I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan E.
Scott
,
Nicola J.
Francis-newton
,
May E.
Marsh
,
Anthony G.
Coyne
,
Gerhard
Fischer
,
Tommaso
Moschetti
,
Andrew R.
Bayly
,
Timothy D.
Sharpe
,
Kalina T.
Haas
,
Lorraine
Barber
,
Chiara R.
Valenzano
,
Rajavel
Srinivasan
,
David J.
Huggins
,
Miyoung
Lee
,
Amy
Emery
,
Bryn
Hardwick
,
Matthias
Ehebauer
,
Claudio
Dagostin
,
Alessandro
Esposito
,
Luca
Pellegrini
,
Trevor
Perrior
,
Grahame
Mckenzie
,
Tom L.
Blundell
,
Marko
Hyvonen
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Chris
Abell
Diamond Proposal Number(s):
[315, 7141]
Open Access
Abstract: BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
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Mar 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Tessa
Keenan
,
Fabio
Parmeggiani
,
Julien
Malassis
,
Clement Q.
Fontenelle
,
Jean-baptiste
Vendeville
,
Wendy
Offen
,
Peter
Both
,
Kun
Huang
,
Andrea
Marchesi
,
Alex
Heyam
,
Carl
Young
,
Simon J.
Charnock
,
Gideon J.
Davies
,
Bruno
Linclau
,
Sabine L.
Flitsch
,
Martin A.
Fascione
Diamond Proposal Number(s):
[13587, 18598]
Abstract: Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.
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Jul 2020
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I03-Macromolecular Crystallography
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Matteo
Marchesini
,
Andrea
Gherli
,
Anna
Montanaro
,
Laura
Patrizi
,
Claudia
Sorrentino
,
Luca
Pagliaro
,
Chiara
Rompietti
,
Samuel
Kitara
,
Sabine
Heit
,
Claus E.
Olesen
,
Jesper V.
Møller
,
Monia
Savi
,
Leonardo
Bocchi
,
Rocchina
Vilella
,
Federica
Rizzi
,
Marilena
Baglione
,
Giorgia
Rastelli
,
Caterina
Loiacono
,
Roberta
La Starza
,
Cristina
Mecucci
,
Kimberly
Stegmaier
,
Franco
Aversa
,
Donatella
Stilli
,
Anne-marie
Lund Winther
,
Paolo
Sportoletti
,
Maike
Bublitz
,
William
Dalby-brown
,
Giovanni
Roti
Diamond Proposal Number(s):
[18069]
Abstract: The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca2+ toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).
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May 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18566]
Open Access
Abstract: Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.
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Dec 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598, 13587]
Open Access
Abstract: Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.
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Nov 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.
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Nov 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Abstract: Poly-ADP-ribose polymerases (PARPs1-16) play pivotal roles in diverse cellular processes. PARPs that catalyze poly-ADP-ribosylation (PARylation) are the best characterized PARP family members because of the availability of potent and selective inhibitors for these PARPs. There has been comparatively little success in developing selective small-molecule inhibitors of PARPs that catalyze mono-ADP-ribosylation (MARylation), limiting our understanding of the cellular role of MARylation. Here we describe the structure-guided design of inhibitors of PARPs that catalyze MARylation. The most selective analog, ITK7, potently inhibits the MARylation activity of PARP11, a nuclear envelope-localized PARP. ITK7 is greater than 200-fold selective over other PARP family members. Using live-cell imaging, we show that ITK7 causes PARP11 to dissociate from the nuclear envelope. These results suggest that the cellular localization of PARP11 is regulated by its catalytic activity.
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Oct 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Andrew D.
Lawrence
,
Emi
Nemoto-smith
,
Evelyne
Deery
,
Joseph A.
Baker
,
Susanne
Schroeder
,
David G.
Brown
,
Jennifer M. A.
Tullet
,
Mark J.
Howard
,
Ian R.
Brown
,
Alison G.
Smith
,
Helena I.
Boshoff
,
Clifton E.
Barry
,
Martin J.
Warren
Abstract: Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes such as mammals, fish, birds, worms, and Protista, including algae. There is still much to learn about how this nutrient is trafficked across the domains of life. Herein, we describe ways to make a number of different corrin analogs with fluorescent groups attached to the main tetrapyrrole-derived ring. A further range of analogs were also constructed by attaching similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of complete and incomplete corrinoids to follow uptake in bacteria, worms, and plants. By using these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis is able to acquire both cobyric acid and cobalamin analogs, that Caenorhabditis elegans takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium sativum are also able to transport B12.
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May 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Riboswitches are structural elements found in mRNA molecules that couple small-molecule binding to regulation of gene expression, usually by controlling transcription or translation. We have determined high-resolution crystal structures of the ykkC guanidine III riboswitch from Thermobifida fusca. The riboswitch forms a classic H-type pseudoknot that includes a triple helix that is continuous with a central core of conserved nucleotides. These form a left-handed helical ramp of inter-nucleotide interactions, generating the guanidinium cation binding site. The ligand is hydrogen bonded to the Hoogsteen edges of two guanine bases. The binding pocket has a side opening that can accommodate a small side chain, shown by structures with bound methylguanidine, aminoguanidine, ethylguanidine, and agmatine. Comparison of the new structure with those of the guanidine I and II riboswitches reveals that evolution generated three different structural solutions for guanidine binding and subsequent gene regulation, although with some common elements.
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Oct 2017
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I02-Macromolecular Crystallography
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Anežka
Tichá
,
Stancho
Stanchev
,
Kutti R.
Vinothkumar
,
David C.
Mikles
,
Petr
Pachl
,
Jakub
Began
,
Jan
Škerle
,
Kateřina
Švehlová
,
Minh T.n.
Nguyen
,
Steven H.l.
Verhelst
,
Darren C.
Johnson
,
Daniel A.
Bachovchin
,
Martin
Lepšík
,
Pavel
Majer
,
Kvido
Strisovsky
Open Access
Abstract: Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.
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Oct 2017
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