I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Heparanase is the only human enzyme responsible for heparan sulfate (HS) breakdown, an activity that remodels the extracellular matrix (ECM) and strongly drives cancer metastasis and angiogenesis. Compelling evidence implies that heparanase promotes essentially all aspects of the tumorigenic process, namely, tumor initiation, vascularization, growth, metastasis, and chemoresistance. A key mechanism by which heparanase accelerates cancer progression is by enabling the release and bioavailability of HS-bound growth factors, chemokines, and cytokines, residing in the tumor microenvironment and supporting tumor growth and metastasis. The currently available heparanase inhibitors are mostly HS/heparin-like compounds that lack specificity and exert multiple off-target side effects. To date, only four such compounds have progressed to clinical trials, and none have been approved for clinical use. We have generated and characterized an anti-heparanase monoclonal antibody (A54 mAb) that specifically inhibits heparanase enzymatic activity (ECM degradation assay) and cellular uptake. Importantly, A54 mAb attenuates xenograft tumor growth and metastasis (myeloma, glioma, pancreatic, and breast carcinomas) primarily when administered (syngeneic or immunocompromised mice) in combination with conventional anti-cancer drugs. Co-crystallization of the A54 Fab fragment and the heparanase enzyme revealed that the interaction between the two proteins takes place adjacent to the enzyme HS/heparin binding domain II (HBDII; Pro271-Ala276), likely hindering heparanase from interacting with HS substrates via steric occlusion of the active site cleft. Collectively, we have generated and characterized a novel mAb that specifically neutralizes heparanase enzymatic activity and attenuates its pro-tumorigenic effects in preclinical models, paving the way for its clinical examination against cancer, inflammation, and other diseases.
|
Sep 2025
|
|
I04-Macromolecular Crystallography
|
Thomas D.
Downes
,
S. Paul
Jones
,
James D.
Firth
,
John F.
Darby
,
Amelia K.
Gilio
,
Hanna F.
Klein
,
Xinyu
Wang
,
David C
Blakemore
,
Claudia
De Fusco
,
Stephen D.
Roughley
,
Lewis R.
Vidler
,
Maria A.
Whatton
,
Alison Jo-Anne
Woolford
,
Gail L.
Wrigley
,
Roderick E.
Hubbard
,
Gideon
Davies
,
Peter
O'Brien
,
Liang
Wu
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Fragment-based drug discovery is widely used in both academia and industry during the early stages of drug discovery. There is a growing interest in the design of 3-D fragments for inclusion in fragment libraries in order to increase chemical space coverage. We present herein the design and synthesis of 58 shape-diverse 3-D fragments that are prepared using just three modular synthetic methodologies. The 3-D fragments comprise a cyclic scaffold (cyclopentane, pyrrolidine, piperidine, tetrahydrofuran or tetrahydropyran) with one aromatic or heteroaromatic ring and possess properties within 'rule-of-three' fragment space. 3-D shape is assessed using principal moments of inertia analysis and conformational diversity is achieved by considering all conformations up to 1.5 kcal mol -1 above the energy of the global minimum energy conformer. Due to the modular nature of the fragment syntheses, these 3-D fragments are synthetically-enabled for fragment elaboration followon work, a key design feature. This modular, shape-diverse 3-D fragment collection has delivered privileged starting points across a spectrum of targets. Fragments from the set have been crystallographically validated in the SARS-CoV-2 main protease (M pro ) and the nonstructural protein 3 (Nsp3) (Mac1) as well as human glycosyltransferase MGATV, a major enzyme in the mammalian N-glycosylation pathway and a promoter of aggressive metastatic cancers, underscoring the breadth of biological space that can be explored.
|
Sep 2025
|
|
I03-Macromolecular Crystallography
|
Gijs
Ruijgrok
,
Wendy A.
Offen
,
Isabelle B.
Pickles
,
Deepa
Raju
,
Thanasis
Patsos
,
Casper
De Boer
,
Tim
Ofman
,
Joep
Rompa
,
Daan
Van Oord
,
Eleanor J.
