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
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Zhanna
Romanyuk
,
Giacomo
Bettin
,
Paul
Brear
,
Sara
Linciano
,
Ylenia
Mazzocato
,
Simone
Bonadies
,
Ilaria
Zanotto
,
Camilla
Mazzucco
,
Alan
Monferone
,
Miguel A.
Soler
,
Gianfranco
Pasut
,
Sara De
Martin
,
Alessandro
Scarso
,
Christian
Heinis
,
Sylvia
Rothenberger
,
Marko
Hyvonen
,
Alessandro
Angelini
Diamond Proposal Number(s):
[33658]
Open Access
Abstract: Macrocyclic peptides (MPs) are valuable molecular formats for drug development, bridging small molecules and larger biologics due to their favorable pharmacological properties. Here, we describe the discovery of low-nanomolar inhibitors of human angiotensin-converting enzyme 2 (hACE2) by quantitatively screening millions of structurally diverse disulfide-cyclized peptide ligands using yeast display technology. The most potent yeast-encoded “one-ring” and “two-ring” MP inhibit hACE2 with Ki values of 1.9 and 1.5 nM, respectively. These inhibitory potencies are comparable to those of other cyclic peptides discovered using well-established in vitro display technologies. Crystal structures of the two MPs in complex with hACE2 reveal the adoption of either a rigid β-hairpin or a cysteine-stabilized α-helix/α-helix motif. Both MPs exhibit binding modes distinct from those of previously reported inhibitors. Thus, yeast display is a valid technology to rapidly generate MPs with desired binding properties for the development of potential therapeutics.
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Mar 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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André
Shamsabadi
,
Adam
Creamer
,
Christy J.
Sadler
,
Aida
Abdelwahed
,
Katherine U.
Gaynor
,
Yuliya
Demydchuk
,
Gabriela
Ivanova-Berndt
,
Katerine
Van Rietschoten
,
Paul
Beswick
,
Liuhong
Chen
,
Gustavo
Arruda Bezerra
,
Aleksei
Lulla
,
Paul
Brear
,
Marko
Hyvonen
,
Michael J.
Skynner
,
Molly M.
Stevens
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Constrained bicyclic peptides (Bicycle molecules) with high affinity for biological targets have emerged as potentially powerful therapeutic agents, particularly for the in vivo targeting of cancer receptors. However, their antibody-mimetic properties have yet to be explored for use in diagnostic immunoassays. These synthetically derived compounds serve as biorecognition scaffolds that allow for facile site-selective modification and large-scale production. A phage display screen against various constructs of the SARS-CoV-2 nucleocapsid (N) protein identified several Bicycle molecules with binding affinities ranging from the micromolar to the low nanomolar range. These Bicycle molecules were validated in the development of enzyme- and nanozyme-linked immunosorbent assays, as well as enzymatic and colorimetric nanoparticle-based lateral flow immunoassays (LFIA) for the detection of ultralow concentrations of the SARS-CoV-2 N protein. We envision that these moieties enable robust, cost-effective, and large-scale development of ultrasensitive biosensors for a diverse range of biomarkers by leveraging their high binding affinity, minimalistic scaffold, and synthetic accessibility.
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Feb 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[25402, 33658]
Open Access
Abstract: Environment-sensitive fluorescent probes are indispensable tools for studying biological systems and advancing drug discovery. This study reports the development of 4-sulfamoyl-7-aminobenzoxadiazole (SBD)-based fluorescent probes for the allosteric site of the liver isoform of pyruvate kinase (PKL). By integrating SBD moieties into known activator scaffolds, such as mitapivat and diarylsulfonamide (DASA) ligands, probes for indicator displacement assays were designed to quantify ligand interactions in the allosteric site. Compound 4a displayed dose-dependent fluorescence enhancement in response to PKL binding and was used in a competitive binding assay with unlabelled ligands: mitapivat, TEPP-46, DASA-58 and reported activator 21. Structure–activity relationship (SAR) analysis revealed key structural features influencing activity and fluorescence sensitivity. The probes report selectively on the allosteric site ligands as the binding was not affected by natural ligands, such as ADP, fructose-1,6-bisphosphate (FBP), phosphoenolpyruvate (PEP), and phenylalanine. These findings provide a practical framework for detecting allosteric ligand engagement in PKL and expand the repertoire of molecular tools for advancing PKL-targeted therapies.
