CHiMP - Deep Learning Tools Trained on Protein Crystallisation Micrographs to Enable Automation of Experiments
2024
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King, O.N.F., Levik, K.E., Sandy, J., and Basham, M.
preprint
BioRxiv
PDF
Abstract
A group of three deep learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate) were created for analysis of micrographs of protein crystallisation experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case, and masks of crystallisation droplets in addition to crystals in the second case, allowing positions and sizes of these entities to be recorded. Creation of these tools used transfer learning, where weights from a pre-trained deep learning network were used as a starting point and re-purposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS where they absolve the need for any user input both for monitoring crystallisation experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug discovery screening platform, also at DLS, to allow automatic targeting of acoustic compound dispensing into crystallisation droplets.
Cite as
King, O.N.F., Levik, K.E., Sandy, J., Basham, M. CHiMP - Deep Learning Tools Trained on Protein Crystallisation Micrographs to Enable Automation of Experiments. BioRxiv. 2024. doi: 10.1101/2024.05.22.595345.
Citations
Online citizen science with the Zooniverse for analysis of biological volumetric data
2023
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Smith, P., King, O.N.F., Pennington, A., Tun, W.M., Basham, M., Jones, M.L., Collinson, L.M., Darrow, M. C., Spiers, H.
open-access
Histochemistry and Cell Biology
doi:10.1007/s00418-023-02204-6
PDF
Abstract
Public participation in research, also known as citizen science, is being increasingly adopted for the analysis of biological volumetric data. Researchers working in this domain are applying online citizen science as a scalable distributed data analysis approach, with recent research demonstrating that non-experts can productively contribute to tasks such as the segmentation of organelles in volume electron microscopy data. This, alongside the growing challenge to rapidly process the large amounts of biological volumetric data now routinely produced, means there is increasing interest within the research community to apply online citizen science for the analysis of data in this context. Here, we synthesise core methodological principles and practices for applying citizen science for analysis of biological volumetric data. We collate and share the knowledge and experience of multiple research teams who have applied online citizen science for the analysis of volumetric biological data using the Zooniverse platform ( www.zooniverse.org ). We hope this provides inspiration and practical guidance regarding how contributor effort via online citizen science may be usefully applied in this domain.
Cite as
Smith, P., King, O.N.F., Pennington, A., Tun, W.M., Basham, M., Jones, M.L., Collinson, L.M., Darrow, M. C., Spiers, H. Online citizen science with the Zooniverse for analysis of biological volumetric data. Histochem Cell Biol. 2023 Jun 7:1-24. doi: 10.1007/s00418-023-02204-6.
Citations
Comparison of Methods to Segment Variable-Contrast XCT Images of Methane-Bearing Sand Using U-Nets Trained on Single Dataset Sub-Volumes
2022
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Alvarez-Borges, F.J.; King, O.N.F.; Madhusudhan, B.N.; Connolley, T.; Basham, M.; Ahmed, S.I.
open-access
Methane
doi:10.3390/methane2010001
PDF
Abstract
Methane (CH4) hydrate dissociation and CH4 release are potential geohazards currently investigated using X-ray computed tomography (XCT). Image segmentation is an important data processing step for this type of research. However, it is often time consuming, computing resource-intensive, operator-dependent, and tailored for each XCT dataset due to differences in greyscale contrast. In this paper, an investigation is carried out using U-Nets, a class of Convolutional Neural Network, to segment synchrotron XCT images of CH4-bearing sand during hydrate formation, and extract porosity and CH4 gas saturation. Three U-Net deployments previously untried for this task are assessed: (1) a bespoke 3D hierarchical method, (2) a 2D multi-label, multi-axis method and (3) RootPainter, a 2D U-Net application with interactive corrections. U-Nets are trained using small, targeted hand-annotated datasets to reduce operator time. It was found that the segmentation accuracy of all three methods surpass mainstream watershed and thresholding techniques. Accuracy slightly reduces in low-contrast data, which affects volume fraction measurements, but errors are small compared with gravimetric methods. Moreover, U-Net models trained on low-contrast images can be used to segment higher-contrast datasets, without further training. This demonstrates model portability, which can expedite the segmentation of large datasets over short timespans.
