I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Nov 2025
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Oct 2025
|
|
I04-Macromolecular Crystallography
|
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.
|
Sep 2025
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
Aug 2025
|
|
I04-Macromolecular Crystallography
|
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.
|
Aug 2024
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
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.
|
Aug 2024
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18548, 25402]
Open Access
Abstract: Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.
|
Nov 2023
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Maria
Reinecke
,
Paul
Brear
,
Larsen
Vornholz
,
Benedict-Tilmann
Berger
,
Florian
Seefried
,
Stephanie
Wilhelm
,
Patroklos
Samaras
,
Laszlo
Gyenis
,
David William
Litchfield
,
Guillaume
Médard
,
Susanne
Müller
,
Jürgen
Ruland
,
Marko
Hyvonen
,
Mathias
Wilhelm
,
Bernhard
Kuster
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound–target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology.
|
Oct 2023
|
|
I04-Macromolecular Crystallography
|
Maximilian A. J.
Harman
,
Steven J.
Stanway
,
Heather
Scott
,
Yuliya
Demydchuk
,
Gustavo
Arruda Bezerra
,
Simone
Pellegrino
,
Liuhong
Chen
,
Paul
Brear
,
Aleksei
Lulla
,
Marko
Hyvonen
,
Paul J.
Beswick
,
Michael J.
Skynner
Diamond Proposal Number(s):
[25402]
Abstract: Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.
|
Jul 2023
|
|
I04-Macromolecular Crystallography
|
Katherine U.
Gaynor
,
Marina
Vaysburd
,
Maximilian A. J.
Harman
,
Anna
Albecka
,
Phillip
Jeffrey
,
Paul
Beswick
,
Guido
Papa
,
Liuhong
Chen
,
Donna
Mallery
,
Brian
Mcguinness
,
Katerine
Van Rietschoten
,
Steven
Stanway
,
Paul
Brear
,
Aleksei
Lulla
,
Katarzyna
Ciazynska
,
Veronica T.
Chang
,
Jo
Sharp
,
Megan
Neary
,
Helen
Box
,
Jo
Herriott
,
Edyta
Kijak
,
Lee
Tatham
,
Eleanor G.
Bentley
,
Parul
Sharma
,
Adam
Kirby
,
Ximeng
Han
,
James P.
Stewart
,
Andrew
Owen
,
John A. G.
Briggs
,
Marko
Hyvonen
,
Michael J.
Skynner
,
Leo C.
James
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles’ inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.
|
Jun 2023
|
|
