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949 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4, 5, 6, 7, 8 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Defining substrate selection by rhinoviral 2A proteinase through its crystal structure with the inhibitor zVAM.fmk Karin M. Deutschmann-Olek , Wyatt W. Yue , Gustavo A. Bezerra , Tim Skern Virology 562, 128 - 141 DOI: 10.1016/j.virol.2021.07.008 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.	Oct 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components Yakubu Princely Abudu , Birendra Kumar Shrestha , Wenxin Zhang , Anthimi Palara , Hanne Britt Brenne , Kenneth Bowitz Larsen , Deanna Lynn Wolfson , Gianina Dumitriu , Cristina Ionica Øie , Balpreet Singh Ahluwalia , Gahl Levy , Christian Behrends , Sharon A. Tooze , Stephane Mouilleron , Trond Lamark , Terje Johansen Journal Of Cell Biology 220 DOI: 10.1083/jcb.202009092 Diamond Proposal Number(s): [18566] Show Abstract ▼ Abstract: Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.	Aug 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc Adam S. B. Jalal , Ngat T. Tran , Ling J. Wu , Karunakaran Ramakrishnan , Martin Rejzek , Giulia Gobbato , Clare E. M. Stevenson , David M. Lawson , Jeff Errington , Tung B. K. Le Molecular Cell 34 DOI: 10.1016/j.molcel.2021.06.025 Diamond Proposal Number(s): [18565, 25108] Open Access Show Abstract ▼ Abstract: ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.	Jul 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition site Rosemary A Bayne , Uma Jayachandran , Aleksandra Kasprowicz , Stefan Bresson , David Tollervey , Edward W J Wallace , Atlanta G. Cook Nucleic Acids Research 4 DOI: 10.1093/nar/gkab615 Diamond Proposal Number(s): [18515] Open Access Show Abstract ▼ Abstract: Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5′ untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.	Jul 2021
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer Stelios Chrysostomou , Rajat Roy , Filippo Prischi , Lucksamon Thamlikitkul , Kathryn L. Chapman , Uwais Mufti , Robert Peach , Laifeng Ding , David Hancock , Christopher Moore , Miriam Molina-Arcas , Francesco Mauri , David J. Pinato , Joel M. Abrahams , Silvia Ottaviani , Leandro Castellano , Georgios Giamas , Jennifer Pascoe , Devmini Moonamale , Sarah Pirrie , Claire Gaunt , Lucinda Billingham , Neil M. Steven , Michael Cullen , David Hrouda , Mathias Winkler , John Post , Philip Cohen , Seth J. Salpeter , Vered Bar , Adi Zundelevich , Shay Golan , Dan Leibovici , Romain Lara , David R. Klug , Sophia N. Yaliraki , Mauricio Barahona , Yulan Wang , Julian Downward , J. Mark Skehel , Maruf M. U. Ali , Michael J. Seckl , Olivier E. Pardo Science Translational Medicine 13 DOI: 10.1126/scitranslmed.aba4627 Diamond Proposal Number(s): [9424] Show Abstract ▼ Abstract: Lung and bladder cancers are mostly incurable due to early development of drug resistance and metastatic dissemination. Hence, better therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, either through RNA interference or CRISPR, sensitised tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail- vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically-engineered mouse models and bladder tumour explants. Through X-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase auto-inhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomised phase 3 SIGNIFICANT Trial had significantly (p =0.048) increased long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.	Jul 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Phosphorylation of the LIR domain of SCOC modulates ATG8 binding affinity and specificity Martina Wirth , Stephane Mouilleron , Wenxin Zhang , Eva Sjøttem , Yakubu Princely Abudu , Ashish Jain , Hallvard Lauritz Olsvik , Jack-Ansgar Bruun , Minoo Razi , Harold B. J. Jefferies , Rebecca Lee , Dhira Joshi , Nicola O'Reilly , Terje Johansen , Sharon A. Tooze Journal Of Molecular Biology 433 DOI: 10.1016/j.jmb.2021.166987 Diamond Proposal Number(s): [9826] Open Access Show Abstract ▼ Abstract: Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.	Jun 2021
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Reviving lost binding sites: Exploring calcium binding‐site transitions between human and murine CD23 Veronica F. Ilkow , Anna M. Davies , Balvinder Dhaliwal , Andrew J. Beavil , Brian J. Sutton , James M. Mcdonnell Febs Open Bio DOI: 10.1002/2211-5463.13214 Diamond Proposal Number(s): [9495] Open Access Show Abstract ▼ Abstract: Immunoglobulin E (IgE) is a central regulatory and triggering molecule of allergic immune responses.IgE’s interaction with CD23 modulates both IgE production and functional activities.CD23 is a non-canonical immunoglobulin receptor, unrelated to receptors of other antibody isotypes. Human CD23 is a calcium-dependent (C-type) lectin-like domain that has apparently lost its carbohydrate binding capability. The calcium binding site classically required for carbohydrate binding in C-type lectins is absent in human CD23 but is present in the murine molecule.To determine if the absence of this calcium binding site affects the structure and function of human CD23, CD23 mutant proteins with increasingly ‘murine-like’ sequences were generated. Restoration of the calcium binding site was confirmed by NMR spectroscopy and structures of mutant human CD23 proteins were determined by X-ray crystallography, although no electron density for calcium was observed.This study offers insights into the evolutionary differences between murine and human CD23, and some of the functional differences between CD23 in different species.	Jun 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	A crystallographic snapshot of SARS-CoV-2 main protease maturation process G. D. Noske , A. M. Nakamura , V. O. Gawriljuk , R. S. Fernandes , G. M. A. Lima , H. V. D. Rosa , H. D. Pereira , A. C. M. Zeri , A. A. F. Z. Nascimento , M. C. L. C. Freire , D. Fearon , A. Douangamath , F. Von Delft , G. Oliva , A. S. Godoy Journal Of Molecular Biology DOI: 10.1016/j.jmb.2021.167118 Diamond Proposal Number(s): [27963] Show Abstract ▼ Abstract: SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.	Jun 2021
I04-1-Macromolecular Crystallography (fixed wavelength)	An automatic pipeline for the design of irreversible derivatives identifies a potent SARS-CoV-2 Mpro inhibitor Daniel Zaidman , Paul Gehrtz , Mihajlo Filep , Daren Fearon , Ronen Gabizon , Alice Douangamath , Jaime Prilusky , Shirly Duberstein , Galit Cohen , C. David Owen , Efrat Resnick , Claire Strain-Damerell , Petra Lukacik , Haim Barr , Martin A. Walsh , Frank Von Delft , Nir London Cell Chemical Biology 63 DOI: 10.1016/j.chembiol.2021.05.018 Diamond Proposal Number(s): [18145, 27963] Show Abstract ▼ Abstract: Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.	Jun 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19 Martin A. Redhead , C. David Owen , Lennart Brewitz , Amelia H. Collette , Petra Lukacik , Claire Strain-Damerell , Sean W. Robinson , Patrick M. Collins , Philipp Schäfer , Mark Swindells , Chris J. Radoux , Iva Navratilova Hopkins , Daren Fearon , Alice Douangamath , Frank Von Delft , Tika R. Malla , Laura Vangeel , Thomas Vercruysse , Jan Thibaut , Pieter Leyssen , Tu-Trinh Nguyen , Mitchell Hull , Anthony Tumber , David J. Hallett , Christopher J. Schofield , David I. Stuart , Andrew L. Hopkins , Martin A. Walsh Scientific Reports 11 DOI: 10.1038/s41598-021-92416-4 Open Access Show Abstract ▼ Abstract: Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.	Jun 2021

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