Cite as
Alvarez-Borges, F.J.; King, O.N.F.; Madhusudhan, B.N.; Connolley, T.; Basham, M.; Ahmed, S.I. Comparison of Methods to Segment Variable-Contrast XCT Images of Methane-Bearing Sand Using U-Nets Trained on Single Dataset Sub-Volumes. Methane 2023, 2, 1-23. https://doi.org/10.3390/methane2010001
Citations
Volume Segmantics: A Python Package for Semantic Segmentation of Volumetric Data Using Pre-trained PyTorch Deep Learning Models
2022
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King, O.N.F, Bellos, D. and Basham, M.
open-access
Journal of Open Source Software
doi:10.21105/joss.04691
PDF
Abstract
Segmentation of 3-dimensional (3D, volumetric) images is a widely used technique that allows
interpretation and quantification of experimental data collected using a number of techniques
(for example, Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Electron
Tomography (ET)). Although the idea of semantic segmentation is a relatively simple one,
giving each pixel a label that defines what it represents (e.g cranial bone versus brain tissue);
due to the subjective and laborious nature of the manual labelling task coupled with the huge
size of the data (multi-GB files containing billions of pixels) this process is often a bottleneck
in imaging workflows. In recent years, deep learning has brought models capable of fast and
accurate interpretation of image data into the toolbox available to scientists. These models
are often trained on large image datasets that have been annotated at great expense. In many
cases however, scientists working on novel samples and using new imaging techniques do not
yet have access to large stores of annotated data. To overcome this issue, simple software
tools that allow the scientific community to create segmentation models using relatively small
amounts of training data are required. Volume Segmantics is a Python package that provides
a command line interface (CLI) as well as an Application Programming Interface (API) for
training 2-dimensional (2D) PyTorch (Paszke et al., 2019) deep learning models on small
amounts of annotated 3D image data. The package also enables applying these models to
new (often much larger) 3D datasets to speed up the process of semantic segmentation.
Cite as
King, O.N.F., Bellos, D. and Basham, M (2022). Volume Segmantics: A Python Package for Semantic Segmentation of Volumetric Data Using Pre-trained PyTorch Deep Learning Models. Journal of Open Source Software, 7(78), 4691, https://doi.org/10.21105/joss.04691
Citations
SuRVoS 2: Accelerating Annotation and Segmentation for Large Volumetric Bioimage Workflows Across Modalities and Scales
2022
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Pennington, A., King, O.N.F., Tun, W.M., Ho, E.M.L., Luengo, I., Darrow, M.C., and Basham, M.
open-access
Frontiers in Cell and Developmental Biology
doi:10.3389/fcell.2022.842342
PDF
Abstract
As sample preparation and imaging techniques have expanded and improved to include a variety of options for larger sized and numbers of samples, the bottleneck in volumetric imaging is now data analysis. Annotation and segmentation are both common, yet difficult, data analysis tasks which are required to bring meaning to the volumetric data. The SuRVoS application has been updated and redesigned to provide access to both manual and machine learning-based segmentation and annotation techniques, including support for crowd sourced data. Combining adjacent, similar voxels (supervoxels) provides a mechanism for speeding up segmentation both in the painting of annotation and by training a segmentation model on a small amount of annotation. The support for layers allows multiple datasets to be viewed and annotated together which, for example, enables the use of correlative data (e.g. crowd-sourced annotations or secondary imaging techniques) to guide segmentation. The ability to work with larger data on high-performance servers with GPUs has been added through a client-server architecture and the Pytorch-based image processing and segmentation server is flexible and extensible, and allows the implementation of deep learning-based segmentation modules. The client side has been built around Napari allowing integration of SuRVoS into an ecosystem for open-source image analysis while the server side has been built with cloud computing and extensibility through plugins in mind. Together these improvements to SuRVoS provide a platform for accelerating the annotation and segmentation of volumetric and correlative imaging data across modalities and scales.
