I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Sarah L.
Kidd
,
Elaine
Fowler
,
Till
Reinhardt
,
Thomas
Compton
,
Natalia
Mateu
,
Hector
Newman
,
Dom
Bellini
,
Romain
Talon
,
Joseph
Mcloughlin
,
Tobias
Krojer
,
Anthony
Aimon
,
Anthony
Bradley
,
Michael
Fairhead
,
Paul
Brear
,
Laura
Diaz-Saez
,
Katherine
Mcauley
,
Hannah F.
Sore
,
Andrew
Madin
,
Daniel H.
O'Donovan
,
Kilian
Huber
,
Marko
Hyvonen
,
Frank
Von Delft
,
Christopher G.
Dowson
,
David R.
Spring
Diamond Proposal Number(s):
[18145, 15649, 14303, 14493]
Open Access
Abstract: Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10–14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.
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May 2020
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I03-Macromolecular Crystallography
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Alexander V.
Strizhak
,
Oleg
Babii
,
Sergii
Afonin
,
Iuliia
Bakanovich
,
Teodors
Pantelejevs
,
Wenshu
Xu
,
Elaine
Fowler
,
Rohan
Eapen
,
Krishna
Sharma
,
Maxim O.
Platonov
,
Vasyl V.
Hurmach
,
Laura
Itzhaki
,
Marko
Hyvonen
,
Anne S.
Ulrich
,
David R.
Spring
,
Igor V.
Komarov
Diamond Proposal Number(s):
[18548]
Open Access
Abstract: Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein–protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the “open” configuration, but up to eight times larger for the corresponding “closed” isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the “closed” isomers is mostly enthalpy-driven, whereas the “open” photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.
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May 2020
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I04-Macromolecular Crystallography
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Krishna
Sharma
,
Alexander V.
Strizhak
,
Elaine
Fowler
,
Wenshu
Xu
,
Ben
Chappell
,
Hannah F.
Sore
,
Warren R. J. D.
Galloway
,
Matthew N.
Grayson
,
Yu Heng
Lau
,
Laura S.
Itzhaki
,
David R.
Spring
Diamond Proposal Number(s):
[14043]
Open Access
Abstract: The Sondheimer dialkyne reagent has previously been employed in strain-promoted double-click cycloadditions with bis-azide peptides to generate stapled peptide inhibitors of protein–protein interactions. The substituted variants of the Sondheimer dialkyne can be used to generate functionalized stapled peptide inhibitors with improved biological properties; however, this remains a relatively underdeveloped field. Herein, we report the synthesis of new substituted variants of Sondheimer dialkyne and their application in the stapling of p53-based diazido peptides to generate potent stapled peptide-based inhibitors of the oncogenic p53-MDM2 interaction. The functionalized stapled peptide formed from a meta-fluoro-substituted Sondheimer dialkyne was found to be the most potent inhibitor. Furthermore, through experimental studies and density functional theory calculations, we investigated the impact of the substituent on the strain-promoted double-click reactivity of Sondheimer dialkyne.
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Jan 2020
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Pooja
Sharma
,
Robert
Mahen
,
Maxim
Rossmann
,
Jamie E.
Stokes
,
Bryn
Hardwick
,
David J.
Huggins
,
Amy
Emery
,
Dominique L.
Kunciw
,
Marko
Hyvonen
,
David R.
Spring
,
Grahame J.
Mckenzie
,
Ashok R.
Venkitaraman
Diamond Proposal Number(s):
[7141, 9537]
Open Access
Abstract: The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.
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Nov 2019
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I04-Macromolecular Crystallography
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Krishna
Sharma
,
Alexander V.
Strizhak
,
Elaine
Fowler
,
Xuelu
Wang
,
Wenshu
Xu
,
Claus
Hatt Jensen
,
Yuteng
Wu
,
Hannah F.
Sore
,
Yu Heng
Lau
,
Marko
Hyvonen
,
Laura S.
Itzhaki
,
David R.
Spring
Diamond Proposal Number(s):
[14043]
Abstract: The Sondheimer dialkyne is extensively used in double strain-promoted azide–alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.
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Aug 2019
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18548]
Open Access
Abstract: The discovery of new Protein–Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, CAM7117, prevents the formation of the holoenzyme assembly in vitro, slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. CAM7117 could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.
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Apr 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9537]
Open Access
Abstract: Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC50 of 44 μM and a molecular weight of only 257 gmol−1 has been identified as the most promising compound. Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.
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May 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9537, 9007]
Open Access
Abstract: CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC50 = 7 μM, GI50 = 10.0 ± 3.6 μM), has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50).
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Feb 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Open Access
Abstract: Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg2+. We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.
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Oct 2017
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I03-Macromolecular Crystallography
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Abstract: Exploitation of synthetic lethality by small-molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND-1 protein hub that links DNA replication, repair and chromosome segregation, represents a novel target for the synthetic lethality approach. Here we report the design, optimization, and validation of double-click stapled peptides encoding the Ctf4-interacting peptide (CIP) of the replicative helicase subunit Sld5. Screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, sub-micromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4-partner DNA polymerase alpha from the replisome in yeast extracts. Our study provides proof-of-principle evidence for the development of small-molecule inhibitors of the human-CTF4 orthologue AND-1.
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Aug 2017
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