I04-1-Macromolecular Crystallography (fixed wavelength)
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Chloe R.
Koulouris
,
Sian E.
Gardiner
,
Tessa K.
Harris
,
Karen T.
Elvers
,
S. Mark
Roe
,
Jason A.
Gillespie
,
Simon E.
Ward
,
Olivera
Grubisha
,
Robert A.
Nicholls
,
John R.
Atack
,
Benjamin D.
Bax
Diamond Proposal Number(s):
[19990]
Open Access
Abstract: Human serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the α-phosphate of ATP. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzyme-fragment structures show Tyr121 flipped out of its pocket in the core of the small domain. Data suggest that this ligandable pocket could be targeted by molecules that inhibit enzyme activity.
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Dec 2022
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I03-Macromolecular Crystallography
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Maria Giulia
Nizi
,
Mirko M.
Maksimainen
,
Sudarshan
Murthy
,
Serena
Massari
,
Juho
Alaviuhkola
,
Barbara E.
Lippok
,
Sven T.
Sowa
,
Albert
Galera-Prat
,
Renata
Prunskaite-Hyyryläinen
,
Bernhard
Lüscher
,
Patricia
Korn
,
Lari
Lehtio
,
Oriana
Tabarrini
Diamond Proposal Number(s):
[19951]
Abstract: While human poly-ADP-ribose chain generating poly-ARTs, PARP1 and 2 and TNKS1 and 2, have been widely characterized, less is known on the pathophysiological roles of the mono-ADP-ribosylating mono-ARTs, partly due to the lack of selective inhibitors. In this context, we have focused on the development of inhibitors for the mono-ART PARP10, whose overexpression is known to induce cell death. Starting from OUL35 (1) and its 4-(benzyloxy)benzamidic derivative (2) we herein report the design and synthesis of new analogues from which the cyclobutyl derivative 3c rescued cells most efficiently from PARP10 induced apoptosis. Most importantly, we also identified 2,3-dihydrophthalazine-1,4-dione as a new suitable nicotinamide mimicking PARP10 inhibitor scaffold. When it was functionalized with cycloalkyl (8a-c), o-fluorophenyl (8h), and thiophene (8l) rings, IC50 values in the 130–160 nM range were obtained, making them the most potent PARP10 inhibitors reported to date. These compounds also inhibited PARP15 with low micromolar IC50s, but none of the other tested poly- and mono-ARTs, thus emerging as dual mono-ART inhibitors. Compounds 8a, 8h and 8l were also able to enter cells and rescue cells from apoptosis. Our work sheds more light on inhibitor development against mono-ARTs and identifies chemical probes to study the cellular roles of PARP10 and PARP15.
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Jul 2022
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10369]
Abstract: The Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilisation, oxidative stress response, and virulence. Here we characterised this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilisation of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystalization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologues suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18145]
Open Access
Abstract: Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme’s substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19458, 23459]
Open Access
Abstract: β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.
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May 2022
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I03-Macromolecular Crystallography
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Tika R.
Malla
,
Lennart
Brewitz
,
Dorian-Gabriel
Muntean
,
Hiba
Aslam
,
C. David
Owen
,
Eidarus
Salah
,
Anthony
Tumber
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.
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May 2022
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I04-Macromolecular Crystallography
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Pamela
Sweeney
,
Ashleigh
Galliford
,
Abhishek
Kumar
,
Dinesh
Raju
,
Naveen B.
Krishna
,
Emmajay
Sutherland
,
Caitlin J.
Leo
,
Gemma
Fisher
,
Roopa
Lalitha
,
Likith
Muthuraj
,
Gladstone
Sigamani
,
Verena
Oehler
,
Silvia
Synowsky
,
Sally L.
Shirran
,
Tracey M.
Gloster
,
Clarissa M.
Czekster
,
Pravin
Kumar
,
Rafael G.
Da Silva
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we characterize the structure and function of S. aureus TrmK using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the S. aureus TrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for S. aureus TrmK-catalysed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In-silico screening of compounds suggested the multi-functional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of S. aureus TrmK, laying a foundation for future structure-based drug discovery.
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May 2022
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I03-Macromolecular Crystallography
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Nikolaos
Georgakopoulos
,
Sandeep
Talapatra
,
Dina
Dikovskaya
,
Sharadha
Dayalan Naidu
,
Maureen
Higgins
,
Jemma
Gatliff
,
Aysel
Ayhan
,
Roxani
Nikoloudaki
,
Marjolein
Schaap
,
Klara
Valko
,
Farideh
Javid
,
Albena T.
Dinkova-Kostova
,
Frank
Kozielski
,
Geoffrey
Wells
Diamond Proposal Number(s):
[12305]
Open Access
Abstract: Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer’s and Parkinson’s diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein–protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct “peptidomimetic” conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16816, 18582, 19190, 25586, 30588]
Open Access
Abstract: Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the ‘invading’ C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Nicky J.
Willis
,
William
Mahy
,
James
Sipthorp
,
Yuguang
Zhao
,
Hannah L.
Woodward
,
Benjamin N.
Atkinson
,
Elliott D.
Bayle
,
Fredrik
Svensson
,
Sarah
Frew
,
Fiona
Jeganathan
,
Amy
Monaghan
,
Stefano
Benvegnù
,
Sarah
Jolly
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Svend
Kjær
,
Steven
Howell
,
Ambrosius P.
Snijders
,
Magda
Bictash
,
Patricia C.
Salinas
,
Jean-Paul
Vincent
,
E. Yvonne
Jones
,
Paul
Whiting
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 19446]
Open Access
Abstract: Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer’s disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
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May 2022
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