I04-1-Macromolecular Crystallography (fixed wavelength)
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Yeojin
Kim
,
Aleksandar
Lučić
,
Christopher
Lenz
,
Frederic
Farges
,
Martin P.
Schwalm
,
Krishna
Saxena
,
Thomas
Hanke
,
Peter G.
Marples
,
Jasmin C.
Aschenbrenner
,
Daren
Fearon
,
Frank
Von Delft
,
Andreas
Kramer
,
Stefan
Knapp
Diamond Proposal Number(s):
[29658]
Open Access
Abstract: Tripartite motif-containing protein 21 (TRIM21), and particularly its PRY-SPRY protein interaction domain, plays a critical role in the immune response by recognizing intracellular antibodies targeting them for degradation. In this study, we performed a crystallographic fragment screening (CFS) campaign to identify potential small molecule binders targeting the PRY-SPRY domain of TRIM21. Our screen identified a total of 109 fragments binding to TRIM21 that were distributed across five distinct binding sites. These fragments have been designed to facilitate straightforward follow-up chemistry, making them ideal starting points for further chemical optimization. A subsequent fragment merging approach demonstrated improved activity. To enable functional validation of compounds with full length human TRIM21, we established a NanoBRET assay suitable for measuring target engagement to the main Fc binding site in life cells. The high-resolution structural data and observed binding modes across the different sites highlight the versatility of the PRY-SPRY domain as a target for small-molecule intervention. The presented data provide a solid foundation for structure-guided ligand design, enabling the rational design of more potent and selective compounds, with the goal to develop bivalent molecules such as Proteolysis Targeting Chimeras (PROTACs).
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Jun 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 28534]
Open Access
Abstract: The spillover of New World (NW) arenaviruses from rodent reservoirs into human populations poses a continued risk to human health. NW arenaviruses present a glycoprotein (GP) complex on the envelope surface of the virion, which orchestrates host cell entry and is a key target of the immune response arising from infection and immunization. Each protomer of the trimeric GP is composed of a stable signal peptide, a GP1 attachment glycoprotein, and a GP2 fusion glycoprotein. To glean insights into the architecture of this key therapeutic target, we determined the crystal structures of NW GP1−GP2 heterodimeric complexes from Junín virus and Machupo virus. Due to the metastability of the interaction between GP1 and GP2, structural elucidation required the introduction of a disulfide bond at the GP1−GP2 complex interface, but no other stabilizing modifications were required. While the overall assembly of NW GP1−GP2 is conserved with that presented by Old World (OW) arenaviruses, including Lassa virus and lymphocytic choriomeningitis virus, NW GP1−GP2 complexes are structurally distinct. Indeed, we note that when compared to the OW GP1−GP2 complex, the globular portion of NW GP1 undergoes limited structural alterations upon detachment from its cognate GP2. We further demonstrate that our engineered GP1−GP2 heterodimers are antigenically relevant and recognized by neutralizing antibodies. These data provide insights into the distinct assemblies presented by NW and OW arenaviruses, as well as provide molecular-level blueprints that may guide vaccine development.
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Jun 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15292]
Abstract: Bradyrhizobium japonicum USDA110 is a widely used microorganism in the formulation of bioinoculants for soybean crops, harboring a copper-containing nitrite reductase with low enzymatic activity. The activity of BjNirK at pH 6.5 was higher compared to that at pH 8.0, regardless of the presence of either physiological or artificial electron donors. Thermal shift assays reveal that the enzyme is more stable at pH 6.5 than at pH 8.0. X-ray structural data reveals that the funnel for substrate entry shows a wider cavity when compared to other class I NirK structures. Furthermore, the presence of an additional channel for proton provision is observed, in addition to the primary and secondary proton channels. The T2Cu active site can accommodate one or two water molecules, resulting in a tetra- or pentacoordinated metal site, respectively. The structural data correlates well with both optical visible and resonance Raman spectroscopies, denoting a strong blue character of the T1Cu site in both solid and solution states. Furthermore, EPR-monitored redox titration reveals that the catalytic rate is not constrained by T1Cu−T2Cu intraprotein electron transfer reaction at either pH 6.5 or pH 8.0. Additionally, bioinformatics studies indicate that the interaction between the enzyme and the electron donor is not pH dependent. These two observations suggest that the low activity of BjNirK is not caused by inefficient donor−enzyme interaction or impaired electron transfer. The present results suggest that the structural architecture and enzyme properties in rhizobia are designed to ensure low activity, a trait that is particularly advantageous for symbiosis.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Christian
Roth
,
Olga V.
