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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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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alena
Kroupova
,
Valentina A.
Spiteri
,
Zoe J.
Rutter
,
Hirotake
Furihata
,
Darren
Darren
,
Sarath
Ramachandran
,
Sohini
Chakraborti
,
Kevin
Haubrich
,
Julie
Pethe
,
Denzel
Gonzales
,
Andre J.
Wijaya
,
Maria
Rodriguez-Rios
,
Manon
Sturbaut
,
Dylan M.
Lynch
,
William
Farnaby
,
Mark A.
Nakasone
,
David
Zollman
,
Alessio
Ciulli
Diamond Proposal Number(s):
[26793, 35324, 33832, 38813]
Open Access
Abstract: The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degrader ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.
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Oct 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[31827]
Open Access
Abstract: Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the “ubiquitination zone.” Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.
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Oct 2024
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I24-Microfocus Macromolecular Crystallography
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Johannes
Popow
,
William
Farnaby
,
Andreas
Gollner
,
Christiane
Kofink
,
Gerhard
Fischer
,
Melanie
Wurm
,
David
Zollman
,
Andre
Wijaya
,
Nikolai
Mischerikow
,
Carina
Hasenoehrl
,
Polina
Prokofeva
,
Heribert
Arnhof
,
Silvia
Arce-Solano
,
Sammy
Bell
,
Georg
Boeck
,
Emelyne
Diers
,
Aileen B.
Frost
,
Jake
Goodwin-Tindall
,
Jale
Karolyi-Oezguer
,
Shakil
Khan
,
Theresa
Klawatsch
,
Manfred
Koegl
,
Roland
Kousek
,
Barbara
Kratochvil
,
Katrin
Kropatsch
,
Arnel A.
Lauber
,
Ross
Mclennan
,
Sabine
Olt
,
Daniel
Peter
,
Oliver
Petermann
,
Vanessa
Roessler
,
Peggy
Stolt-Bergner
,
Patrick
Strack
,
Eva
Strauss
,
Nicole
Trainor
,
Vesna
Vetma
,
Claire
Whitworth
,
Siying
Zhong
,
Jens
Quant
,
Harald
Weinstabl
,
Bernhard
Kuster
,
Peter
Ettmayer
,
Alessio
Ciulli
Diamond Proposal Number(s):
[14980]
Abstract: Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.
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Sep 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Proteolysis targeting chimeras (PROTACs) are heterobifunctional small-molecule degraders made of a linker connecting a target-binding moiety to a ubiquitin E3 ligase-binding moiety. The linker unit is known to influence the physicochemical and pharmacokinetic properties of PROTACs, as well as the properties of ternary complexes, in turn impacting on their degradation activity in cells and in vivo. Our LRRK2 PROTAC XL01126, bearing a trans-cyclohexyl group in the linker, is a better and more cooperative degrader than its corresponding cis- analogue despite its much weaker binary binding affinities. Here, we investigate how this subtle stereocenter alteration in the linker affects the ligand binding affinity to the E3 ligase VHL. We designed a series of molecular matched pairs, truncating from the full PROTACs down to the VHL ligand, and find that across the series the trans-cyclohexyl compounds showed consistently weaker VHL-binding affinity compared to the cis- counterparts. High-resolution co-crystal structures revealed that the trans linker exhibits a rigid stick-out conformation, while the cis linker collapses into a folded-back conformation featuring a network of intramolecular contacts and long-range interactions with VHL. These observations are noteworthy as they reveal how a single stereochemical inversion within a PROTAC linker impacts conformational rigidity and binding mode, in turn fine-tuning differentiated propensity to binary and ternary complex formation, and ultimately cellular degradation activity.
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Jun 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sarath
Ramachandran
,
Nikolai
Makukhin
,
Kevin
Haubrich
,
Manjula
Nagala
,
Beth
Forrester
,
Dylan M.
Lynch
,
Ryan
Casement
,
Andrea
Testa
,
Elvira
Bruno
,
Rosaria
Gitto
,
Alessio
Ciulli
Open Access
Abstract: The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.
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Oct 2023
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I04-Macromolecular Crystallography
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Alessio
Ciulli
,
Michael
Gütschow
,
Lan Phuong
Vu
,
Claudia J.
Diehl
,
Ryan
Casement
,
Adam G.