Cite as
Pennington, A., King, O.N.F., Tun, W.M., Ho, E.M.L., Luengo, I., Darrow, M.C., and Basham, M. SuRVoS 2: Accelerating Annotation and Segmentation for Large Volumetric Bioimage Workflows Across Modalities and Scales. Frontiers in Cell and Developmental Biology. 2022 ;10:842342. DOI: https://doi.org/10.3389/fcell.2022.842342.
Citations
Beamline K11 DIAD: A new instrument for dual imaging and diffraction at Diamond Light Source
2021
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Reinhard C, Drakopoulos M, Ahmed SI, Deyhle H, James A, Charlesworth CM, Burt M, Sutter J, Alexander S, Garland P, Yates T, Marshall R, Kemp B, Warrick E, Pueyos A, Bradnick B, Nagni M, Winter AD, Filik J, Basham M, Wadeson N, King ONF, Aslani N and Dent AJ.
open-access
Journal of Synchrotron Radiation
doi:10.1107/S1600577521009875
PDF
Abstract
The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source is a new dual-beam instrument for full-field imaging/tomography and powder diffraction. This instrument provides the user community with the capability to dynamically image 2D and 3D complex structures and perform phase identification and/or strain mapping using micro-diffraction. The aim is to enable in situ and in operando experiments that require spatially correlated results from both techniques, by providing measurements from the same specimen location quasi-simultaneously. Using an unusual optical layout, DIAD has two independent beams originating from one source that operate in the medium energy range (7-38 keV) and are combined at one sample position. Here, either radiography or tomography can be performed using monochromatic or pink beam, with a 1.4 mm × 1.2 mm field of view and a feature resolution of 1.2 µm. Micro-diffraction is possible with a variable beam size between 13 µm × 4 µm and 50 µm × 50 µm. One key functionality of the beamline is image-guided diffraction, a setup in which the micro-diffraction beam can be scanned over the complete area of the imaging field-of-view. This moving beam setup enables the collection of location-specific information about the phase composition and/or strains at any given position within the image/tomography field of view. The dual beam design allows fast switching between imaging and diffraction mode without the need of complicated and time-consuming mode switches. Real-time selection of areas of interest for diffraction measurements as well as the simultaneous collection of both imaging and diffraction data of (irreversible) in situ and in operando experiments are possible.
Cite as
Reinhard C, Drakopoulos M, Ahmed SI, Deyhle H, James A, Charlesworth CM, Burt M, Sutter J, Alexander S, Garland P, Yates T, Marshall R, Kemp B, Warrick E, Pueyos A, Bradnick B, Nagni M, Winter AD, Filik J, Basham M, Wadeson N, King ONF, Aslani N, Dent AJ. Beamline K11 DIAD: a new instrument for dual imaging and diffraction at Diamond Light Source. J Synchrotron Radiat. 2021 Nov 1;28(Pt 6):1985-1995. doi: https://doi.org/10.1107/S1600577521009875. Epub 2021 Oct 22.
Citations
A massively multi-scale approach to characterising tissue architecture by synchrotron micro-CT applied to the human placenta
2021
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Tun WM, Poologasundarampillai G, Bischof H, Nye G, King ONF, Basham M, Tokudome Y, Lewis RM, Johnstone ED, Brownbill P, Darrow M and Chernyavsky IL.
open-access
Journal of the Royal Society Interface
doi:10.1098/rsif.2021.0140
PDF
Abstract
Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure–function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure–function relationships in both normal and diseased placentas, allowing us to optimize therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in the characterization of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.
Cite as
Tun WM, Poologasundarampillai G, Bischof H, Nye G, King ONF, Basham M, Tokudome Y, Lewis RM, Johnstone ED, Brownbill P, Darrow M, Chernyavsky IL. A massively multi-scale approach to characterizing tissue architecture by synchrotron micro-CT applied to the human placenta. J R Soc Interface. 2021 Jun;18(179):20210140. doi: https://doi.org/10.1098/rsif.2021.0140. Epub 2021 Jun 2.