Dodson
,
Alexander
Beekers
,
Thijs
Voskuilen
,
Michela
Ferrari
,
Liang
Wu
,
Antonius P. A.
Janssen
,
Jeroen D. C.
Codée
,
P. Lynne
Howell
,
Gideon J.
Davies
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[32736]
Open Access
Abstract: During infection, the human opportunistic pathogen Pseudomonas aeruginosa forms protective biofilms, whose matrix consists of proteins, nucleic acids, and polysaccharides such as alginate, Psl, and Pel. Psl, a polymeric pentasaccharide composed of mannose, rhamnose, and glucose, is produced during the early stages of biofilm formation, serving as a protective barrier against antibiotics and the immune system. The Psl biosynthesis gene cluster, besides encoding various glycosyltransferases, also includes an endoglycosidase, PslG. Here, we show, by activity-based protein profiling, structural studies on enzyme–inhibitor complexes, and defined substrate processing, that PslG is not, as previously suggested, an endo-β-mannosidase but instead a retaining endo-β-glucosidase. This insight allows the design of both competitive and covalent PslG inhibitors, as we show for repeating pentasaccharide mimetics featuring either a reducing end deoxynojirimycin or cyclophellitol moiety. This work provides valuable tools to deepen the understanding of Psl biosynthesis, its function in biofilm formation, and its contribution to antibiotic resistance. We demonstrate the enzyme’s actual endo−β–glucosidase activity, a means to monitor PslG activity in P. aeruginosa biofilms, and a blueprint for inhibitor design.
|
Feb 2025
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[9948, 13587, 24948]
Open Access
Abstract: Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, β-d-glucuronidases, and N-acetyl-glucosaminidases. The mode of inhibition of glucuronidases, in contrast to sialidases, has long been enigmatic as siastatin B appears too bulky and incorrectly substituted to be accommodated within a β-d-glucuronidase active site pocket. Herein, we show through crystallographic analysis of protein-inhibitor complexes that siastatin B generates both a hemiaminal and a 3-geminal diol iminosugar (3-GDI) that are, rather than the parent compound, directly responsible for enzyme inhibition. The hemiaminal product is the first observation of a natural product that belongs to the noeuromycin class of inhibitors. Additionally, the 3-GDI represents a new and potent class of the iminosugar glycosidase inhibitor. To substantiate our findings, we synthesized both the gluco- and galacto-configured 3-GDIs and characterized their binding both structurally and kinetically to exo-β-d-glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-β-d-glucuronidases and an unprecedented binding mode by this new class of inhibitor. Our results reveal the mechanism by which siastatin B acts as a broad-spectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.
|
Dec 2023
|
|
|
|
Open Access
Abstract: An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.
|
Mar 2023
|
|
|
|
Maud
Dumoux
,
Thomas
Glen
,
Jake L. R.
Smith
,
Elaine M. L.
Ho
,
Luis Ma
Perdigão
,
Avery
Pennington
,
Sven
Klumpe
,
Neville B. Y.
Yee
,
David A.
Farmer
,
Pui Y. A.
Lai
,
William
Bowles
,
Ron
Kelley
,
Jürgen M
Plitzko
,
Liang
Wu
,
Mark
Basham
,
Daniel K.
Clare
,
C. Alistair
Siebert
,
Michele C.
Darrow
,
James H.
Naismith
,
Michael
Grange
Open Access
Abstract: Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of sub-cellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in-situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM (cryo-pFIB/SEM). We evaluated the choice of plasma ion source and imaging regimes to produce high quality SEM images of a range of different biological samples. Using an automated workflow we produced three dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20 to 50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in-situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.
|
Feb 2023
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
Casper
De Boer
,
Zachary
Armstrong
,
Vincent A. J.
Lit
,
Uri
Barash
,
Gijs
Ruijgrok
,
Ilanit
Boyango
,
Merle M.
Weitzenberg
,
Sybrin P.
Schröder
,
Alexi J. C.
