I03-Macromolecular Crystallography
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Christopher
Lenz
,
Lewis
Elson
,
Johannes
Dopfer
,
Frederic
Farges
,
Andreas
Kramer
,
Frank
Löhr
,
Susanne
Müller
,
Stéphanie M.
Guéret
,
Herbert
Waldmann
,
Volker
Dötsch
,
Krishna
Saxena
,
Stefan
Knapp
Open Access
Abstract: Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein–protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent TE.
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Dec 2025
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I03-Macromolecular Crystallography
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Emily C.
Hank
,
Nicholas D.
D'Arcy-Evans
,
Emma R.
Scaletti
,
Carlos
Benítez-Buelga
,
Olov
Wallner
,
Florian
Ortis
,
Kaixin
Zhou
,
Liuzhen
Meng
,
Alicia
Del Prado
,
Patricia
Calvo
,
Ingrid
Almlöf
,
Elisée
Wiita
,
Karen
Nierlin
,
Sara
Kosenina
,
Andreas
Krämer
,
Alice
Eddershaw
,
Mario
Kehler
,
Maeve
Long
,
Ann-Sofie
Jemth
,
Holly
Dawson
,
Josephine
Stewart
,
Adam
Dickey
,
Mikhael E.
Astorga
,
Marek
Varga
,
Evert J.
Homan
,
Martin
Scobie
,
Stefan
Knapp
,
Leandro
Sastre
,
Pal
Stenmark
,
Miguel
De Vega
,
Thomas
Helleday
,
Maurice
Michel
Diamond Proposal Number(s):
[15806, 21625]
Open Access
Abstract: Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.
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Oct 2025
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I03-Macromolecular Crystallography
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Laura
Hillebrand
,
Guiqun
Wang
,
Alexander
Rasch
,
Benedikt
Masberg
,
Apirat
Chaikuad
,
Thales
Kronenberger
,
Ellen
Günther
,
Michael
Forster
,
Antti
Poso
,
Michael
Lämmerhofer
,
Stefan A.
Laufer
,
Stefan
Knapp
,
Matthias
Gehringer
Diamond Proposal Number(s):
[36057]
Open Access
Abstract: While cysteine targeting in kinases is well established and widely used, covalent interactions with other amino acids remain much less explored. We aimed to develop covalent inhibitors targeting tyrosine residues in the protein kinases JAK3 and MK2 using structure-based design principles to generate small sets of ligands containing tyrosine-reactive sulfonyl fluoride and the less-explored fluorosulfate warheads. While the JAK3 inhibitors failed to achieve covalent binding, the fluorosulfate-bearing MK2 inhibitor 42, which had been designed as an allosteric binder, unexpectedly formed a bond with the “catalytic” lysine, additionally uncovering a unique interaction at the hinge region. This highlights the untapped potential of fluorosulfates and provides a rare example of the use of this electrophile for lysine targeting in kinases. Our results highlight the limitations of traditional design methods and support the integration of fragment/lead-like covalent library screening to discover unanticipated interactions.
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Aug 2025
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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
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Open Access
Abstract: Differential scanning fluorimetry screening of the Library of Pharmacologically Active Compounds (LOPAC) identified four hits for the PRYSPRY domain of the human E3 ligase tripartite motif-containing protein 21 (TRIM21). Isothermal titration calorimetry subsequently confirmed suramin as a binder with micromolar affinity. To further investigate the binding mechanism, mouse TRIM21 was used as a structural surrogate due to its improved protein stability and high sequence similarity to the human counterpart. A crystal structure of the complex refined at 1.3 Å resolution revealed a unique binding mode, providing new avenues for targeting TRIM21 and for the development of proteolysis-targeting chimeras (PROTACs).
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Mar 2025
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Claudia
Tredup
,
Suzanne
Ackloo
,
Hartmut
Beck
,
Peter J.
Brown
,
Alex N.
Bullock
,
Alessio
Ciulli
,
Ivan
Dikic
,
Kristina
Edfeldt
,
Aled M.
Edwards
,
Jonathan M.
Elkins
,
Henner F.
Farin
,
Edward A.
Fon
,
Matthias
Gstaiger
,
Judith
Günther
,
Anna-Lena
Gustavsson
,
Sandra
Häberle
,
Laura
Isigkeit
,
Kilian V. M.
Huber
,
Andras
Kotschy
,
Oliver
Krämer
,
Andrew R.
Leach
,
Brian D.
Marsden
,
Hisanori
Matsui
,
Daniel
Merk
,
Florian
Montel
,
Monique P. C.
Mulder
,
Susanne
Müller
,
Dafydd R.
Owen
,
Ewgenij
Proschak
,
Sandra
Röhm
,
Alexandra
Stolz
,
Michael
Sundström
,
Frank
Von Delft
,
Timothy M.
Willson
,
Cheryl H.