Citations
Non-competitive cyclic peptides for targeting enzyme-substrate complexes
2018
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McAllister TE, Yeh TL, Abboud MI, Leung IKH, Hookway ES, King ONF, Bhushan B, Williams ST, Hopkinson RJ, Münzel M, Loik ND, Chowdhury R, Oppermann U, Claridge TDW, Goto Y, Suga H, Schofield CJ and Kawamura A.
open-access
Chemical Science
doi:10.1039/c8sc00286j
PDF
Abstract
Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) – enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein–protein interactions.
Cite as
McAllister TE, Yeh TL, Abboud MI, Leung IKH, Hookway ES, King ONF, Bhushan B, Williams ST, Hopkinson RJ, Münzel M, Loik ND, Chowdhury R, Oppermann U, Claridge TDW, Goto Y, Suga H, Schofield CJ, Kawamura A. Non-competitive cyclic peptides for targeting enzyme-substrate complexes. Chem Sci. 2018 Apr 23;9(20):4569-4578. doi: https://doi.org/10.1039/c8sc00286j.
Citations
Highly selective inhibition of histone demethylases by de novo macrocyclic peptides
2017
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Kawamura A, Münzel M, Kojima T, Yapp C, Bhushan B, Goto Y, Tumber A, Katoh T, King ON, Passioura T, Walport LJ, Hatch SB, Madden S, Müller S, Brennan PE, Chowdhury R, Hopkinson RJ, Suga H and Schofield CJ.
open-access
Nature Communications
doi:10.1038/ncomms14773
PDF
Abstract
The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Cite as
Kawamura A, Münzel M, Kojima T, Yapp C, Bhushan B, Goto Y, Tumber A, Katoh T, King ON, Passioura T, Walport LJ, Hatch SB, Madden S, Müller S, Brennan PE, Chowdhury R, Hopkinson RJ, Suga H, Schofield CJ. Highly selective inhibition of histone demethylases by de novo macrocyclic peptides. Nat Commun 8, 14773 (2017). https://doi.org/10.1038/ncomms14773
Citations
Pan-histone demethylase inhibitors simultaneously targeting Jumonji C and lysine-specific demethylases display high anticancer activities
2014
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Rotili D, Tomassi S, Conte M, Benedetti R, Tortorici M, Ciossani G, Valente S, Marrocco B, Labella D, Novellino E, Mattevi A, Altucci L, Tumber A, Yapp C, King ON, Hopkinson RJ, Kawamura A, Schofield CJ and Mai A.
open-access
Journal of Medicinal Chemistry
doi:10.1021/jm4012802
PDF
Abstract
In prostate cancer, two different types of histone lysine demethylases (KDM), LSD1/KDM1 and JMJD2/KDM4, are coexpressed and colocalize with the androgen receptor. We designed and synthesized hybrid LSD1/JmjC or "pan-KDM" inhibitors 1-6 by coupling the skeleton of tranylcypromine 7, a known LSD1 inhibitor, with 4-carboxy-4'-carbomethoxy-2,2'-bipyridine 8 or 5-carboxy-8-hydroxyquinoline 9, two 2-oxoglutarate competitive templates developed for JmjC inhibition. Hybrid compounds 1-6 are able to simultaneously target both KDM families and have been validated as potential antitumor agents in cells. Among them, 2 and 3 increase H3K4 and H3K9 methylation levels in cells and cause growth arrest and substantial apoptosis in LNCaP prostate and HCT116 colon cancer cells. When tested in noncancer mesenchymal progenitor (MePR) cells, 2 and 3 induced little and no apoptosis, respectively, thus showing cancer-selective inhibiting action.
Cite as
Rotili D, Tomassi S, Conte M, Benedetti R, Tortorici M, Ciossani G, Valente S, Marrocco B, Labella D, Novellino E, Mattevi A, Altucci L, Tumber A, Yapp C, King ON, Hopkinson RJ, Kawamura A, Schofield CJ, Mai A. Pan-histone demethylase inhibitors simultaneously targeting Jumonji C and lysine-specific demethylases display high anticancer activities. J Med Chem. 2014 Jan 9;57(1):42-55. doi: https://doi.org/10.1021/jm4012802.
