I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34035]
Open Access
Abstract: The discovery of tubulysins sparked considerable interest due to their high cytotoxic activity against multidrug resistant tumors. Total synthesis of these complex natural products—peptidic metabolites from myxobacteria Archangium gephyra and Angiococcus disciformis—demonstrated the power of organic chemistry and paved the way for the development of simpler, more stable, and selective derivatives. Despite these significant achievements, tubulysins are not used as stand-alone drugs due to their toxic side effects. In this study, we outline the design, synthesis, and evaluation of photoswitchable tubulysin analogues, which could be starting points for development of photopharmacological therapies aimed at addressing the toxicity challenges associated with tubulysins. The cytotoxic activity of one of the key analogues was shown to be light-controllable. For the first time, we provide a comparative analysis of the crystal structures of both photoisomers of a diarylethene-containing compound complexed with target proteins. This comparison enables a mechanistic explanation for the experimentally observed differences in the target binding efficiency and respective activity between the two photoisomers of the photoswitchable tubulysin analogue.
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Aug 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043, 25402, 33658, 40158]
Open Access
Abstract: Bruton’s Tyrosine Kinase (BTK) is a validated target for hematological malignancies, with numerous FDA-approved inhibitors on the market. Current therapies target the highly conserved ATP binding site and hence limit the therapeutic index given the site’s highly conserved nature across the kinome. We explore a novel approach for BTK inhibition by targeting the PH domain-mediated membrane recruitment and activation of BTK. We have identified a fragment which covalently modifies a lysine in the inositol phosphate (PIP3) binding site and inhibits the binding of a soluble PIP3 headgroup analog to the PH domain. Fragment growth and an extensive structure-binding relationship study uncovered 27 crystal structures and a best-in-class analog, 24. Evaluation of pKa values of the targeted lysine in BTK and other PH domains suggests this as a more general approach to PH domain inhibition.
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May 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Human protein kinase CK2 is a constitutively active serine/threonine kinase implicated in numerous cancers. Although ATP-competitive inhibitors such as CX-4945 show therapeutic potential, they are limited by off-target effects and incomplete or transient CK2 suppression. PROTACs offer an alternative strategy by inducing proteasome-mediated degradation, with potential advantages in potency, selectivity, and duration of action. Herein, a series of CK2-targeting PROTACs has been designed and synthesised. By conjugating a CAM4066-derived warhead to CRBN or VHL ligands, four VHL-recruiting PROTACs, were prepared using PEG and alkyl linkers, alongside two CRBN-recruiting analogues featuring constrained linkers. A ligand–linker analogue in which a linker is projected from the solvent-exposed region of CK2α retained binding affinity comparable to CAM4066, confirming that linker installation is tolerated and preserves key interactions in the αD and ATP sites.
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Apr 2026
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043, 18548, 25402]
Open Access
Abstract: Casein kinase 2α (CK2α) is an oncology drug target that acts as a positive regulator of many tumorigenic signaling pathways. We previously reported that CK2α has a unique cryptic binding site, the αD pocket, that offers the potential for inhibitors with improved kinase selectivity. The prototype bivalent molecule CAM4066 (6) confirmed that improved selectivity could be achieved while binding in both the ATP-binding site and the αD pocket. A drug discovery project to develop a new series of bivalent CK2α inhibitors with increased cell potency and selectivity identified 61f (APL-5125), a highly potent, ATP-competitive CK2α inhibitor with exquisite kinase selectivity and cellular potency. Compound 61f demonstrates in vivo inhibition of p-AKT S129 in tumors (HCT116) following once-daily oral administration and shows a clear PK–PD relationship with unbound drug exposure. 61f has a superior preclinical profile to existing CK2α inhibitors and is currently under evaluation in patients with advanced solid tumors.
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Oct 2025
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I19-Small Molecule Single Crystal Diffraction
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Andrew W.
Heard
,
Luca
Pesce
,
Peter T.
Gierth
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Simone
Adorinni
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Tanya K.
Ronson
,
Barbara
Rossi
,
John D.
Thoburn
,
Tomas
Deingruber
,
Martin
Welch
,
David R.
Spring
,
Silvia
Marchesan
,
Giovanni M.
Pavan
,
Jonathan R.
Nitschke
Diamond Proposal Number(s):
[29890]
Open Access
Abstract: Metal ions with well-defined coordination geometries can serve as fixed joints within self-assembled architectures, defining the relative orientations of ligands within higher-order superstructures. The exchange of ligands and metal ions between different positions is slow, involving disruption or distortion. Here we report a series of AgI12X4L6 3 × 3 metal–organic grid-like structures, where the core AgI12X4 nanocluster is in dynamic motion, with AgI ions moving between different binding sites, with concomitant conformational changes of the organic ligands, which continue to occupy well-defined positions nevertheless. The identity of the incorporated halide anion governs the activation barrier for silver ion exchange, thus enabling rate control in response to two distinct stimuli: by changing the temperature, and by exchanging one halide for another. The dynamic nanocluster within these grids thus provides a new mode of using metal ions in coordination-driven self-assembly, establishing that the mobile AgI ions behave in similar ways to Ag0 atoms in surface-bound clusters and in silver nanoparticles. The kinetic parameters determined in this work, and the techniques developed to measure them, could serve the scientific community to provide additional insight into dynamic metal nanoclusters.
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Aug 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Meng
Wang
,
Prasanthi
Medarametla
,
Thales
Kronenberger
,
Tomas
Deingruber
,
Paul
Brear
,
Wendy
Figueroa
,
Pok-Man
Ho
,
Thomas
Krueger
,
James C.