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Nov 2025
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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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I04-Macromolecular Crystallography
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Nai-Shu
Hsu
,
Cong
Tang
,
Raquel V.
Mendes
,
Carlos
Labão-Almeida
,
Caio V.
Dos Reis
,
Ana R.
Coelho
,
Marta C.
Marques
,
Mar
Cabeza Cabrerizo
,
Roman
Misteli
,
Timothy P. C.
Rooney
,
Marko
Hyvonen
,
Francisco
Corzana
,
Rita
Fior
,
Gonçalo J. L.
Bernardes
Diamond Proposal Number(s):
[35365]
Open Access
Abstract: Pharmacological activation of STING holds promise in cancer treatment. A recent trend is the development of tumour-specific or conditionally activated STING agonists for enhanced safety and efficacy. Here we explore an unconventional prodrug activation strategy for on-tumour synthesis of a potent agonist. Leveraging the unique mechanism of MSA2, a small-molecule agonist that dimerizes non-covalently before binding to STING, we showed that its analogues bearing reactive functional groups readily and selectively form covalent dimers under mild conditions and in complex environments. We identified a reacting pair that led to a thioether-linked dimer with submicromolar potency in cell-based assays. Caging one of the reactants with a self-immolative β-glucuronide moiety resulted in a two-component prodrug system that near-exclusively formed the active compounds in tumours overexpressing β-glucuronidase. These results exemplify the use of small-molecule recognition for on-site generation of active compounds from benign precursors.
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Sep 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[33658, 40148]
Open Access
Abstract: Pyruvate kinases (PKs) are highly allosterically regulated enzymes that play a central role in cellular metabolism and are increasingly recognized as valuable therapeutic targets in cancer, metabolic diseases, and diabetes. Despite their biological and clinical significance, methods to directly assess allosteric ligand engagement of PK isoforms remain limited. Here, we report the development of LumiPK, a novel, environment-sensitive fluorescent tracer designed to monitor allosteric binding to the liver isoform of pyruvate kinase (PKL). LumiPK integrates an environment-sensitive 4-sulfamonyl-7-aminobenzoxadiazole fluorophore into a potent allosteric modulator scaffold. It emerged as the lead compound from a small ligand series, showing high affinity for PKL (KD = 37 ± 5 nM) in recombinant assays; the most potent fluorescent PK reporter reported to date. A NanoBRET assay using a PKL-Nluc fusion (PKLNluc) enabled intracellular monitoring of unlabeled ligand engagement. LumiPK maintained high potency (EC50 = 18.4 nM) in cellular experiments. Competitive NanoBRET and fluorescence titration assays confirmed binding of known PKL activators (mitapivat, TEPP-46, DASA-58) in both cellular and recombinant settings, with KD values remaining consistent across these methods. LumiPK thus provides a robust tool for probing PKL allosteric modulation and fills a key gap in target engagement technologies for PKL.
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Aug 2025
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I04-Macromolecular Crystallography
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Katrin
Fischer
,
Aleksei
Lulla
,
Tsz Y.
So
,
Pehuén
Pereyra-Gerber
,
Matthew I. J.
Raybould
,
Timo N.
Kohler
,
Juan Carlos
Yam-Puc
,
Tomasz S.
Kaminski
,
Robert
Hughes
,
Gwendolyn L.
Pyeatt
,
Florian
Leiss-Maier
,
Paul
Brear
,
Nicholas J.
Matheson
,
Charlotte M.
Deane
,
Marko
Hyvonen
,
James E. D.
Thaventhiran
,
Florian
Hollfelder
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Monoclonal antibodies are increasingly used to prevent and treat viral infections and are pivotal in pandemic response efforts. Antibody-secreting cells (ASCs; plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methods to study antigen-specific ASCs either have low throughput, require expensive and labor-intensive screening or are technically demanding and therefore not widely accessible. Here we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs. Our approach combines microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. With our technology, we screened millions of mouse and human ASCs and obtained monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 with high affinity (<1 pM) and neutralizing capacity (<100 ng ml−1) in 2 weeks with a high hit rate (>85% of characterized antibodies bound the target). By facilitating access to the underexplored ASC compartment, the approach enables efficient antibody discovery and immunological studies into the generation of protective antibodies.
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Aug 2024
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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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