Sarris
,
Nico J.
Meeuwenoord
,
Pedro
Bule
,
Yasmine
Kayal
,
Neta
Ilan
,
Jeroen D. C.
Codée
,
Israel
Vlodavsky
,
Herman S.
Overkleeft
,
Gideon J.
Davies
,
Liang
Wu
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.
|
Aug 2022
|
|
I04-Macromolecular Crystallography
|
Ken
Kok
,
Chi-Lin
Kuo
,
Rebecca E.
Katzy
,
Lindsey T.
Lelieveld
,
Liang
Wu
,
Véronique
Roig-Zamboni
,
Gijsbert A.
Van Der Marel
,
Jeroen D. C.
Codée
,
Gerlind
Sulzenbacher
,
Gideon J.
Davies
,
Herman S.
Overkleeft
,
Johannes M. F. G.
Aerts
,
Marta
Artola
Open Access
Abstract: α-Glucosidase inhibitors are potential therapeutics for the treatment of diabetes, viral infections, and Pompe disease. Herein, we report a 1,6-epi-cyclophellitol cyclosulfamidate as a new class of reversible α-glucosidase inhibitors that displays enzyme inhibitory activity by virtue of its conformational mimicry of the substrate when bound in the Michaelis complex. The α-d-glc-configured cyclophellitol cyclosulfamidate 4 binds in a competitive manner the human lysosomal acid α-glucosidase (GAA), ER α-glucosidases, and, at higher concentrations, intestinal α-glucosidases, displaying an excellent selectivity over the human β-glucosidases GBA and GBA2 and glucosylceramide synthase (GCS). Cyclosulfamidate 4 stabilizes recombinant human GAA (rhGAA, alglucosidase alfa, Myozyme) in cell medium and plasma and facilitates enzyme trafficking to lysosomes. It stabilizes rhGAA more effectively than existing small-molecule chaperones and does so in vitro, in cellulo, and in vivo in zebrafish, thus representing a promising therapeutic alternative to Miglustat for Pompe disease.
|
Aug 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Gideon J.
Davies
,
Rhianna J.
Rowland
,
Yurong
Chen
,
Imogen
Breen
,
Liang
Wu
,
Wendy A.
Offen
,
Thomas
Beenakker
,
Qin
Su
,
Adrianus M. C. H.
Van Den Nieuwendijk
,
Johannes M. F. G.
Aerts
,
Marta
Artola
,
Herman S.
Overkleeft
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Gaucher disease (GD) is a lysosomal storage disorder caused by inherited deficiencies in β-glucocerebrosidase (GBA). Current treatments require rapid disease diagnosis and a means of monitoring therapeutic efficacy, both of which may be supported by the use of GBA-targeting activity-based probes (ABPs). Here, we report the synthesis and structural analysis of a range of cyclophellitol epoxide and aziridine inhibitors and ABPs for GBA. We demonstrate their covalent mechanism-based mode of action and uncover binding of the new N- functionalised aziridines to the ligand binding cleft. These inhibitors became scaffolds for the development of ABPs; the O6-fluorescent tags of which bind in an allosteric site at the dimer interface. Considering GBA’s preference for O6- and N -functionalised reagents, we synthesised a bi-functional aziridine ABP which we hoped would offer a more powerful imaging agent. Whilst this ABP binds to two unique active site clefts of GBA, no further benefit in potency was achieved over our first generation ABPs. Nevertheless, such ABPs should serve useful in the study of GBA in relation to GD and inform the design of future probes.
|
Sep 2021
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18598]
Abstract: A synthetic heparan sulfate disaccharide has been assessed as a fluorogenic heparanase substrate, enabling enzyme turnover and inhibition kinetics measurements despite slow turnover. Crystal structures with human heparanase also provide the first ever observation of a substrate in an activated 1S3 conformation, highlighting previously unknown interactions involved in enzymatic processing. Our data provide insights into the heparanase catalytic mechanism, and will inform the design of improved heparanase substrates and inhibitors.
|
Oct 2020
|
|