Citations
5-Carboxy-8-hydroxyquinoline is a broad spectrum 2-oxoglutarate oxygenase inhibitor which causes iron translocation
2013
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Hopkinson RJ, Tumber A, Yapp C, Chowdhury R, Aik WS, Che KH, Li XS, Kristensen JBL, King ONF, Chan MC, Yeoh KK, Choi H, Walport LJ, Thinnes CC, Bush JT, Lejeune C, Rydzik AM, Rose NR, Bagg EA, McDonough MA, Krojer, TJ, Yue WW, Ng SS, Olsen L, Brennan PE, Oppermann U, Muller S, Klose RJ, Ratcliffe PJ, Schofield CJ and Kawamura A.
open-access
Chemical Science
doi:10.1039/C3SC51122G
PDF
Abstract
2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.
Cite as
Hopkinson RJ, Tumber A, Yapp C, Chowdhury R, Aik W, Che KH, Li XS, Kristensen JBL, King ONF, Chan MC, Yeoh KK, Choi H, Walport LJ, Thinnes CC, Bush JT, Lejeune C, Rydzik AM, Rose NR, Bagg EA, McDonough MA, Krojer T, Yue WW, Ng SS, Olsen L, Brennan PE, Oppermann U, Muller-Knapp S, Klose RJ, Ratcliffe PJ, Schofield CJ, Kawamura A. 5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation. Chem Sci. 2013 Aug 1;4(8):3110-3117. doi: https://doi.org/10.1039/C3SC51122G.
Citations
Discovery of ML324, a JMJD2 demethylase inhibitor with demonstrated antiviral activity
2012
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Rai G, Kawamura A, Tumber A, Liang Y, Vogel JL, Arbuckle JH, Rose NR, Dexheimer TS, Foley TL, King ON, Quinn A, Mott BT, Schofield CJ, Oppermann U, Jadhav A, Simeonov A, Kristie TM and Maloney DJ.
open-access
Probe Reports from the NIH Molecular Libraries Program [Internet].
PDF
Abstract
A critical and dynamic epigenetic post-translational modification involves N(ε)-methylation of histone lysine residues by histone methyltransferases. This process was originally thought to be an irreversible epigenetic mark, yet two representative classes of histone lysine demethylases which reverse this process are LSD1/2 and the Jumonji domain containing proteins (JMJD) have emerged. Despite an increased interest in these enzymes as a result of their suspected role in a variety of diseases (e.g. cancer and virus infection), a dearth of potent and cell-permeable inhibitors of the JMJD2 enzymes remain. As such, we sought to discover novel small molecule inhibitors of the JMJD2 family of histone demethylases via a quantitative high throughput screen and subsequent medicinal chemistry optimization campaign. Herein, we describe the discovery and optimization of N-(3-(dimethyamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide, ML324, a probe molecule that displays submicromolar inhibitory activity toward JMJD2E (in vitro) and possesses excellent in vitro ADME properties. In contrast to previously reported inhibitors of the JMJD proteins, ML324 displays excellent cell permeability providing an opportunity for more extensive cell-based studies of JMJD2 enzymes to be undertaken. In addition, ML324 demonstrates potent anti-viral activity against both herpes simplex virus (HSV) and human cytomegalovirus (hCMV) infection via inhibition viral IE gene expression. ML324 suppresses the formation of HSV plaques, even at high MOI, and blocks HSV-1 reactivation in a mouse ganglia explant model of latently infected mice. The studies described herein provide the basis for the use of JMJD2 inhibitors in proof-of-concept animal models for treatment of herpes virus infections and recurrence.
Cite as
Rai G, Kawamura A, Tumber A, Liang Y, Vogel JL, Arbuckle JH, Rose NR, Dexheimer TS, Foley TL, King ON, Quinn A, Mott BT, Schofield CJ, Oppermann U, Jadhav A, Simeonov A, Kristie TM, Maloney DJ. Discovery of ML324, a JMJD2 demethylase inhibitor with demonstrated antiviral activity. 2012 Dec 17 [updated 2013 Sep 16]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010–.