Moroz
,
Suzan A. D.
Miranda
,
Lucas
Jahn
,
Elena V.
Blagova
,
Andrey A.
Lebedev
,
Dorotea R.
Segura
,
Mary A.
Stringer
,
Esben P.
Friis
,
Joao P. L.
Franco Cairo
,
Gideon J.
Davies
,
Keith S.
Wilson
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Endo-galactosaminidases are an underexplored family of enzymes involved in the degradation of galactosaminogalactan (GAG) and other galactosamine-containing cationic exopolysaccharides produced by fungi and bacteria. These exopolysaccharides are part of the cell wall and extracellular matrix of microbial communities. Currently, these galactosaminidases are found in three distinct CAZy families: GH114, GH135 and GH166. Despite the widespread occurrence of these enzymes in nearly all bacterial and fungal clades, only limited biochemical and structural data are available for these three groups. To expand our knowledge of endo-galactosaminidases, we selected several sequences predicted to encode endo-galactosaminidases and produced them recombinantly for structural and functional studies. Only very few predicted proteins could be produced in soluble form, and activity against bacterial Pel (pellicle) polysaccharide could only be confirmed for one enzyme. Here, we report the structures of two bacterial and one fungal enzyme. Whereas the fungal enzyme belongs to family GH114, the two bacterial enzymes do not lie in the current GH families but instead define a new family, GH191. During structure solution we realized that crystals of all three enzymes had various defects including twinning and partial disorder, which in the case of a more severe pathology in one of the structures required the design of a specialized refinement/model-building protocol. Comparison of the structures revealed several features that might be responsible for the described activity pattern and substrate specificity compared with other GAG-degrading enzymes.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: The discovery of new drugs is essential for human well-being, as effective treatments are crucial for combating diseases, addressing emerging health threats, and improving overall quality of life. The continuous development of novel therapeutics is particularly important in response to evolving pathogens, drug resistance, and unmet medical needs. However, drug discovery is a complex and resource-intensive process, given the intricacy of biological systems and the vast chemical space of potential drug candidates. Traditional wet-lab experiments are costly and time-consuming, making the search for novel drugs akin to finding a needle in a haystack.
In this thesis, we investigate how machine learning systems can support early-stage drug discovery in scenarios where experimental data is scarce. First, in a practical study addressing a real-world low-data challenge - namely, the search for potential drug candidates during the COVID-19 pandemic - we apply and evaluate a zero-shot learning method. Furthermore, the thesis introduces a novel few-shot learning architecture, MHNfs, which enhances molecular representations through a memory-based mechanism that we refer to as context enrichment, where contextual information is retrieved from an external memory. The work also connects recent few-shot learning approaches in drug discovery with the concept of in-context learning, originally introduced for large language models. Finally, to support real-world applications, the thesis presents an interactive interface that enables chemists and drug designers to easily provide prompts to and interact with the MHNfs model, facilitating the integration of few-shot learning into their workflows.
Overall, the thesis contributes to the advancement of few-shot learning methods for drug discovery and their practical applications in real-world scenarios.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction which favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to the AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction.
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May 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[30489]
Open Access
Abstract: Crystallographic fragment screening is a powerful methodology that enables the identification of low molecular weight ligands and has shown great promises in drug discovery. In this work we report the results of a fragment screening carried out in an effort to further map the cavities of trypanothione reductase from Trypanosoma brucei (TbTR), a critical target for drug design against human African trypanosomiases (HAT), for which efficient and non-toxic trypanocidal drugs are lacking. Moreover, the conservation of trypanothione reductase among trypanosomatids, including Leishmania, could facilitate the design of a wide-spectrum drug against many parasitic diseases. At the XCHEM facility (Diamond Light Sources, United Kingdom) we performed the soaking of TbTR monoclinic crystals with fragments from DSIpoised and EubOPEN DSIp libraries and we identified eight new hits binding to different cavities of TR including the trypanothione and the NADPH binding cavities. These fragments exhibited affinities ranging from submillimolar to millimolar, as determined by surface plasmon resonance (SPR). While the newly identified fragments did not significantly alter TbTR’s enzymatic activity—consistent with the nature of low-affinity ligands—they provide valuable insights into key interactions of fragments with TR and, together with prior fragment screening campaigns, pave the way towards follow-up chemical optimization into lead compounds.