Pearce
,
Antti
Poso
,
James G.
Wakefield
,
David R.
Spring
,
Martin
Welch
Diamond Proposal Number(s):
[14043]
Open Access
Abstract: Fatty acids are a primary source of carbon for Pseudomonas aeruginosa (PA) in the airways of people with cystic fibrosis (CF). Here, we use tandem mass-tag proteomics to analyse the protein expression profile of a CF clinical isolate grown on different fatty acids. Two fatty acyl-CoA dehydrogenases (designated FadE1 and FadE2) are strongly induced during growth on fatty acids. FadE1 displays a strong preference for long-chain acyl-CoAs, whereas FadE2 exclusively utilizes medium-chain acyl-CoAs. Structural analysis of the enzymes enables us to identify residues comprising the substrate selectivity filter in each. Engineering these residues enables us to invert the substrate specificity of each enzyme. Mutants in fadE1 displayed impaired virulence in an infection model, and decreased growth on long chain fatty acids. The unique features of the substrate binding pocket enable us to identify an inhibitor that is differentially active against FadE1 and FadE2.
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Mar 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Open Access
Abstract: Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with human airway infections. Based on its amino acid sequence similarity with Pyrococcus furiosus protease I, P. aeruginosa PfpI was originally annotated as an intracellular protease. In this work, we show that PfpI is a methylglyoxalase. The X-ray crystal structure of the purified protein was solved to 1.4 Å resolution. The structural data indicated that PfpI shares the same constellation of active site residues (including the catalytic Cys112 and His113) as those seen in a well-characterized bacterial methylglyoxalase from Escherichia coli, YhbO. Using NMR, we confirmed that PfpI qualitatively converted methylglyoxal into lactic acid. Quantitation of lactate produced by the methylglyoxalase activity of PfpI yielded a kcat of 102 min-1 and a KM of 369 μM. Mutation of Cys112 and His113 in PfpI led to complete loss of methylglyoxalase activity. To investigate the functional impact of PfpI in vivo, a ΔpfpI deletion mutant was made. Quantitative proteomic analyses revealed a pattern of changes consistent with perturbation of ribosomal function, Zn2+ limitation, C1 metabolism, and glutathione metabolism. These findings are consistent with PfpI being a glutathione-independent methylglyoxalase. Previously, transposon insertion (pfpI::Tn) mutants have been reported to exhibit phenotypes associated with antibiotic resistance, motility and the response to oxidative stress. However, the ΔpfpI mutant generated in this study displayed none of these phenotypes. Whole-genome sequencing of the previously described pfpI::Tn mutants revealed that they also contain a variety of other genetic changes that likely account for their observed phenotypes.
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Mar 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Open Access
Abstract: The 2-methylcitrate cycle (2-MCC) and the glyoxylate cycle are central metabolic pathways in Pseudomonas aeruginosa, enabling the organism to utilize organic acids such as propionate and acetate during infection. Here, we show that these cycles are linked through enzymatic redundancy, with isocitrate lyase (AceA) exhibiting secondary 2-methylisocitrate lyase (2-MICL) activity. Furthermore, we use a combination of structural analyses, enzyme kinetics, metabolomics, and targeted mutation of PrpBPa to demonstrate that whereas loss of PrpB function impairs growth on propionate, the promiscuous 2-MICL activity of AceA compensates for this by mitigating the accumulation of toxic 2-MCC intermediates. Our findings suggest that simultaneous inhibition of PrpB and AceA could present a robust antimicrobial strategy to target P. aeruginosa in propionate-rich environments, such as the cystic fibrosis airways. Our results emphasize the importance of understanding pathway interconnections in the development of novel antimicrobial agents.
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Feb 2025
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I04-Macromolecular Crystallography
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Rohan
Eapen
,
Cynthia
Okoye
,
Christopher
Stubbs
,
Marianne
Schimpl
,
Thomas
Tischer
,
Eileen
Mccall
,
Maria
Zacharopoulou
,
Fernando
Ferrer
,
David
Barford
,
David
Spring
,
Cath
Lindon
,
Christopher
Phillips
,
Laura S.
Itzhaki
Diamond Proposal Number(s):
[20015]
Open Access
Abstract: E3 ubiquitin ligases engage their substrates via ‘degrons’ - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are hard to drug via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the ‘Destruction box’ (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20-peptide complexes. Using a cellular thermal shift assay we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/CCdc20 and are more potent than the small molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially also for harnessing APC/C for targeted protein degradation.
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Jan 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Simon R.
Stockwell
,
Duncan E.
Scott
,
Gerhard
Fischer
,
Estrella
Guarino
,
Timothy P. C.
Rooney
,
Tzu-Shean
Feng
,
Tommaso
Moschetti
,
Rajavel
Srinivasan
,
Esther
Alza
,
Alice
Asteian
,
Claudio
Dagostin
,
Anna
Alcaide
,
Mathieu
Rocaboy
,
Beata
Blaszczyk
,
Alicia
Higueruelo
,
Xuelu
Wang
,
Maxim
Rossmann
,
Trevor R.
Perrior
,
Tom L.
Blundell
,
David R.
Spring
,
Grahame
Mckenzie
,
Chris
Abell
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Marko
Hyvonen
Diamond Proposal Number(s):
[9537, 14043]
Open Access
Abstract: Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein–protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.
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Aug 2024
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