I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[35587]
Abstract: Coronaviruses, including SARS-CoV-2, possess an mRNA 5' capping apparatus capable of mimicking the natural eukaryotic capping signature. Two SAM-dependent methylating enzymes play important roles in this process: nsp14 methylates the N7 of the guanosine cap, and nsp16-nsp10 methylates the 2'-O- of subsequent nucleotides of viral mRNA. The 2'-O-methylation performed by nsp16-nsp10 is crucial for the escape of the viral RNA from innate immunity. Inhibition of this enzymatic activity has been proposed as a way to combat coronaviruses. In this study, we employed X-ray crystallography to analyze the binding of the SAM analogues to the active site of nsp16-nsp10. We obtained eleven 3D crystal structures of the nsp16-nsp10 complexes with SAM-derived inhibitors, demonstrated different conformations of the methionine substituting part of the molecules, and confirmed that simultaneous dual-site targeting of both SAM and RNA sites correlates with higher inhibitory potential.
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Sep 2024
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I03-Macromolecular Crystallography
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Abstract: Prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3) are 2-oxoglutarate (2OG)-dependent oxygenases catalysing C-4 hydroxylation of prolyl residues in α-subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), modifications that promote HIF-α degradation via the ubiquitin-proteasome pathway. Pharmacological inhibition of the PHDs induces HIF-α stabilisation, so promoting HIF target gene transcription. PHD inhibitors are used to treat anaemia caused by chronic kidney disease (CKD) due to their ability to stimulate erythropoietin (EPO) production. We report studies on the effects of the approved PHD inhibitors Desidustat and Enarodustat, and the clinical candidate TP0463518, on activities of a representative set of isolated recombinant human 2OG oxygenases. The three molecules manifest selectivity for PHD inhibition over that of the other 2OG oxygenases evaluated. We obtained crystal structures of Desidustat and Enarodustat in complex with the human 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), which, together with modelling studies, inform on the binding modes of Desidustat and Enarodustat to active site Fe(II) in 2OG oxygenases, including PHD1-3. The results will help in the design of selective inhibitors of both the PHDs and other 2OG oxygenases, which are of medicinal interest due to their involvement inter alia in metabolic regulation, epigenetic signalling, DNA-damage repair, and agrochemical resistance.
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Sep 2024
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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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
Open Access
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.
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Aug 2024
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I03-Macromolecular Crystallography
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Stuart
Lang
,
Dan A.
Fletcher
,
Alain-Pierre
Petit
,
Nicola
Luise
,
Paul K.
Fyfe
,
Fabio
Zuccotto
,
David
Porter
,
Anthony
Hope
,
Fiona
Bellany
,
Catrina
Kerr
,
Claire J.
Mackenzie
,
Paul G.
Wyatt
,
David W.
Gray
Diamond Proposal Number(s):
[19844]
Open Access
Abstract: Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pKi values of 5.3 and 5.4 respectively.
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Apr 2024
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Dihydrofolate reductase (DHFR) is a key enzyme involved in the folate pathway that has been heavily targeted for the development of therapeutics against cancer and bacterial and protozoa infections amongst others. Despite being an essential enzyme for Mycobacterium tuberculosis (Mtb) viability, DHFR remains an underexploited target for tuberculosis (TB) treatment. Herein, we report the preparation and evaluation of a series of compounds against Mtb DHFR (MtbDHFR). The compounds have been designed using a merging strategy of traditional pyrimidine-based antifolates with a previously discovered unique fragment hit against MtbDHFR. In this series, four compounds displayed a high affinity against MtbDHFR, with sub-micromolar affinities. Additionally, we determined the binding mode of six of the best compounds using protein crystallography, which revealed occupation of an underutilised region of the active site.
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May 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Degradation of the extracellular matrix (ECM) supports tissue integrity and homeostasis, but is also a key factor in cancer metastasis. Heparanase (HPSE) is a mammalian ECM-remodeling enzyme with β-D-endo-glucuronidase activity overexpressed in several malignancies, and is thought to facilitate tumor growth and metastasis. By this virtue, HPSE is considered an attractive target for the development of cancer therapies, yet to date no HPSE inhibitors have progressed to the clinic. Here we report on the discovery of glucurono-configured cyclitol derivatives featuring simple substituents at the 4-O-position as irreversible HPSE inhibitors. We show that these compounds, unlike glucurono-cyclophellitol, are selective for HPSE over β-D-exo-glucuronidase (GUSB), also in platelet lysate. The observed selectivity is induced by steric and electrostatic interactions of the substituents at the 4-O-position. Crystallographic analysis supports this rationale for HPSE selectivity, and computer simulations provide insights in the conformational preferences and binding poses of the inhibitors, which we believe are good starting points for the future development of HPSE-targeting antimetastatic cancer drugs.