Citations
Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases
2012
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Rose NR, Woon EC, Tumber A, Walport LJ, Chowdhury R, Li XS, King ON, Lejeune C, Ng SS, Krojer T, Chan MC, Rydzik AM, Hopkinson RJ, Che KH, Daniel M, Strain-Damerell C, Gileadi C, Kochan G, Leung IK, Dunford J, Yeoh KK, Ratcliffe PJ, Burgess-Brown N, von Delft F, Muller S, Marsden B, Brennan PE, McDonough MA, Oppermann U, Klose RJ, Schofield CJ and Kawamura A.
open-access
Journal of Medicinal Chemistry
doi:10.1021/jm300677j
PDF
Abstract
The JmjC oxygenases catalyse the N-demethylation of Nε-methyl lysine residues in histones and are current therapeutic targets. A set of 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.
Cite as
Rose NR, Woon EC, Tumber A, Walport LJ, Chowdhury R, Li XS, King ON, Lejeune C, Ng SS, Krojer T, Chan MC, Rydzik AM, Hopkinson RJ, Che KH, Daniel M, Strain-Damerell C, Gileadi C, Kochan G, Leung IK, Dunford J, Yeoh KK, Ratcliffe PJ, Burgess-Brown N, von Delft F, Muller S, Marsden B, Brennan PE, McDonough MA, Oppermann U, Klose RJ, Schofield CJ, Kawamura A. Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases. J Med Chem. 2012 Jul 26;55(14):6639-43. doi: https://doi.org/10.1021/jm300677j. Epub 2012 Jul 11.
Citations
The Cryptosporidium parvum kinome
2011
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Artz JD, Wernimont AK, Allali-Hassani A, Zhao Y, Amani M, Lin YH, Senisterra G, Wasney GA, Fedorov O, King O, Roos A, Lunin VV, Qiu W, Finerty P Jr, Hutchinson A, Chau I, von Delft F, MacKenzie F, Lew J, Kozieradzki I, Vedadi M, Schapira M, Zhang C, Shokat K, Heightman T and Hui R.
open-access
BMC Genomics
doi:10.1186/1471-2164-12-478
PDF
Abstract
Background:
Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.
Results:
The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35% could only be classified as OPK (other protein kinases). In addition, about 25% of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening Cp CDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of < 10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of Cp CDPK1. In addition, structural analysis of Cp CDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.
Conclusions:
Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.
Cite as
Artz JD, Wernimont AK, Allali-Hassani A, Zhao Y, Amani M, Lin YH, Senisterra G, Wasney GA, Fedorov O, King O, Roos A, Lunin VV, Qiu W, Finerty P Jr, Hutchinson A, Chau I, von Delft F, MacKenzie F, Lew J, Kozieradzki I, Vedadi M, Schapira M, Zhang C, Shokat K, Heightman T, Hui R. The Cryptosporidium parvum kinome. BMC Genomics. 2011 Sep 30;12:478. doi: https://doi.org/10.1186/1471-2164-12-478.
Citations
Inhibition of 2-oxoglutarate dependent oxygenases
2011
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Rose NR, McDonough MA, King ON, Kawamura A and Schofield CJ.
Chemical Society Reviews
doi:10.1039/c0cs00203h
PDF
Abstract
2-Oxoglutarate (2OG) dependent oxygenases are ubiquitous iron enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. In humans their roles include collagen biosynthesis, fatty acid metabolism, DNA repair, RNA and chromatin modifications, and hypoxic sensing. Commercial applications of 2OG oxygenase inhibitors began with plant growth retardants, and now extend to a clinically used pharmaceutical compound for cardioprotection. Several 2OG oxygenases are now being targeted for therapeutic intervention for diseases including anaemia, inflammation and cancer. In this critical review, we describe studies on the inhibition of 2OG oxygenases, focusing on small molecules, and discuss the potential of 2OG oxygenases as therapeutic targets (295 references).
