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

Instruments / Technical Area	Publication Details	Published
	Outcomes of the EMDataResource cryo-EM ligand modeling challenge Catherine L. Lawson , Andriy Kryshtafovych , Grigore D. Pintilie , Stephen Burley , Jiří Černý , Vincent B. Chen , Paul Emsley , Alberto Gobbi , Andrzej Joachimiak , Sigrid Noreng , Michael G. Prisant , Randy J. Read , Jane S. Richardson , Alexis L. Rohou , Bohdan Schneider , Benjamin D. Sellers , Chenghua Shao , Elizabeth Sourial , Chris I. Williams , Christopher J. Williams , Ying Yang , Venkat Abbaraju , Pavel V. Afonine , Matthew L. Baker , Paul S. Bond , Tom L. Blundell , Tom Burnley , Arthur Campbell , Renzhi Cao , Jianlin Cheng , Grzegorz Chojnowski , Kevin D. Cowtan , Frank Dimaio , Reza Esmaeeli , Nabin Giri , Helmut Grubmüller , Soon Wen Hoh , Jie Hou , Corey F. Hryc , Carola Hunte , Maxim Igaev , Agnel P. Joseph , Wei-Chun Kao , Daisuke Kihara , Dilip Kumar , Lijun Lang , Sean Lin , Sai R. Maddhuri Venkata Subramaniya , Sumit Mittal , Arup Mondal , Nigel W. Moriarty , Andrew Muenks , Garib N. Murshudov , Robert A. Nicholls , Mateusz Olek , Colin M. Palmer , Alberto Perez , Emmi Pohjolainen , Karunakar R. Pothula , Christopher N. Rowley , Daipayan Sarkar , Luisa U. Schäfer , Christopher J. Schlicksup , Gunnar F. Schröder , Mrinal Shekhar , Dong Si , Abhishek Singharoy , Oleg V. Sobolev , Genki Terashi , Andrea C. Vaiana , Sundeep C. Vedithi , Jacob Verburgt , Xiao Wang , Rangana Warshamanage , Martyn Winn , Simone Weyand , Keitaro Yamashita , Minglei Zhao , Michael F. Schmid , Helen M. Berman , Wah Chiu Nature Methods 24 DOI: 10.1038/s41592-024-02321-7 Show Abstract ▼ Abstract: The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein–nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9–2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.	Jun 2024
I02-Macromolecular Crystallography	Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase H. Leonardo Silvestre , J. L. Asensio , T. L. Blundell , A. Bastida , V. M. Bolanos-Garcia International Journal Of Biological Macromolecules 263 DOI: 10.1016/j.ijbiomac.2024.130348 Diamond Proposal Number(s): [9007, 14043] Open Access Show Abstract ▼ Abstract: Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, Nα-acetyl transferase from Bacillus cereus (RimLBC), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimLBC belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimLBC. To gain further insight into RimLBC biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimLBC affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimLBC. Last, we advance a mechanistic model of RimLBC mode of recognition of its protein substrates. Taken together, our studies confirmed RimLBC as a new ribosomal Nα-acetyltransferase and provide structural and functional insights into substrate recognition by Nα-acetyltransferases and protein acetylation in bacteria.	Apr 2024
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structural characterization of Mycobacterium abscessus phosphopantetheine adenylyl transferase ligand interactions: implications for fragment-based drug design Sherine E. Thomas , William J. Mccarthy , Jamal El Bakali , Karen P. Brown , So Yeon Kim , Michal Blaszczyk , Vitor Mendes , Chris Abell , R. Andres Floto , Anthony G. Coyne , Tom L. Blundell Frontiers In Molecular Biosciences 9 DOI: 10.3389/fmolb.2022.880432 Diamond Proposal Number(s): [9537, 14043, 18548] Open Access Show Abstract ▼ Abstract: Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (Kd 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.	May 2022
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Development of inhibitors of SAICAR synthetase (PurC) from Mycobacterium abscessus using a fragment-based approach Sitthivut Charoensutthivarakul , Sherine E. Thomas , Amy Curran , Karen P. Brown , Juan M. Belardinelli , Andrew J. Whitehouse , Marta Acebron-Garcia-De-Eulate , Jaspar Sangan , Subramanian G. Gramani , Mary Jackson , Vitor Mendes , R. Andres Floto , Tom L. Blundell , Anthony G. Coyne , Chris Abell Acs Infectious Diseases DOI: 10.1021/acsinfecdis.1c00432 Diamond Proposal Number(s): [9537, 14043, 18548] Show Abstract ▼ Abstract: Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.	