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May 2025
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B23-Circular Dichroism
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: A Capillary Zone Electrophoresis (CZE) fragment screening methodology was developed and applied to the human plasma protein Transthyretin (TTR), normally soluble, but could misfold and aggregate, causing amyloidosis. Termed Free Probe Peak Height Restoration (FPPHR), it monitors changes in the level of free ligand known to bind TTR (the Probe Ligand) in the presence of competing fragments. 129 fragments were screened, 12 of the 16 initial hits (12.4% hit rate) were co-crystallised with TTR, 11 were found at the binding site (92% confirmation rate). Subsequent analogue screens have identified a novel TTR-binding scaffold 4-(3H-pyrazol-4-yl)quinoline and its derived compounds were further studied by crystallography, circular dichroism (CD), isothermal titration calorimetry (ITC) and radiolabelled 125I-Thyroxine displacement assay in neat plasma. Two lead molecules had similar ITC Kd and 125I-Thyroxine displacement IC50 values to that of Tafamidis, adding another potential pipeline for transthyretin amyloidosis. The methodology is reproducible, procedurally simple, automatable, label-free without target immobilisation, non-fluorescence based and site-specific with low false positive rate, which could be applicable to fragment screening of many drug targets.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Krishna M.
Padmanabha Das
,
Jun
Chen
,
Paul S.
Charifson
,
Jeremy
Green
,
Henry
Tang
,
Sanjay
Panchal
,
Fan
Pu
,
Alla
Korepanova
,
Abhinav
Dubey
,
Gustavo
Afanador
,
Vladimir
Stojkovic
,
Boguslaw
Nocek
,
Lance
Bigelow
,
Sarah H.
Stubbs
,
Robert A.
Davey
,
David A.
Degoey
,
Haribabu
Arthanari
,
Mark N.
Namchuk
Diamond Proposal Number(s):
[32394]
Open Access
Abstract: SARS-CoV-2 Mpro is a cysteine protease, that acts as a symmetrical dimer and displays positive cooperativity for substrate turnover. A series of potent reversible covalent peptidomimetic aldehydes and nitriles was designed as Mpro inhibitors. To better understand the observed structure activity relationships (SAR) binding potency and mechanism was examined by enzyme activity assay, surface plasmon resonance, X-ray crystallography, matrix-assisted laser desorption electrospray ionization and NMR. Potent aldehydes bind Mpro cooperativity but bind covalently to only one subunit of the dimer. The analogous nitriles do not bind cooperatively, and the degree of covalent binding observed varied depending on the assay method employed. The NMR studies support that potent inhibition of Mpro by the nitriles does not require covalent binding. The data highlight the caveats in using orthogonal assays to confirm compound mechanism, particularly in cooperative systems.
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May 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[21773]
Open Access
Abstract: There is an urgent need for new antibiotics. FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target. Very few inhibitors of FabF are known and most are derived from natural products. In an effort to further explore the chemical space of FabF ligands, we have carried out fragment screening by X-ray crystallography against an intermediated state-mimicking variant of P. aeruginosa FabF (PaFabF C164Q). This screen has resulted in 48 hits out of which 16 bind in or close to the malonyl-CoA or fatty acid binding site or an adjacent dimer interface. None of the closer investigated fragments were active in a binding assay, but the same was the case for fragments derived from a potent FabF inhibitor. For hit optimization, we focused on the two fragments binding close to the catalytic residues of FabF. Different strategies were followed in the optimization process: exploration of commercially available analogues, fragment merging, virtual screening of a combinatorial make-on-demand space, and design and in-house synthesis of analogues. In total, more than 90 analogues of the hit compounds were explored, and for 10 of those co-crystal structures could be determined. The most potent ligand was discovered using manual structure-based design and has a binding affinity of 65 μM. This data package forms a strong foundation for the development of more potent and diverse FabF inhibitors.
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Apr 2025
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