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Dec 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Olov
Wallner
,
Armando
Cázares-Körner
,
Emma R.
Scaletti
,
Geoffrey
Masuyer
,
Tove
Bekkhus
,
Torkild
Visnes
,
Kirill
Mamonov
,
Florian
Ortis
,
Thomas
Lundbäck
,
Maria
Volkova
,
Tobias
Koolmeister
,
Elisée
Wiita
,
Olga
Loseva
,
Monica
Pandey
,
Evert
Homan
,
Carlos
Benítez-Buelga
,
Jonathan
Davies
,
Martin
Scobie
,
Ulrika Warpman
Berglund
,
Christina
Kalderén
,
Pal
Stenmark
,
Thomas
Helleday
,
Maurice
Michel
Diamond Proposal Number(s):
[15806, 21625]
Open Access
Abstract: 8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G:C→T:A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N -piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of different members of the class. Potent members adopt a chair within the N -Piperidinyl-linker, while a boat conformation was found for weaker analogues.
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Sep 2022
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I03-Macromolecular Crystallography
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Arathy
Jose
,
Daniel
Guest
,
Remi
Legay
,
Graham J.
Tizzard
,
Simon
Coles
,
Mariliza
Derveni
,
Edward
Wright
,
Lester
Marrison
,
Alpha A.
Lee
,
Aaron
Morris
,
Matt
Robinson
,
Frank
Von Delft
,
Daren
Fearon
,
Lizbe
Koekemoer
,
Tetiana
Matviuk
,
Anthony
Aimon
,
Christopher J.
Schofield
,
Tika R.
Malla
,
Nir
London
,
Barnaby W.
Greenland
,
Mark C.
Bagley
,
John
Spencer
Diamond Proposal Number(s):
[19301]
Open Access
Abstract: The pentafluorosulfanyl (-SF5) functional group is of increasing interest as a bioisostere in medicinal chemistry. A library of SF5-containing compounds, including amide, isoxazole, and oxindole derivatives, was synthesised using a range of solution-based and solventless methods, including microwave and ball-mill techniques. The library was tested against targets including human dihydroorotate dehydrogenase (HDHODH). A subsequent focused approach led to synthesis of analogues of the clinically used disease modifying anti-rheumatic drugs (DMARDs), Teriflunomide and Leflunomide, considered for potential COVID-19 use, where SF5 bioisostere deployment led to improved inhibition of HDHODH compared with the parent drugs. The results demonstrate the utility of the SF5 group in medicinal chemistry.
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Feb 2022
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I04-Macromolecular Crystallography
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Federica
Verdirosa
,
Laurent
Gavara
,
Laurent
Sevaille
,
Giusy
Tassone
,
Giuseppina
Corsica
,
Alice
Legru
,
Georges
Feller
,
Giulia
Chelini
,
Paola S.
Mercuri
,
Silvia
Tanfoni
,
Filomena
Sannio
,
Manuela
Benvenuti
,
Giulia
Cerboni
,
Filomena
De Luca
,
Ezeddine
Bouajila
,
Yen
Vo Hoang
,
Patricia
Licznar-Fajardo
,
Moreno
Galleni
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Jean-Denis
Docquier
,
Jean-François
Hernandez
Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Metallo-β-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need . We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, they were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we developed compounds where the hydrazone-like bond of the Schiff bases was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but some showed a significantly better activity on VIM-type enzymes, with K i values in the μM to sub-μM range. The resolution of the crystallographic structure of VIM-2 in complex with one inhibitor yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the β-lactam susceptibility of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cells at high concentration, thus showing promising safety.
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Jan 2022
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Open Access
Abstract: The two SARS-CoV-2 proteases, i.e. the main protease (M pro ) and the papain-like protease (PL pro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL pro catalysis in vitro . The assay was applied to investigate the effect of reported small-molecule PL pro inhibitors and selected M pro inhibitors on PL pro catalysis. The results reveal that some, but not all, PL pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL pro . Less selective M pro inhibitors, e.g. auranofin, inhibit PL pro , highlighting the potential for dual PL pro /M pro inhibition. MS-based PL pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.
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Jan 2022
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