Cite as
Rose NR, McDonough MA, King ON, Kawamura A, Schofield CJ. Inhibition of 2-oxoglutarate dependent oxygenases. Chem Soc Rev. 2011 Aug;40(8):4364-97. doi: https://doi.org/10.1039/c0cs00203h. Epub 2011 Mar 10.
Citations
The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases
2011
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Chowdhury R, Yeoh KK, Tian YM, Hillringhaus L, Bagg EA, Rose NR, Leung IK, Li XS, Woon EC, Yang M, McDonough MA, King ON, Clifton IJ, Klose RJ, Claridge TD, Ratcliffe PJ, Schofield CJ and Kawamura A.
open-access
EMBO Reports
doi:10.1038/embor.2011.43
PDF
Abstract
Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(−)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC50) values for the R-form of 2HG varied from approximately 25 μM for the histone Nε-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.
Cite as
Chowdhury R, Yeoh KK, Tian YM, Hillringhaus L, Bagg EA, Rose NR, Leung IK, Li XS, Woon EC, Yang M, McDonough MA, King ON, Clifton IJ, Klose RJ, Claridge TD, Ratcliffe PJ, Schofield CJ, Kawamura A. The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases. EMBO Rep. 2011 May;12(5):463-9. doi: https://doi.org/10.1038/embor.2011.43. Epub 2011 Apr 1.
Citations
Inhibition of histone demethylases by 4-carboxy-2,2'-bipyridyl compounds
2011
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Chang KH*, King ON*, Tumber A*, Woon EC, Heightman TD, McDonough MA, Schofield CJ and Rose NR.
ChemMedChem
doi:10.1002/cmdc.201100026
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Abstract
Exploiting epigenetics: 2-Oxoglutarate (2OG)-dependent histone lysine demethylases, such as JMJD2E, are potential therapeutic targets in a range of diseases. Through structure–activity relationship studies and analyses, we identified a potent 4-carboxy-2,2′-bipyridyl compound, which inhibits JMJD2E with an IC50 value of 110 nM, representing a 66-fold improvement over the lead compound. These bipyridyl derivatives bind in the 2-oxoglutarate binding site.
Cite as
Chang KH, King ONF, Tumber A, Woon ECY, Heightman TD, McDonough MA, Schofield CJ, Rose NR. Inhibition of histone demethylases by 4-carboxy-2,2'-bipyridyl compounds. ChemMedChem. 2011 May 2;6(5):759-764. doi: https://doi.org/10.1002/cmdc.201100026. Epub 2011 Mar 15.
Citations
A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation
2011
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Lucic B, Zhang Y, King O, Mendoza-Maldonado R, Berti M, Niesen FH, Burgess-Brown NA, Pike AC, Cooper CD, Gileadi O and Vindigni A.
open-access
Nucleic Acids Research
doi:10.1093/nar/gkq1031
PDF
Abstract
RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1 49–616 ) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ1 49–616 and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1 49–616 dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein–protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.
Cite as
Lucic B, Zhang Y, King O, Mendoza-Maldonado R, Berti M, Niesen FH, Burgess-Brown NA, Pike AC, Cooper CD, Gileadi O, Vindigni A. A prominent β-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation. Nucleic Acids Res. 2011 Mar;39(5):1703-17. doi: https://doi.org/10.1093/nar/gkq1031.
Citations
Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing
2011
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Fedorov O, Huber K, Eisenreich A, Filippakopoulos P, King O, Bullock AN, Szklarczyk D, Jensen LJ, Fabbro D, Trappe J, Rauch U, Bracher F and Knapp S.
open-access
Chemistry & Biology
doi:10.1016/j.chembiol.2010.11.009
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Abstract
There is a growing recognition of the importance of protein kinases in the control of alternative splicing. To define the underlying regulatory mechanisms, highly selective inhibitors are needed. Here, we report the discovery and characterization of the dichloroindolyl enaminonitrile KH-CB19, a potent and highly specific inhibitor of the CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4). Co-crystal structures of KH-CB19 with CLK1 and CLK3 revealed a non-ATP mimetic binding mode, conformational changes in helix αC and the phosphate binding loop and halogen bonding to the kinase hinge region. KH-CB19 effectively suppressed phosphorylation of SR (serine/arginine) proteins in cells, consistent with its expected mechanism of action. Chemical inhibition of CLK1/CLK4 generated a unique pattern of splicing factor dephosphorylation and had at low nM concentration a profound effect on splicing of the two tissue factor isoforms flTF (full-length TF) and asHTF (alternatively spliced human TF).
