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1,136 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
B21-High Throughput SAXS I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Evolutionary and molecular basis of ADP-ribosylation reversal by zinc-dependent macrodomains Antonio Ariza , Qiang Liu , Nathan Cowieson , Ivan Ahel , Dmitri V. Filippov , Johannes Gregor Matthias Rack Journal Of Biological Chemistry 20 DOI: 10.1016/j.jbc.2024.107770 Diamond Proposal Number(s): [9306, 12346, 15613] Open Access Show Abstract ▼ Abstract: Dynamic ADP-ribosylation signalling is a crucial pathway that controls fundamental cellular processes, in particular, the response to cellular stresses such as DNA damage, reactive oxygen species and infection. In some pathogenic microbes the response to oxidative stress is controlled by a SirTM/zinc-containing macrodomain (Zn-Macro) pair responsible for establishment and removal of the modification, respectively. Targeting this defence mechanism against the host’s innate immune response may lead to novel approaches to support the fight against emerging antimicrobial resistance. Earlier studies suggested that Zn-Macros play a key role in the activation of this defence. Therefore, we used phylogenetic, biochemical, and structural approaches to elucidate the functional properties of these enzymes. Using the substrate mimetic asparagine-ADP-ribose as well as the ADP-ribose product, we characterise the catalytic role of the zinc ion in the removal of the ADP-ribosyl modification. Furthermore, we determined structural properties that contribute to substrate selectivity within the different Zn-Macro branches. Together, our data not only give new insights into the Zn-Macro family but also highlight their distinct features that may be exploited for the development of future therapies.	Sep 2024
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	BRCA2 stabilises RAD51 and DMC1 nucleoprotein filaments through a conserved interaction mode James M. Dunce , Owen R. Davies Nature Communications 15 DOI: 10.1038/s41467-024-52699-3 Diamond Proposal Number(s): [13587, 18598, 27649] Open Access Show Abstract ▼ Abstract: BRCA2 is essential for DNA repair by homologous recombination in mitosis and meiosis. It interacts with recombinases RAD51 and DMC1 to facilitate the formation of nucleoprotein filaments on resected DNA ends that catalyse recombination-mediated repair. BRCA2’s BRC repeats bind and disrupt RAD51 and DMC1 filaments, whereas its PhePP motifs bind recombinases and stabilise their nucleoprotein filaments. However, the mechanism of filament stabilisation has hitherto remained unknown. Here, we report the crystal structure of a BRCA2-DMC1 complex, revealing how core interaction sites of PhePP motifs bind to recombinases. The interaction mode is conserved for RAD51 and DMC1, which selectively bind to BRCA2’s two distinct PhePP motifs via subtly divergent binding pockets. PhePP motif sequences surrounding their core interaction sites protect nucleoprotein filaments from BRC-mediated disruption. Hence, we report the structural basis of how BRCA2’s PhePP motifs stabilise RAD51 and DMC1 nucleoprotein filaments for their essential roles in mitotic and meiotic recombination.	Sep 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Selective Aurora A-TPX2 interaction inhibitors have in vivo efficacy as targeted antimitotic agents Simon R. Stockwell , Duncan E. Scott , Gerhard Fischer , Estrella Guarino , Timothy P. C. Rooney , Tzu-Shean Feng , Tommaso Moschetti , Rajavel Srinivasan , Esther Alza , Alice Asteian , Claudio Dagostin , Anna Alcaide , Mathieu Rocaboy , Beata Blaszczyk , Alicia Higueruelo , Xuelu Wang , Maxim Rossmann , Trevor R. Perrior , Tom L. Blundell , David R. Spring , Grahame Mckenzie , Chris Abell , John Skidmore , Ashok R. Venkitaraman , Marko Hyvonen Journal Of Medicinal Chemistry 9 DOI: 10.1021/acs.jmedchem.4c01165 Diamond Proposal Number(s): [9537, 14043] Open Access Show Abstract ▼ Abstract: Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein–protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.	Aug 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The role of water networks in phosphodiesterase inhibitor dissociation and kinetic selectivity Antoni R. Blaazer , Abhimanyu K. Singh , Lorena Zara , Pierre Boronat , Lady J. Bautista , Steve Irving , Maciej Majewski , Xavier Barril , Maikel Wijtmans , U. Helena Danielson , Geert Jan Sterk , Rob Leurs , Jacqueline E. Van Muijlwijk-Koezen , David G. Brown , Iwan De Esch Chemmedchem DOI: 10.1002/cmdc.202400417 Open Access Show Abstract ▼ Abstract: In search of new opportunities to develop Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) inhibitors that have selectivity over the off-target human PDE4 (hPDE4), different stages of a fragment-growing campaign were studied using a variety of biochemical, structural, thermodynamic, and kinetic binding assays. Remarkable differences in binding kinetics were identified and this kinetic selectivity was explored with computational methods, including molecular dynamics and interaction fingerprint analyses. These studies indicate that a key hydrogen bond between GlnQ.50 and the inhibitors is exposed to a water channel in TbrPDEB1, leading to fast unbinding. This water channel is not present in hPDE4, leading to inhibitors with a longer residence time. The computer-aided drug design protocols were applied to a recently disclosed TbrPDEB1 inhibitor with a different scaffold and our results confirm that shielding this key hydrogen bond through disruption of the water channel represents a viable design strategy to develop more selective inhibitors of TbrPDEB1. Our work shows how computational protocols can be used to understand the contribution of solvent dynamics to inhibitor binding, and our results can be applied in the design of selective inhibitors for homologous PDEs found in related parasites.	