Jan 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Discovery of novel inhibitors of uridine diphosphate-n-acetylenolpyruvylglucosamine reductase (MurB) from Pseudomonas aeruginosa, an opportunistic infectious agent causing death in cystic fibrosis patients Marta Acebron-Garcia-De-Eulate , Joan Mayol-Llinàs , Matthew T. O. Holland , So Yeon Kim , Karen P. Brown , Chiara Marchetti , Jeannine Hess , Ornella Di Pietro , Vitor Mendes , Chris Abell , R. Andres Floto , Anthony G. Coyne , Tom L. Blundell Journal Of Medicinal Chemistry 48 DOI: 10.1021/acs.jmedchem.1c01684 Diamond Proposal Number(s): [18548] Show Abstract ▼ Abstract: Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 μM (LE 0.35).	Jan 2022
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis Pooja Gupta , Sherine E. Thomas , Shaymaa A. Zaidan , Maria A. Pasillas , James Cory-Wright , Víctor Sebastián-Pérez , Ailidh Burgess , Emma Cattermole , Clio Meghir , Chris Abell , Anthony G. Coyne , William R. Jacobs , Tom L. Blundell , Sangeeta Tiwari , Vitor Mendes Computational And Structural Biotechnology Journal 19, 3491 - 3506 DOI: 10.1016/j.csbj.2021.06.006 Diamond Proposal Number(s): [14043, 18548] Open Access Show Abstract ▼ Abstract: The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.	Jun 2021
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death Duncan E. Scott , Nicola J. Francis-Newton , May E. Marsh , Anthony G. Coyne , Gerhard Fischer , Tommaso Moschetti , Andrew R. Bayly , Timothy D. Sharpe , Kalina T. Haas , Lorraine Barber , Chiara R. Valenzano , Rajavel Srinivasan , David J. Huggins , Miyoung Lee , Amy Emery , Bryn Hardwick , Matthias Ehebauer , Claudio Dagostin , Alessandro Esposito , Luca Pellegrini , Trevor Perrior , Grahame Mckenzie , Tom L. Blundell , Marko Hyvonen , John Skidmore , Ashok R. Venkitaraman , Chris Abell Cell Chemical Biology 66 DOI: 10.1016/j.chembiol.2021.02.006 Diamond Proposal Number(s): [315, 7141] Open Access Show Abstract ▼ Abstract: BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.	Mar 2021
Krios II-Titan Krios II at Diamond	SAP domain forms a flexible part of DNA aperture in Ku70/80 Ales Hnizda , Petr Tesina , Than-Binh Nguyen , Zdeněk Kukačka , Lukas Kater , Amanda K. Chaplin , Roland Beckmann , David B. Ascher , Petr Novák , Tom L. Blundell The Febs Journal DOI: 10.1111/febs.15732 Diamond Proposal Number(s): [17057] Open Access Show Abstract ▼ Abstract: Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double‐strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C‐terminal domain, known as the SAP domain. Using single‐particle cryo‐electron microscopy, mass spectrometric analysis of intermolecular cross‐linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA‐bound state. The second position, which was observed in both apo and DNA‐bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA.	Feb 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site Vitor Mendes , Simon R. Green , Joanna C. Evans , Jeannine Hess , Michael Blaszczyk , Christina Spry , Owain Bryant , James Cory-Wright , Daniel S-H. Chan , Pedro H. M. Torres , Zhe Wang , Navid Nahiyaan , Sandra O’neill , Sebastian Damerow , John Post , Tracy Bayliss , Sasha L. Lynch , Anthony G. Coyne , Peter C. Ray , Chris Abell , Kyu Y. Rhee , Helena I. M. Boshoff , Clifton E. Barry , Valerie Mizrahi , Paul G. Wyatt , Tom L. Blundell Nature Communications 12 DOI: 10.1038/s41467-020-20224-x Diamond Proposal Number(s): [9537, 14043, 18548] Open Access Show Abstract ▼ Abstract: Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.	Jan 2021
I03-Macromolecular Crystallography	Using a fragment-based approach to identify alternative chemical scaffolds targeting dihydrofolate reductase from mycobacterium tuberculosis João A. Ribeiro , Alexander Hammer , Gerardo A. Libreros-Zúñiga , Sair M. Chavez-Pacheco , Petros Tyrakis , Gabriel S. De Oliveira , Timothy Kirkman , Jamal El Bakali , Silvana A. Rocco , Mauricio L. Sforça , Roberto Parise-Filho , Anthony G. Coyne , Tom L. Blundell , Chris Abell , Marcio V. B. Dias Acs Infectious Diseases DOI: 10.1021/acsinfecdis.0c00263 Show Abstract ▼ Abstract: Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.	Jul 2020

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