Arrowsmith
,
Stefan
Knapp
Open Access
Abstract: Target 2035 is a global initiative that seeks to identify a pharmacological modulator of most human proteins by the year 2035. As part of an ongoing series of annual updates of this initiative, we summarise here the efforts of the EUbOPEN project whose objectives and results are making a strong contribution to the goals of Target 2035. EUbOPEN is a public–private partnership with four pillars of activity: (1) chemogenomic library collections, (2) chemical probe discovery and technology development for hit-to-lead chemistry, (3) profiling of bioactive compounds in patient-derived disease assays, and (4) collection, storage and dissemination of project-wide data and reagents. The substantial outputs of this programme include a chemogenomic compound library covering one third of the druggable proteome, as well as 100 chemical probes, both profiled in patient derived assays, as well as hundreds of data sets deposited in existing public data repositories and a project-specific data resource for exploring EUbOPEN outputs.
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Nov 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Martin P.
Schwalm
,
Johannes
Dopfer
,
Adarsh
Kumar
,
Francesco A.
Greco
,
Nicolas
Bauer
,
Frank
Löhr
,
Jan
Heering
,
Sara
Cano-Franco
,
Severin
Lechner
,
Thomas
Hanke
,
Ivana
Jaser
,
Viktoria
Morasch
,
Christopher
Lenz
,
Daren
Fearon
,
Peter G.
Marples
,
Charles W. E.
Tomlinson
,
Lorene
Brunello
,
Krishna
Saxena
,
Nathan B. P.
Adams
,
Frank
Von Delft
,
Susanne
Müller
,
Alexandra
Stolz
,
Ewgenij
Proschak
,
Bernhard
Kuster
,
Stefan
Knapp
,
Vladimir V.
Rogov
Diamond Proposal Number(s):
[29658]
Open Access
Abstract: Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development.
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Nov 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[36097, 36057]
Open Access
Abstract: Casein kinase-2 (CK2) are serine/threonine kinases with dual co-factor (ATP and GTP) specificity, that are involved in the regulation of a wide variety of cellular functions. Small molecules targeting CK2 have been described in the literature targeting different binding pockets of the kinase with a focus on type I inhibitors such as the recently published chemical probe SGC-CK2-1. In this study, we investigated whether known allosteric inhibitors binding to a pocket adjacent to helix αD could be combined with ATP mimetic moieties defining a novel class of ATP competitive compounds with a unique binding mode. Linking both binding sites requires a chemical linking moiety that would introduce a 90-degree angle between the ATP mimetic ring system and the αD targeting moiety, which was realized using a sulfonamide. The synthesized inhibitors were highly selective for CK2 with binding constants in the nM range and low micromolar activity. While these inhibitors need to be further improved, the present work provides a structure-based design strategy for highly selective CK2 inhibitors.
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Jul 2024
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I03-Macromolecular Crystallography
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Václav
Němec
,
Marek
Remeš
,
Petr
Beňovský
,
Michael C.
Böck
,
Eliška
Šranková
,
Jong Fu
Wong
,
Julien
Cros
,
Eleanor
Williams
,
Lap Hang
Tse
,
David
Smil
,
Deeba
Ensan
,
Methvin B.
Isaac
,
Rima
Al-Awar
,
Regina
Gomolková
,
Vlad-Constantin
Ursachi
,
Bohumil
Fafílek
,
Zuzana
Kahounová
,
Ráchel
Víchová
,
Ondřej
Vacek
,
Benedict-Tilman
Berger
,
Carrow I.
Wells
,
Cesear R.
Corona
,
James D.
Vasta
,
Matthew B.
Robers
,
Pavel
Krejci
,
Karel
Souček
,
Alex N.
Bullock
,
Stefan
Knapp
,
Kamil
Paruch
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Activin receptor-like kinases 1–7 (ALK1–7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Fernando H.
De Souza Gama
,
Luiz A.
Dutra
,
Michael
Hawgood
,
Caio Vinícius
Dos Reis
,
Ricardo A. M.
Serafim
,
Marcos A.
Ferreira
,
Bruno V. M.
Teodoro
,
Jéssica Emi
Takarada
,
André S.
Santiago
,
Dimitrios-Ilias
Balourdas
,
Ann-Sofie
Nilsson
,
Bruno
Urien
,
Vitor M.
Almeida
,
Carina
Gileadi
,
Priscila Z.
Ramos
,
Anita
Salmazo
,
Stanley N. S.
Vasconcelos
,
Micael R.
Cunha
,
Susanne
Mueller
,
Stefan
Knapp
,
Katlin B.
Massirer
,
Jonathan M.
Elkins
,
Opher
Gileadi
,
Alessandra
Mascarello
,
Bennie B. L. G.
Lemmens
,
Cristiano R. W.
Guimarães
,
Hatylas
Azevedo
,
Rafael M.
Counago
Abstract: Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.
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May 2024
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