Krios II-Titan Krios II at Diamond
Krios IV-Titan Krios IV at Diamond
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Zhuoyao
Chen
,
Gamma
Chi
,
Timea
Balo
,
Xiangrong
Chen
,
Beatriz Ralsi
Montes
,
Steven C.
Clifford
,
Vincenzo
D'Angiolella
,
Timea
Szabo
,
Arpad
Kiss
,
Tibor
Novak
,
András
Herner
,
András
Kotschy
,
Alex N.
Bullock
Diamond Proposal Number(s):
[34631]
Open Access
Abstract: Neomorphic mutations and drugs can elicit unanticipated effects that require mechanistic understanding to inform clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. Here, we identify that these neomorphic interactions are mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 capture 2:1 and 2:2 KBTBD4-HDAC2 complexes, as well as a 2:1:1 KBTBD4-HDAC2-CoREST1 complex, at resolutions spanning 2.7 to 3.3 Å. The mutant and drug-induced complexes adopt similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 is identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshapes the same surface of KBTBD4 providing an example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4, while structure-activity relationship (SAR) analysis of UM171 reveals analog S234984 as a more potent molecular glue for future studies.
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Apr 2025
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Imre
Fejes
,
Piroska
Markacz
,
Janos
Tatai
,
Monika
Rudas
,
Petra
Dunkel
,
Mario
Gyuris
,
Miklos
Nyerges
,
Nicolas
Provost
,
Valérie
Duvivier
,
Philippe
Delerive
,
Virginie
Martiny
,
Alexandra
Bristiel
,
Brice
Vidal
,
William
Richardson
,
Elisabeth M.
Rothweiler
,
Jeppe
Tranberg-Jensen
,
Charlotte E.
Manning
,
Melissa
Sweeney
,
Rod
Chalk
,
Kilian V. M.
Huber
,
Alex N.
Bullock
,
Andras
Herner
,
Klaus
Seedorf
,
Cedric
Vinson
,
Csaba
Weber
,
Andras
Kotschy
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: The NRF2-KEAP1 interaction is central for cytoprotection against stresses, giving it high clinical significance. Covalent modification of KEAP1 is an efficient approach, but the covalent inhibitors used in the clinic carry undesired side effects originating in their moderate selectivity. Starting with a phenotypic screen, we identified a new covalent inhibitor chemotype that was optimized to deliver a series of potent and highly selective KEAP1 binders. While the developed compounds showed both cellular and in vivo activity, upregulating antioxidant response element-dependent target genes, they showed no genotoxicity in vitro. The lead compound exhibited broad selectivity in activity-based protein profiling and showed no significant interaction with a panel of commonly studied receptors nor with a broad panel of kinases. The nature of its interaction with KEAP1 and the origin of its selectivity were revealed by X-ray crystallography.
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Nov 2024
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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-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433, 19301]
Open Access
Abstract: KCTD family proteins typically assemble into cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of human KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes to both its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the GTP cyclohydrolase I feedback regulatory protein (GFRP) despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD1 BTB domain fold and identifies an unexpected ion-binding site for future study of KCTD1’s function in the ectoderm, neural crest, and kidney.
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Aug 2024
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I04-Macromolecular Crystallography
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Frances M.
Bashore
,
Sophia M.
Min
,
Xiangrong
Chen
,
Stefanie
Howell
,
Caroline H.
Rinderle
,
Gabriel
Morel
,
Josie A.
Silvaroli
,
Carrow I.
Wells
,
Bruce A.
Bunnell
,
David H.
Drewry
,
Navjot S.
Pabla
,
Sila K.
Ultanir
,
Alex N.
Bullock
,
Alison D.
Axtman
Open Access
Abstract: Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved cocrystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial–mesenchymal transition.
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Aug 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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I03-Macromolecular Crystallography
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Héctor
González-Álvarez
,
Deeba
Ensan
,
Tao
Xin
,
Jong Fu
Wong
,
Carlos A.
Zepeda-Velázquez
,
Julien
Cros
,
Laurent
Hoffer
,
Taira
Kiyota
,
Brian J.
Wilson
,
Ahmed
Aman
,
Owen
Roberts
,
Methvin B.
Isaac
,
Alex N.
Bullock
,
David
Smil
,
Rima
Al-Awar
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Despite decades of research on new diffuse intrinsic pontine glioma (DIPG) treatments, little or no progress has been made on improving patient outcomes. In this work, we explored novel scaffold modifications of M4K2009, a 3,5-diphenylpyridine ALK2 inhibitor previously reported by our group. Here we disclose the design, synthesis, and evaluation of a first-in-class set of 5- to 7-membered ether-linked and 7-membered amine-linked constrained inhibitors of ALK2. This rigidification strategy led us to the discovery of the ether-linked inhibitors M4K2308 and M4K2281 and the amine-linked inhibitors M4K2304 and M4K2306, each with superior potency against ALK2. Notably, M4K2304 and M4K2306 exhibit exceptional selectivity for ALK2 over ALK5, surpassing the reference compound. Preliminary studies on their in vivo pharmacokinetics, including blood–brain barrier penetration, revealed that these constrained scaffolds have favorable exposure and do open a novel chemical space for further optimization and future evaluation in orthotopic models of DIPG.
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Mar 2024
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I03-Macromolecular Crystallography
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Kerry A.
Miller
,
David A.
Cruz Walma
,
Daniel M.
Pinkas
,
Rebecca S.
Tooze
,
Joshua C.
Bufton
,
William
Richardson
,
Charlotte E.
Manning
,
Alice E.
Hunt
,
Julien
Cros
,
Verity
Hartill
,
Michael J
Parker
,
Simon J.
Mcgowan
,
Stephen R. F.
Twigg
,
Rod
Chalk
,
David
Staunton
,
David
Johnson
,
Andrew O M
Wilkie
,
Alex N.
Bullock
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Introduction: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. Methods: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. Results: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. Conclusion: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.
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Jan 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10619]
Open Access
Abstract: KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function.
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May 2023
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I04-Macromolecular Crystallography
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Han Wee
Ong
,
Yi
Liang
,
William
Richardson
,
Emily R.
Lowry
,
Carrow I.
Wells
,
Xiangrong
Chen
,
Margaux
Silvestre
,
Kelvin
Dempster
,
Josie A.
Silvaroli
,
Jeffery L.
Smith
,
Hynek
Wichterle
,
Navjot S.
Pabla
,
Sila K.
Ultanir
,
Alex N.
Bullock
,
David H.
Drewry
,
Alison D.
Axtman
Diamond Proposal Number(s):
[28172]
Open Access
Abstract: Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/β affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/β signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/β, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/β promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.
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Apr 2023
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