15 14 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 2 / 2 3 Macromolecular Crystallography Group Beamline I04-1 / XChem Fragment screening offers new leads in the search for Alzheimer’s treatments Neurodegenerative Diseases – Non-Communicable Diseases – Health &Wellbeing – Biochemistry – Neurology – Chemistry – Structural Biology – Organic Chemistry – Drug Discovery – Life Sciences & Biotech In the nervous system, Wnt signalling is important for neuronal differentiation, development, and stem cell maintenance. Overall, neuronal Wnt signalling tends to decrease with ageing. At the same time, the Wnt inhibiting molecules, such as Notum, may increase, resulting in a reduction of neurogenesis and contributing to neurodegenerative diseases, such as Alzheimer’s disease. Notum has recently been identified as a negative regulator of Wnt signalling through the removal of an essential palmitoleate group from Wnt proteins, thus deactivating Wnts. Notum inhibitors could make more active Wnt protein available for signalling, thus offering therapeutic benefits for Wnt insufficient pathological states, such as Alzheimer’s disease. However, some previously discovered Notum inhibitors cannot penetrate the blood-brain barrier, so researchers are engaged in crystallographic fragment screening for novel Notum inhibitors. They carried out these experiments at Diamond’s XChem platform. Built around MX beamline I04-1, XChem specialises in screening fragments directly in crystals by X-ray crystallography, using synthesis-aligned fragment libraries. With the sample preparation laboratory lab34 co-located with the beamline, it offers a highly streamlined process allowing up to 1,000 compounds to be screened individually in less than a week, including ~24 hours of unattended beamtime. The process covers soaking, harvesting, automatic data collection, and data analysis, and tailored software and automated systems enable researchers to record and track data seamlessly from initial crystal cultivation to data analysis. Fragment screening has become increasingly popular over the last decade as it promises a step-change in early drug discovery process. It provides valuable and cost-effective insights for rational drug design, enabling scientists to identify high-quality lead candidates in the early stages of discovery. Rather than focusing on a few large complex compounds, it explores a larger part of the existing chemical space from the drug targets of interest, providing new entry routes for developing lead compounds. This research involved soaking over a thousand compounds into Notum crystals and collecting X-ray diffraction data. After analysing 768 datasets, they found 59 compounds that bind to the Notum enzyme pocket with different potencies. Six hits were chosen for further development, and one (1-phenyl- 1,2,3-triazole) has shown promising properties and the capability to penetrate the brain. The lead compound, 1-phenyl-1,2,3-triazole, is now being used to assess when and in which tissues blocking the action of Notum could have beneficial effects by re-balancing the level of Wnt signalling. Related publication: Willis, NJ. et al. Design of a potent, selective, and brain-penetrant inhibitor of Wnt-deactivating enzyme Notum by optimization of a crystallographic fragment hit. Journal of Medicinal Chemistry 65 , 7212-7230 (2022). DOI: 10.1021/acs.jmedchem.2c00162. Funding acknowledgement: Alzheimer’s Research UK (520909) Alzheimer’s Drug Discovery Foundation (ADDF-Harrington Scholar Award to P.V.F.) Cancer Research UK (Programme Grant C375/A17721) Wellcome Centre for Human Genetics, University of Oxford (Centre Grant 203141/Z/16/Z). Corresponding authors: Prof Paul V. Fish, University College London,
[email protected]. Prof E. Yvonne Jones, University of Oxford,
[email protected] The Notum structure and the fragment hits within the enzyme pocket. (A) Notum structure shown as cartoon and fragment hits from complex structures were superimposed alongside the natural substrate (PAM)-bound structure. (B) Close-up view of the enzyme pocket hits. (C–F) The electron density maps for the fragment 1 and its derivatives with their potencies. 1c shows lead like properties with good brain penetrating capability. Macromolecular Crystallography Group Beamline I23 Structural studies of the enhanced binding affinity of therapeutic nucleic acids to proteins Health &Wellbeing – Biochemistry – Chemistry – Structural Biology – Drug Discovery – Life Sciences & Biotech Introducing phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids significantly increases their stability and potency. The phosphorothioate backbone is the most widely used modification in therapeutic nucleic acids, including antisense oligonucleotides (ASOs). This modification involves a replacement of one of the two oxygen atoms in the repeating phosphate groups of the DNA with sulphur. PS-modified nucleic acids show improved properties, such as metabolic stability from nuclease-mediated degradation. One of the hallmarks of PS ASOs is enhanced interactions with cellular proteins. This property, on the one hand, facilitates cellular uptake of nucleic acid drugs and their cell retention. However, on the other hand, it might contribute to the cytotoxic properties of the drug molecule. The molecular mechanisms of interactions between PS nucleic acids and proteins have not been fully established. To better understand how PS ASOs interact with cellular proteins, researchers solved two crystal structures of PS ASO bound to annexin A2 (AnxA2), a calcium-binding protein previously implicated in the release of PS ASOs from endo-lysosomal compartments. The high quality of their sample crystals allowed them to use a unique experimental setup at the I23 beamline to perform long-wavelength X-ray diffraction experiments. These experiments led to precise localisation of the sulphur atoms in the structure and allowed the identification of PS stereoisomers in the DNA bound to the protein. Their results unambiguously confirmed, for thefirst time, that vanderWaals contacts between the sulphur atom and hydrophobic parts of arginine and lysine side chains are the driving force for enhanced interaction of PS ASO with proteins. Interestingly, stereoisomer preference at a given phosphorothioate in the DNA oligonucleotide is determined by the hydrophobic environment around the PS linkage coming not only from the protein but also from adjacent structural features within the ASO such as 5-Methyl groups on cytosine nucleobases. Overall, their results provide valuable insights into the general mechanism of the enhanced binding of PS ASOs to cellular proteins and indicate that the interaction between PS linkages and lysine and arginine residues is a general phenomenon that is observed not only for nucleic acid-binding proteins but may also account for the association of ASO with proteins that are not known to bind DNA. This work provides information that will be instrumental in the rational design of improved nucleic acid-based drugs. Related publication: Hyjek-Składanowska M. et al . Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding. Nucleic Acids Research 51, 1409–1423 (2023). DOI: 10.1093/nar/ gkac774 Funding acknowledgement: Ionis Pharmaceuticals iNEXT-Discovery [H2020 - 871037] Corresponding authors: Marcin Nowotny, International Institute of Molecular and Cell Biology,
[email protected] (A) Crystal structure of AnxA2 in complex with PS ASO. Annexin domains are coloured pink, violet, purple, and indigo. PS backbone is shown in yellow, 2'-MOE nucleotides are shown in green, DNA nucleotides are shown in dark grey. (B) Close-up view on the phosphorothioate-binding surface. Polar interactions are shown as red dotted lines. van der Waals interactions are shown as yellow dotted lines. (C) Difference Fourier anomalous map calculated based on long-wavelength X-ray diffraction data (λ = 2.7552 Å), shown as teal mesh. The preferred occupancy of Rp PS stereoisomer is facilitated by the hydrophobic interactions with surrounding amino acids.