Grazyna T.
Kochan
,
Nadia J.
Kershaw
,
Kirsty S.
Hewitson
,
Luke A.
Mcneill
,
Holger
Kramer
,
Martin
Münzel
,
Richard J.
Hopkinson
,
Udo
Oppermann
,
Penny A.
Handford
,
Michael A.
Mcdonough
,
Christopher J.
Schofield
Open Access
Abstract: AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
|
Oct 2019
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Efrat
Resnick
,
Anthony
Bradley
,
Jinrui
Gan
,
Alice
Douangamath
,
Tobias
Krojer
,
Ritika
Sethi
,
Paul P.
Geurink
,
Anthony
Aimon
,
Gabriel
Amitai
,
Dom
Bellini
,
James
Bennett
,
Michael
Fairhead
,
Oleg
Fedorov
,
Ronen
Gabizon
,
Jin
Gan
,
Jingxu
Guo
,
Alexander
Plotnikov
,
Nava
Reznik
,
Gian Filippo
Ruda
,
Laura
Diaz-saez
,
Verena M.
Straub
,
Tamas
Szommer
,
Srikannathasan
Velupillai
,
Daniel
Zaidman
,
Yanling
Zhang
,
Alun R.
Coker
,
Christopher G.
Dowson
,
Haim
Barr
,
Chu
Wang
,
Kilian V. M.
Huber
,
Paul E.
Brennan
,
Huib
Ovaa
,
Frank
Von Delft
,
Nir
London
Abstract: Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
|
May 2019
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[1221, 7864, 9948, 15433]
Abstract: Biosynthesis of 6-deoxy sugars, including L-fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hex-ose nucleotide precursors as the first committed step. Here, we determined pre- and post-catalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5’’ and C6’’, coupled to NADP-mediated hydride transfer from C4’’ to C6’’. We show that concerted acid-base catalysis from only two active-site groups, Tyr179 and Glu157, promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves least position changes of enzyme and substrate groups; and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.
|
Mar 2019
|
|
|
|
Abstract: 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 program 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.
|
Oct 2018
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Crystallographic fragment screening uses low molecular weight compounds to probe the protein surface and although individual protein-fragment interactions are high quality, fragments commonly bind at low occupancy, historically making identification difficult. However, our new Pan-Dataset Density Analysis method readily identifies binders missed by conventional analysis: for fragment screening data of lysine-specific demethylase 4D (KDM4D), the hit rate increased from 0.9% to 10.6%. Previously unidentified fragments reveal multiple binding sites and demonstrate: the versatility of crystallographic fragment screening; that surprisingly large conformational changes are possible in crystals; and that low crystallographic occupancy does not by itself reflect a protein-ligand complex's significance.
|
May 2017
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Nicholas M.
Pearce
,
Tobias
Krojer
,
Anthony R.
Bradley
,
Patrick
Collins
,
Radoslaw P.
Nowak
,
Romain
Talon
,
Brian D.
Marsden
,
Sebastian
Kelm
,
Jiye
Shi
,
Charlotte M.
Deane
,
Frank
Von Delft
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: In macromolecular crystallography, the rigorous detection of changed states (for example, ligand binding) is difficult unless signal is strong. Ambiguous ('weak' or 'noisy') density is experimentally common, since molecular states are generally only fractionally present in the crystal. Existing methodologies focus on generating maximally accurate maps whereby minor states become discernible; in practice, such map interpretation is disappointingly subjective, time-consuming and methodologically unsound. Here we report the PanDDA method, which automatically reveals clear electron density for the changed state-even from inaccurate maps-by subtracting a proportion of the confounding 'ground state'; changed states are objectively identified from statistical analysis of density distributions. The method is completely general, implying new best practice for all changed-state studies, including the routine collection of multiple ground-state crystals. More generally, these results demonstrate: the incompleteness of atomic models; that single data sets contain insufficient information to model them fully; and that accuracy requires further map-deconvolution approaches.
|
Apr 2017
|
|
Data acquisition
|
Open Access
Abstract: The steady expansion in the capacity of modern beamlines for high-throughput data collection, enabled by increasing X-ray brightness, capacity of robotics and detector speeds, has pushed the bottleneck upstream towards sample preparation. Even in ligand-binding studies using crystal soaking, the experiment best able to exploit beamline capacity, a primary limitation is the need for gentle and nontrivial soaking regimens such as stepwise concentration increases, even for robust and well characterized crystals. Here, the use of acoustic droplet ejection for the soaking of protein crystals with small molecules is described, and it is shown that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 min. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables the targeted placement of the compound/solvent away from crystals and towards drop edges, allowing gradual diffusion of solvent across the drop. This ensures both an improvement in the reproducibility of X-ray diffraction and increased solvent tolerance of the crystals, thus enabling higher effective compound-soaking concentrations. The technique is detailed here with examples from the protein target JMJD2D, a histone lysine demethylase with roles in cancer and the focus of active structure-based drug-design efforts.
|
Mar 2017
|
|
