I03-Macromolecular Crystallography
|
Han Wee
Ong
,
Xuan
Yang
,
Jeffery L.
Smith
,
Sharon
Taft-Benz
,
Stefanie
Howell
,
Rebekah J.
Dickmander
,
Tammy M.
Havener
,
Marcia K.
Sanders
,
Jason W.
Brown
,
Rafael M.
Counago
,
Edcon
Chang
,
Andreas
Kramer
,
Nathaniel J.
Moorman
,
Mark
Heise
,
Alison D.
Axtman
,
David H.
Drewry
,
Timothy M.
Willson
Diamond Proposal Number(s):
[36057]
Open Access
Abstract: The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition.
|
Sep 2025
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[34241, 37661]
Open Access
Abstract: The development of molecules that interact with G-quadruplex (G4) sequences requires effective evaluation methods. Several techniques are currently available, including nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS), fluorescence using FRET-melting, G4-fluorescent intercalator displacement assay (G4-FID) and affinity chromatography. Among these, CD spectroscopy is gaining prominence due to its lower material requirements, faster experimentation and quicker data processing. However, conventional CD methods have limitations, such as higher sample volume required and the inability to handle high-throughput analysis efficiently. The use of synchrotron radiation in high-throughput analysis methods (HT-SRCD) has further advanced the investigation of small-molecule interactions with DNA G4 structures in the presence of various monovalent cations. HT-SRCD offers the capability to analyze multiple samples simultaneously, overcoming the limitations of conventional CD methods. To validate this approach, three biologically relevant G4 sequences—HTelo1, G3T3 and T95-2T—were investigated. Their interactions with a library of small tetrazole-based molecules, synthesized via a four-component Ugi reaction, and with a peptide sequence deriving from RHAU helicases (Rhau25), were evaluated. The results demonstrate that this method not only effectively discriminates between different ligands but also provides valuable insights into the selectivity and the modes of interaction of these ligands with the G4 sequences.
|
Aug 2025
|
|
I15-1-X-ray Pair Distribution Function (XPDF)
|
Diamond Proposal Number(s):
[34748]
Open Access
Abstract: n the quest for greener alternatives to conventional organic solvents, Deep Eutectic Solvents (DESs) have gained significant attention due to their sustainability, biodegradability, and tunability. The use of water as an active and genuine component has recently led to the emergence of water-based DESs (wb-DESs). Here, a careful experimental characterization was performed on choline acetate (ChAc)/water mixtures across a range of water:ChAc molar ratios (n = 2–6). Differential Scanning Calorimetry (DSC) revealed glass transitions between 150 and 180 K, with no first-order transitions, leading to a classification of these mixtures as Low Transition-Temperature Mixtures (LTTMs). Physicochemical measurements, including density, viscosity, electrical conductivity, and refractive index, were conducted over a broad temperature range. NMR analyses provided insights into dynamics and solvation environments, with 1H T1slow relaxation times reaching their lowest value at n = 2, consistent with the formation of a strong hydrogen-bonding network. The n = 2 mixture was further investigated using Small and Wide-Angle X-ray Scattering (S-WAXS) and ab initio molecular dynamics (AIMD). These studies, jointly with 1H NMR choline diffusion coefficient, directly challenge previous claims of the existence of aggregation phenomena in wb-DES. The simulation revealed a well-organized solvation structure, where acetate and water synergistically stabilize the choline cation through a cooperative hydrogen-bonding network. These findings highlight the impact and significance of an integrated physicochemical study in guiding the rational development of new sustainable systems, such as wb-DESs.
|
Aug 2025
|
|
I20-Scanning-X-ray spectroscopy (XAS/XES)
|
Ajeesh Kumar
Somakumar
,
Ivo
Romet
,
Agnieszka
Grabias
,
Marcin
Kruk
,
Shusaku
Hayama
,
Damian
Wlodarczyk
,
Justyna
Barzowska
,
Yadhu Krishnan
Edathumkandy
,
Eduard
Feldbach
,
Puxian
Xiong
,
Yaroslav
Zhydachevskyy
,
Monika
Trzaskowska
,
Hanka
Przybylinska
,
Andrzej
Suchocki
Open Access
Abstract: An extensive experimental study of trivalent iron (Fe3+) ions in orthorhombic lithium gallate nanocrystals was undertaken. Various spectroscopic methods, such as Raman spectroscopy, extended X-ray absorption fine structure, the Mössbauer effect, electron paramagnetic resonance, photoluminescence, thermoluminescence, and cathodoluminescence were used to investigate the synthesized phosphor. This study revealed the existence of multiple Fe3+ sites, out of which the tetrahedral sites are preferentially occupied. Extensive optical studies showed that the Fe3+ doped lithium gallate phosphor is a promising candidate for various luminescence and thermoluminescence-related applications in the near-infrared regime.
|
May 2025
|
|
I03-Macromolecular Crystallography
|
Open Access
Abstract: Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.
|
Jan 2024
|
|
B23-Circular Dichroism
|
Diamond Proposal Number(s):
[29075, 31797]
Open Access
Abstract: Cyanine dyes are known to form H- and J-aggregates in aqueous solutions. Here we show that the cyanine dye, S0271, assembles in water into vortex induced chiral J-aggregates. The chirality of the J-aggregates depends on the directionality of the vortex. This study utilised both conventional benchtop CD spectropolarimeters and Mueller matrix polarimetry. It was found that J-aggregates have real chirality alongside linear dichroism and linear and circular birefringence. We identify the factors that are key to the formation of metastable chiral J-aggregates and propose a mechanism for their assembly.
|
Feb 2023
|
|
|
|
Open Access
Abstract: Perovskite-related materials show very promising properties in many fields. Pb-free perovskites are particularly interesting, because of the toxicity of Pb. In this study, hybrid double perovskite MA2KBiCl6 (MA = methylammonium cation) was found to have interesting variable temperature behaviours. Both variable temperature single crystal X-ray diffraction, synchrotron powder diffraction, and Raman spectroscopy were conducted to reveal a rhombohedral to cubic phase transition at around 330 K and an order to disorder transition for inorganic cage below 210 K.
|
Dec 2022
|
|
I24-Microfocus Macromolecular Crystallography
|
Suzanne
O'Connor
,
Yann-Vai
Le Bihan
,
Isaac M.
Westwood
,
Manjuan
Liu
,
Oi Wei
Mak
,
Gabriel
Zazeri
,
Ana P. R.
Povinelli
,
Alan M.
Jones
,
Rob
Van Montfort
,
Jóhannes
Reynisson
,
Ian
Collins
Diamond Proposal Number(s):
[6385]
Open Access
Abstract: Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.
|
Jan 2022
|
|
I11-High Resolution Powder Diffraction
|
Diamond Proposal Number(s):
[17415]
Open Access
Abstract: Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous medium. On the other hand, a higher loading factor at physiological temperature enhances the formation of an amorphous phase instead of magnetite crystallization. It was confirmed that the iron-overloading effect alone (observed during pathological events) cannot contribute to the formation of magnetite.
|
Nov 2021
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Olivier E.
Nonga
,
Darja
Lavogina
,
Erki
Enkvist
,
Katrin
Kestav
,
Apirat
Chaikuad
,
Sarah E.
Dixon-Clarke
,
Alex N.
Bullock
,
Sergei
Kopanchuk
,
Taavi
Ivan
,
Ramesh
Ekambaram
,
Kaido
Viht
,
Stefan
Knapp
,
Asko
Uri
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
|
Jul 2021
|
|