Cite as
Fedorov O, Huber K, Eisenreich A, Filippakopoulos P, King O, Bullock AN, Szklarczyk D, Jensen LJ, Fabbro D, Trappe J, Rauch U, Bracher F, Knapp S. Specific CLK inhibitors from a novel chemotype for regulation of alternative splicing. Chem Biol. 2011 Jan 28;18(1):67-76. doi: https://doi.org/10.1016/j.chembiol.2010.11.009.
Citations
Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors
2010
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King ON*, Li XS*, Sakurai M*, Kawamura A, Rose NR, Ng SS, Quinn AM, Rai G, Mott BT, Beswick P, Klose RJ, Oppermann U, Jadhav A, Heightman TD, Maloney DJ, Schofield CJ and Simeonov A.
open-access
PLoS One
doi:10.1371/journal.pone.0015535
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Abstract
Background: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal findings: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.
Cite as
King ON, Li XS, Sakurai M, Kawamura A, Rose NR, Ng SS, Quinn AM, Rai G, Mott BT, Beswick P, Klose RJ, Oppermann U, Jadhav A, Heightman TD, Maloney DJ, Schofield CJ, Simeonov A. Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors. PLoS One. 2010 Nov 23;5(11):e15535. doi: https://doi.org/10.1371/journal.pone.0015535.
Citations
Development of homogeneous luminescence assays for histone demethylase catalysis and binding.
2010
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Kawamura A, Tumber A, Rose NR, King ON, Daniel M, Oppermann U, Heightman TD and Schofield CJ.
Analytical Biochemistry
doi:10.1016/j.ab.2010.04.030
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Abstract
Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.
Cite as
Kawamura A, Tumber A, Rose NR, King ON, Daniel M, Oppermann U, Heightman TD, Schofield C. Development of homogeneous luminescence assays for histone demethylase catalysis and binding. Anal Biochem. 2010 Sep 1;404(1):86-93. doi: https://doi.org/10.1016/j.ab.2010.04.030.
Citations
Selective inhibitors of the JMJD2 histone demethylases: Combined nondenaturing mass spectrometric screening and crystallographic approaches
2010
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Rose NR, Woon EC, Kingham GL, King ON, Mecinović J, Clifton IJ, Ng SS, Talib-Hardy J, Oppermann U, McDonough MA, and Schofield CJ.
open-access
Journal of Medicinal Chemistry
doi:10.1021/jm901680b
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Abstract
Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of Nε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.
Cite as
Nathan R. Rose, Esther C. Y. Woon, Guy L. Kingham, Oliver N. F. King, Jasmin Mecinović, Ian J. Clifton, Stanley S. Ng, Jobina Talib-Hardy, Udo Oppermann, Michael A. McDonough, and Christopher J. Schofield. Selective inhibitors of the JMJD2 histone demethylases: Combined nondenaturing mass spectrometric screening and crystallographic approaches. Journal of Medicinal Chemistry 2010 53 (4), 1810-1818. https://doi.org/10.1021/jm901680b
Citations
Large Scale Structural Analysis of the Classical Human Protein Tyrosine Phosphatome
2009
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Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S and Knapp S.
open-access
Cell
doi:10.1016/j.cell.2008.11.038
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Abstract
Protein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a “head-to-toe” dimerization model for RPTPγ/ζ that is distinct from the “inhibitory wedge” model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.
Cite as
Barr AJ, Ugochukwu E, Lee WH, King ON, Filippakopoulos P, Alfano I, Savitsky P, Burgess-Brown NA, Müller S, Knapp S. Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell. 2009 Jan 23;136(2),352-63. https://doi.org/10.1016/j.cell.2008.11.038.
Citations