Aug 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them Subhadra Dalwani , Alexander Metz , Franziska U. Huschmann , Manfred S. Weiss , Rik K. Wierenga , Rajaram Venkatesan Acta Crystallographica Section D Structural Biology 80 DOI: 10.1107/S2059798324006557 Diamond Proposal Number(s): [19951, 24732] Open Access Show Abstract ▼ Abstract: The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE–fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.	Aug 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Design, synthesis, and in vitro biological evaluation of meta-sulfonamidobenzamide-based antibacterial LpxH inhibitors Andrea Benediktsdottir , Sanjeewani Sooriyaarachchi , Sha Cao , Nina E. Ottosson , Stefan Lindström , Bo Lundgren , Katharina Klöditz , Daina Lola , Olga Bobileva , Einars Loza , Diarmaid Hughes , T. Alwyn Jones , Sherry L. Mowbray , Edouard Zamaratski , Anja Sandström , Anders Karlén European Journal Of Medicinal Chemistry 66 DOI: 10.1016/j.ejmech.2024.116790 Diamond Proposal Number(s): [24689, 23773] Open Access Show Abstract ▼ Abstract: New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.	Aug 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A fragment based competitive 19F LB‐NMR platform for hotspot directed ligand profiling William J. Mccarthy , Sherine E. Thomas , Tayo Olaleye , Jennifer A Boland , Rodrigo Andres Floto , Glyn Williams , Tom L. Blundell , Anthony G Coyne , Chris Abell Angewandte Chemie International Edition DOI: 10.1002/anie.202406846 Diamond Proposal Number(s): [18548, 25402] Open Access Show Abstract ▼ Abstract: Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment-based competitive 19F Ligand Based-NMR (LB-NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand-binding hotspots. As a proof of concept, the method was applied to 4’-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X-ray crystallographic characterisation of the hits from a 960-member fragment screen identified three ligand-binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19F LB-NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X-ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment-based competitive 19F LB-NMR screening platform for hotspot-directed ligand profiling.	Jun 2024
I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Broad protection against invasive fungal disease from a nanobody targeting the active site of fungal b‐1,3‐glucanosyltransferases Sergio Redrado-Hernández , Javier Macías-León , Jorge Castro-López , Ana Belén Sanz , Elena Dolader , Maykel Arias , Andrés Manuel González-Ramírez , David Sánchez-Navarro , Yuliya Petryk , Vladimir Farkaš , Cecile Vincke , Serge Muyldermans , Irene García-Barbazán , Celia Del Agua , Oscar Zaragoza , Javier Arroyo , Julián Pardo , Eva Gálvez , Ramon Hurtado-Guerrero Angewandte Chemie International Edition DOI: 10.1002/anie.202405823 Diamond Proposal Number(s): [20229] Open Access Show Abstract ▼ Abstract: Invasive fungal disease accounts for ~3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal β-1,3-glucanosyltransferases (Gel) involved in β-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for β-1,3-glucan remodelling in C. deneoformans survival. These findings add new insights about the role of b-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.	Jun 2024
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy Lucas Do Amaral Martins , Priscila S. Ferreira , Otávio Augusto Leitão Dos Santos , Leticia Oliveira Martins , Luiz Gabriel Cabral Fernandes Barroso , Humberto M. Pereira , Márcia Waddington-Cruz , Fernando Lucas Palhano , Debora Foguel Journal Of Biological Chemistry 36 DOI: 10.1016/j.jbc.2024.107495 Open Access Show Abstract ▼ Abstract: Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.	Jun 2024
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Fragment-based discovery of a series of allosteric-binding site modulators of β-glucocerebrosidase Nick Palmer , Christopher Agnew , Caroline Benn , William J. Buffham , Joan N. Castro , Gianni Chessari , Mellissa Clark , Benjamin D. Cons , Joseph E. Coyle , Lee A. Dawson , Christopher C. F. Hamlett , Charlotte Hodson , Finn Holding , Christopher N. Johnson , John W. Liebeschuetz , Pravin Mahajan , James M. Mccarthy , Christopher W. Murray , Marc O’reilly , Torren Peakman , Amanda Price , Magdalini Rapti , Judith Reeks , Patrick Schöpf , Jeffrey D. St-Denis , Chiara Valenzano , Nicola G. Wallis , Reto Walser , Heather Weir , Nicola E. Wilsher , Andrew Woodhead , Carla F. Bento , Dominic Tisi Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.4c00702 Diamond Proposal Number(s): [23276, 20014, 17184] Show Abstract ▼ Abstract: β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher’s disease and are a major genetic risk factor for Parkinson’s disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.	Jun 2024

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