I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Simon R.
Green
,
Susan H.
Davis
,
Sebastian
Damerow
,
Curtis A.
Engelhart
,
Michael
Mathieson
,
Beatriz
Baragaña
,
David A.
Robinson
,
Jevgenia
Tamjar
,
Alice
Dawson
,
Fabio K.
Tamaki
,
Kirsteen I.
Buchanan
,
John
Post
,
Karen
Dowers
,
Sharon M.
Shepherd
,
Chimed
Jansen
,
Fabio
Zuccotto
,
Ian H.
Gilbert
,
Ola
Epemolu
,
Jennifer
Riley
,
Laste
Stojanovski
,
Maria
Osuna-Cabello
,
Esther
Pérez-Herrán
,
María José
Rebollo
,
Laura
Guijarro López
,
Patricia
Casado Castro
,
Isabel
Camino
,
Heather C.
Kim
,
James M.
Bean
,
Navid
Nahiyaan
,
Kyu Y.
Rhee
,
Qinglan
Wang
,
Vee Y.
Tan
,
Helena I. M.
Boshoff
,
Paul J.
Converse
,
Si-Yang
Li
,
Yong S.
Chang
,
Nader
Fotouhi
,
Anna M.
Upton
,
Eric L.
Nuermberger
,
Dirk
Schnappinger
,
Kevin D.
Read
,
Lourdes
Encinas
,
Robert H.
Bates
,
Paul G.
Wyatt
,
Laura A. T.
Cleghorn
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
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Oct 2022
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I03-Macromolecular Crystallography
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Rachel
Milne
,
Natalie
Wiedemar
,
Victoriano
Corpas-Lopez
,
Eoin
Moynihan
,
Richard J.
Wall
,
Alice
Dawson
,
David A.
Robinson
,
Sharon M.
Shepherd
,
Robert J.
Smith
,
Irene
Hallyburton
,
John M.
Post
,
Karen
Dowers
,
Leah S.
Torrie
,
Ian H.
Gilbert
,
Beatriz
Baragaña
,
Stephen
Patterson
,
Susan
Wyllie
Diamond Proposal Number(s):
[19844]
Open Access
Abstract: There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
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Aug 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Low-nanomolar binding constants were recorded for a series of six 2′-fluoro-(carbamoylpyridinyl)deschloroepibatidine analogues with acetylcholine-binding protein (AChBP). The crystal structures of three complexes with AChBP reveal details of molecular recognition in the orthosteric binding site and imply how the other three ligands bind. Comparisons exploiting AChBP as a surrogate for α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the key interactions are conserved. The ligands interact with the same residues as the archetypal nAChR agonist nicotine yet display greater affinity, thereby rationalizing their in vivo activity as potent antagonists of nicotine-induced antinociception. An oxyanion-binding site is formed on the periphery of the AChBP orthosteric site by Lys42, Asp94, Glu170 and Glu210. These residues are highly conserved in the human α4, β2 and α7 nAChR sequences. However, specific sequence differences are discussed that could contribute to nAChR subtype selectivity and in addition may represent a point of allosteric modulation. The ability to engage with this peripheral site may explain, in part, the function of a subset of ligands to act as agonists of α7 nAChR.
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Mar 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Protein-engineering methods have been exploited to produce a surrogate system for the extracellular neurotransmitter-binding site of a heteromeric human ligand-gated ion channel, the glycine receptor. This approach circumvents two major issues: the inherent experimental difficulties in working with a membrane-bound ion channel and the complication that a heteromeric assembly is necessary to create a key, physiologically relevant binding site. Residues that form the orthosteric site in a highly stable ortholog, acetylcholine-binding protein, were selected for substitution. Recombinant proteins were prepared and characterized in stepwise fashion exploiting a range of biophysical techniques, including X-ray crystallography, married to the use of selected chemical probes. The decision making and development of the surrogate, which is termed a glycine-binding protein, are described, and comparisons are provided with wild-type and homomeric systems that establish features of molecular recognition in the binding site and the confidence that the system is suited for use in early-stage drug discovery targeting a heteromeric α/β glycine receptor.
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Nov 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980]
Abstract: The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.
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Apr 2019
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I04-Macromolecular Crystallography
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Pasquale
Linciano
,
Alice
Dawson
,
Ina
Pöhner
,
David M.
Costa
,
Monica S.
Sá
,
Anabela
Cordeiro-Da-Silva
,
Rosaria
Luciani
,
Sheraz
Gul
,
Gesa
Witt
,
Bernhard
Ellinger
,
Maria
Kuzikov
,
Philip
Gribbon
,
Jeanette
Reinshagen
,
Markus
Wolf
,
Birte
Behrens
,
Véronique
Hannaert
,
Paul A. M.
Michels
,
Erika
Nerini
,
Cecilia
Pozzi
,
Flavio
Di Pisa
,
Giacomo
Landi
,
Nuno
Santarem
,
Stefania
Ferrari
,
Puneet
Saxena
,
Sandra
Lazzari
,
Giuseppe
Cannazza
,
Lucio H.
Freitas-Junior
,
Carolina B.
Moraes
,
Bruno S.
Pascoalino
,
Laura M.
Alcântara
,
Claudia P.
Bertolacini
,
Vanessa
Fontana
,
Ulrike
Wittig
,
Wolfgang
Müller
,
Rebecca C.
Wade
,
William N.
Hunter
,
Stefano
Mangani
,
Luca
Costantino
,
Maria P.
Costi
Diamond Proposal Number(s):
[8574]
Open Access
Abstract: Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
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Sep 2017
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I03-Macromolecular Crystallography
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Open Access
Abstract: The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[11088]
Abstract: The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway (PPP) and is considered a promising target for the discovery of a new drug against Chagas diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate β-d-glucose-6-phosphate (G6P) and the reduced ‘catalytic’ cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP+ competitive inhibitors targeting the parasite enzyme.
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Aug 2016
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I04-Macromolecular Crystallography
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Open Access
Abstract: The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit-subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization.
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Aug 2010
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NONE-No attached Diamond beamline
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Abstract: Two high-pressure polymorphs of sulfuric acid monohydrate (oxonium hydrogensulfate) have been obtained at ambient temperature by crystallisation at high pressure from the liquid at 1.3 GPa (form III) and by direct compression of the ambient-pressure form I first to 1.26 GPa (form II) and then to 1.72 GPa (form III). The structure of form III was solved by single crystal X-ray diffraction and this structure was used as the basis for the refinement of hydrogen positions using high-pressure neutron powder diffraction data. Form III crystallises in the orthorhombic crystal system at 1.97 GPa, and features parallel chains of hydrogensulfate ions linked by oxonium ions to form a three-dimensional hydrogen-bonded network. On further compression to 3.05 GPa, the direction of maximum compressibility is found to be along the a-axis and is associated with the shortening of a hydrogen bond between a hydrogensulfate ion and an oxonium ion. The structure of form II remains elusive although at ambient temperature it is stable (or metastable) at pressures as low as 0.42 GPa, perhaps indicating that it could be recoverable to ambient-pressure at low temperature.
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Jan 2008
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