ORCID Profile
0000-0003-1969-4949
Current Organisation
University of Oxford
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Publisher: Springer Science and Business Media LLC
Date: 10-04-2017
DOI: 10.1038/NG.3835
Publisher: American Chemical Society (ACS)
Date: 05-12-0001
Publisher: Proceedings of the National Academy of Sciences
Date: 06-04-2022
Abstract: The emergence of SARS-CoV-2 triggering the COVID-19 pandemic ranks as arguably the greatest medical emergency of the last century. COVID-19 has highlighted health disparities both within and between countries and will leave a lasting impact on global society. Nonetheless, substantial investment in life sciences over recent decades has facilitated a rapid scientific response with innovations in viral characterization, testing, and sequencing. Perhaps most remarkably, this permitted the development of highly effective vaccines, which are being distributed globally at unprecedented speed. In contrast, drug treatments for the established disease have delivered limited benefits so far. Innovative and rapid approaches in the design and execution of large-scale clinical trials and repurposing of existing drugs have saved many lives however, many more remain at risk. In this review we describe challenges and unmet needs, discuss existing therapeutics, and address future opportunities. Consideration is given to factors that have hindered drug development in order to support planning for the next pandemic challenge and to allow rapid and cost-effective development of new therapeutics with equitable delivery.
Publisher: Oxford University Press (OUP)
Date: 06-10-2015
Publisher: Elsevier BV
Date: 04-2021
Publisher: Springer Science and Business Media LLC
Date: 20-10-2021
DOI: 10.1038/S41467-021-25649-6
Abstract: Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.
Publisher: Public Library of Science (PLoS)
Date: 14-03-2016
Publisher: Elsevier BV
Date: 02-2022
Publisher: eLife Sciences Publications, Ltd
Date: 03-09-2019
DOI: 10.7554/ELIFE.42463
Abstract: Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV ersity IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism.
Publisher: Cold Spring Harbor Laboratory
Date: 27-12-2019
DOI: 10.1101/2019.12.25.888438
Abstract: UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecule’s flexibility. Principal component analysis of the MD trajectories affords a description of UGGT’s overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and cl ing. These inter-domain motions modify the accessible surface area of the enzyme’s central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name “Parodi limit” the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 Å. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a “one-size-fits-all adjustable spanner” substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzyme’s active site.
Publisher: Proceedings of the National Academy of Sciences
Date: 24-07-2017
Abstract: A dedicated endoplasmic reticulum quality control (ERQC) machinery ensures the correct fold of secreted proteins bearing N-linked glycans, which constitute around a fifth of the whole proteome and are essential for many important cellular processes such as signaling, immunity, adhesion, transport, and metabolism. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the sole checkpoint enzyme of ERQC, flagging incorrectly folded glycoproteins for ER retention. Here, we describe crystal structures of full-length UGGT. We show that enzymatic activity depends on interdomain conformational mobility, indicating that the intrinsic flexibility of UGGT may endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates.
Publisher: Proceedings of the National Academy of Sciences
Date: 26-07-2016
Abstract: Most pathogenic enveloped viruses crucially depend on the quality control (QC) machinery in the endoplasmic reticulum (ER) of the host cell. ERQC inhibitors therefore have the double potential benefit of targeting a wide variety of viruses (“broad-spectrum antivirals”) without the risk of losing efficacy due to escape mutations in the viral genome. Our recent work has proven that inhibition of the central enzyme of ERQC, α-glucosidase II (α-GluII), is sufficient for antiviral activity against dengue fever in vitro and in vivo. Here, we show how antiviral inhibitors bind to portions of α-GluII that are unique to this enzyme, and we open the way to the development of potent and selective antivirals against existing and emerging infectious disease.
Publisher: Cold Spring Harbor Laboratory
Date: 07-2020
DOI: 10.1101/2020.06.30.179523
Abstract: Mammalian protein N - linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition and function. N - linked glycans are synthesized from Glc 3 Man 9 GlcNAc 2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo -acting glycoside hydrolase involved in N - glycan trimming and unusually is located within the Golgi, where it allows ER escaped glycoproteins to bypass the classical N - glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur upon substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. The structures inspired the development of new inhibitors that disrupted host protein N - glycan processing of viral glycans and reduced infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts of developing broad-spectrum antiviral agents and bring about a more detailed view of the biology of mammalian glycosylation. The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play a key role in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, and one with great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase, MANEA, provides an alternative processing pathway to evade glucosidase inhibitors. We report the 3D structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Publisher: American Chemical Society (ACS)
Date: 31-03-2020
Publisher: Springer Singapore
Date: 2018
DOI: 10.1007/978-981-10-8727-1_19
Abstract: Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.
Publisher: Elsevier BV
Date: 05-2016
Publisher: Proceedings of the National Academy of Sciences
Date: 05-11-2020
Abstract: The glycosylation of proteins is a major protein modification that occurs extensively in eukaryotes. Glycosidases in the secretory pathway that trim N-linked glycans play key roles in protein quality control and in the specific modifications leading to mature glycoproteins. Inhibition of glucosidases in the secretory pathway is a proven therapeutic strategy, that holds great promise in the treatment of viral disease. The enzyme endo-α-1,2-mannosidase (MANEA) provides an alternative processing pathway to evade glucosidase inhibitors. We report the three-dimensional structure of human MANEA and complexes with enzyme inhibitors that we show act as antivirals for bovine viral diarrhea and human dengue viruses. The structure of MANEA will support inhibitor optimization and the development of more potent antivirals.
Publisher: Cold Spring Harbor Laboratory
Date: 31-05-2023
DOI: 10.1101/2023.05.30.542711
Abstract: Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity (“responsive mutant”). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1 -/- and UGGT1/2 -/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula . The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. Deletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1 -/- single and UGGT1/2 -/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate.
Publisher: Frontiers Media SA
Date: 14-12-2022
DOI: 10.3389/FMOLB.2022.960248
Abstract: None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP–glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals—with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA ( c rystal p o lymorph a nd l igand l ikelihood-based a ssignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple / REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement R free statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.
Publisher: Cold Spring Harbor Laboratory
Date: 30-10-2020
DOI: 10.1101/2020.10.29.339317
Abstract: The COVID-19 pandemic was a stark reminder that a barren global antiviral pipeline has grave humanitarian consequences. Pandemics could be prevented in principle by accessible, easily deployable broad-spectrum oral antivirals. Here we report the results of the COVID Moonshot , a fully open-science, crowd sourced, structure-enabled drug discovery c aign targeting the SARS-CoV-2 main protease. We discovered a novel chemical series that is differentiated from current Mpro inhibitors in that it maintains a new non-covalent, non-peptidic scaffold with nanomolar potency. Our approach leveraged crowdsourcing, high-throughput structural biology, machine learning, and exascale molecular simulations and high-throughput chemistry. In the process, we generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. In a first for a structure-based drug discovery c aign, all compound designs ( ,000 designs), crystallographic data ( ligand-bound X-ray structures), assay data ( ,000 measurements), and synthesized molecules ( ,400 compounds) for this c aign were shared rapidly and openly, creating a rich open and IP-free knowledgebase for future anti-coronavirus drug discovery.
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Nicole Zitzmann.