Bond
,
Christian
Steinebach
,
Nika
Strašek
,
Aleša
Bricelj
,
Andrej
Perdih
,
Gregor
Schnakenburg
,
Izidor
Sosič
Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Hypoxia-inducible factor-1α (HIF-1α) constitutes the principal mediator of cellular adaptation to hypoxia in humans. The HIF-1α protein level and activity are tightly regulated by the ubiquitin E3 ligase von Hippel–Lindau (VHL). Here, we performed a structure-guided and bioactivity-driven design of new VHL inhibitors. Our iterative and combinatorial strategy focused on chemical variability at the phenylene unit and encompassed further points of diversity. The exploitation of tailored phenylene fragments and the stereoselective installation of the benzylic methyl group provided potent VHL ligands. Three high-resolution structures of VHL–ligand complexes were determined, and bioactive conformations of these ligands were explored. The most potent inhibitor (30) exhibited dissociation constants lower than 40 nM, independently determined by fluorescence polarization and surface plasmon resonance and an enhanced cellular potency, as evidenced by its superior ability to induce HIF-1α transcriptional activity. Our work is anticipated to inspire future efforts toward HIF-1α stabilizers and new ligands for proteolysis-targeting chimera (PROTAC) degraders.
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Sep 2023
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I24-Microfocus Macromolecular Crystallography
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Alexander
Hanzl
,
Ryan
Casement
,
Hana
Imrichova
,
Scott J.
Hughes
,
Eleonora
Barone
,
Andrea
Testa
,
Sophie
Bauer
,
Jane
Wright
,
Matthias
Brand
,
Alessio
Ciulli
,
Georg E.
Winter
Diamond Proposal Number(s):
[14980]
Abstract: Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined ‘functional hotspots’. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.
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Nov 2022
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I24-Microfocus Macromolecular Crystallography
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Christiane
Kofink
,
Nicole
Trainor
,
Barbara
Mair
,
Simon
Wöhrle
,
Melanie
Wurm
,
Nikolai
Mischerikow
,
Michael J.
Roy
,
Gerd
Bader
,
Peter
Greb
,
Géraldine
Garavel
,
Emelyne
Diers
,
Ross
Mclennan
,
Claire
Whitworth
,
Vesna
Vetma
,
Klaus
Rumpel
,
Maximilian
Scharnweber
,
Julian E.
Fuchs
,
Thomas
Gerstberger
,
Yunhai
Cui
,
Gabriela
Gremel
,
Paolo
Chetta
,
Stefan
Hopf
,
Nicole
Budano
,
Joerg
Rinnenthal
,
Gerhard
Gmaschitz
,
Moriz
Mayer
,
Manfred
Koegl
,
Alessio
Ciulli
,
Harald
Weinstabl
,
William
Farnaby
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.
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Oct 2022
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Susanne
Müller
,
Suzanne
Ackloo
,
Arij
Al Chawaf
,
Bissan
Al-Lazikani
,
Albert
Antolin
,
Jonathan B.
Baell
,
Hartmut
Beck
,
Shaunna
Beedie
,
Ulrich A. K.
Betz
,
Gustavo
Arruda Bezerra
,
Paul E.
Brennan
,
David
Brown
,
Peter J.
Brown
,
Alex N.
Bullock
,
Adrian J.
Carter
,
Apirat
Chaikuad
,
Mathilde
Chaineau
,
Alessio
Ciulli
,
Ian
Collins
,
Jan
Dreher
,
David
Drewry
,
Kristina
Edfeldt
,
Aled M.
Edwards
,
Ursula
Egner
,
Stephen V.
Frye
,
Stephen M.
Fuchs
,
Matthew D.
Hall
,
Ingo V.
Hartung
,
Alexander
Hillisch
,
Stephen H.
Hitchcock
,
Evert
Homan
,
Natarajan
Kannan
,
James R.
Kiefer
,
Stefan
Knapp
,
Milka
Kostic
,
Stefan
Kubicek
,
Andrew S.
Leach
,
Sven
Lindemann
,
Brian D.
Marsden
,
Hisanori
Matsui
,
Jordan L.
Meier
,
Daniel
Merk
,
Maurice
Michel
,
Maxwell R.
Morgan
,
Anke
Mueller-Fahrnow
,
Dafydd R.
Owen
,
Benjamin G.
Perry
,
Saul H.
Rosenberg
,
Kumar Singh
Saikatendu
,
Matthieu
Schapira
,
Cora
Scholten
,
Sujata
Sharma
,
Anton
Simeonov
,
Michael
Sundström
,
Giulio
Superti-Furga
,
Matthew H.
Todd
,
Claudia
Tredup
,
Masoud
Vedadi
,
Frank
Von Delft
,
Timothy M.
Willson
,
Georg E.
Winter
,
Paul
Workman
,
Cheryl H.
Arrowsmith
Open Access
Abstract: Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.
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Dec 2021
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