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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Thomas
Hanke
,
Sebastian
Mathea
,
Julia
Woortman
,
Eidarus
Salah
,
Benedict-Tilman
Berger
,
Anthony
Tumber
,
Risa
Kashima
,
Akiko
Hata
,
Bernhard
Kuster
,
Susanne
Müller
,
Stefan
Knapp
Abstract: LIMKs are important regulators of actin and microtubule dynamics, and they play essential roles in many cellular processes. Deregulation of LIMKs has been linked to the development of diverse diseases, including cancers and cognitive disabilities, but well-characterized inhibitors known as chemical probes are still lacking. Here, we report the characterization of three highly selective LIMK1/2 inhibitors covering all canonical binding modes (type I/II/III) and the structure-based design of the type II/III inhibitors. Characterization of these chemical probes revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed excellent selectivity in a comprehensive scanMAX kinase selectivity panel. Phosphoproteomics revealed remarkable differences between type I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models of fragile X-chromosome, 15 showed promising activity, suggesting the potential application of allosteric LIMK inhibitors treating this orphan disease.
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Sep 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Sandra
Röhm
,
Benedict-Tilman
Berger
,
Martin
Schröder
,
Deep
Chatterjee
,
Sebastian
Mathea
,
Andreas C.
Joerger
,
Daniel M.
Pinkas
,
Joshua C.
Bufton
,
Amelie
Tjaden
,
Lohitesh
Kovooru
,
Mark
Kudolo
,
Christian
Pohl
,
Alex N.
Bullock
,
Susanne
Müller
,
Stefan
Laufer
,
Stefan
Knapp
Abstract: Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.
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Sep 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Ricardo A. M.
Serafim
,
Fiona J.
Sorrell
,
Benedict-Tilman
Berger
,
Ross J.
Collins
,
Stanley N. S.
Vasconcelos
,
Katlin B.
Massirer
,
Stefan
Knapp
,
James
Bennett
,
Oleg
Fedorov
,
Hitesh
Patel
,
William J.
Zuercher
,
Jonathan M.
Elkins
Diamond Proposal Number(s):
[15433]
Abstract: SLK (STE20-like kinase) and STK10 (serine/threonine kinase 10) are closely related kinases whose enzymatic activity is linked to the regulation of ezrin, radixin, and moesin function and to the regulation of lymphocyte migration and the cell cycle. We identified a series of 3-anilino-4-arylmaleimides as dual inhibitors of SLK and STK10 with good kinome-wide selectivity. Optimization of this series led to multiple SLK/STK10 inhibitors with nanomolar potency. Crystal structures of exemplar inhibitors bound to SLK and STK10 demonstrated the binding mode of the inhibitors and rationalized their selectivity. Cellular target engagement assays demonstrated the binding of the inhibitors to SLK and STK10 in cells. Further selectivity analyses, including analysis of activity of the reported inhibitors against off-targets in cells, identified compound 31 as the most potent and selective inhibitor of SLK and STK10 yet reported.
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Aug 2021
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I04-Macromolecular Crystallography
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Abstract: Covalent kinase inhibitors account for some of the most successful drugs that have recently entered the clinic and many others are in preclinical development. A common strategy is to target cysteines in the vicinity of the ATP binding site using an acrylamide electrophile. To increase the tissue selectivity of kinase inhibitors, it could be advantageous to control the reactivity of these electrophiles with light. Here, we introduce covalent inhibitors of the kinase JNK3 that function as photoswitchable affinity labels (PALs). Our lead compounds contain a diazocine photoswitch, are poor non-covalent inhibitors in the dark, and becomes effective covalent inhibitors after irradiation with visible light. Our proposed mode of action is supported by X-ray structures that explain why these compounds are unreactive in the dark and undergo proximity-based covalent attachment following exposure to light.
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Jun 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.
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Jun 2021
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I03-Macromolecular Crystallography
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Sandra
Röhm
,
Martin
Schroeder
,
Jessica E.
Dwyer
,
Caroline S.
Widdowson
,
Apirat
Chaikuad
,
Benedict-Tilman
Berger
,
Andreas C.
Joerger
,
Andreas
Krämer
,
Jule
Harbig
,
Daniel
Dauch
,
Mark
Kudolo
,
Stefan
Laufer
,
Mark C.
Bagley
,
Stefan
Knapp
Diamond Proposal Number(s):
[10619]
Abstract: The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.
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Dec 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Sandra
Röhm
,
Benedict-Tilman
Berger
,
Martin
Schroeder
,
Apirat
Chaikuad
,
Rob
Winkel
,
Koen F. W.
Hekking
,
Jorg J. C.
Benningshof
,
Gerhard
Müller
,
Roberta
Tesch
,
Mark
Kudolo
,
Michael
Forster
,
Stefan
Laufer
,
Stefan
Knapp
Diamond Proposal Number(s):
[10619]
Abstract: p38 mitogen-activated protein kinases are key mediators of environmental stress response and are promising targets for treatment of inflammatory diseases and cancer. Numerous efforts have led to the discovery of several potent inhibitors; however, so far no highly selective type-II inhibitors have been reported. We previously identified VPC-00628 as a potent and selective type-II inhibitor of p38α/β with few off-targets. Here we analyzed the chemical building blocks of VPC-00628 that played a key role in achieving potency and selectivity through targeting an inactive state of the kinases induced by a unique folded P-loop conformation. Using a rapid, systematic combinatorial synthetic approach, we identified compound 93 (SR-318) with excellent potency and selectivity for p38α/β, which potently inhibited the TNF-α release in whole blood. SR-318 therefore presents a potent and selective type-II inhibitor of p38α/β that can be used as a chemical probe for targeting this particular inactive state of these two p38 isoforms.
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Oct 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Václav
Němec
,
Michaela
Hylsová
,
Lukáš
Maier
,
Jana
Flegel
,
Sonja
Sievers
,
Slava
Ziegler
,
Martin
Schroeder
,
Benedict-Tilman
Berger
,
Apirat
Chaikuad
,
Barbora
Valčíková
,
Stjepan
Uldrijan
,
Stanislav
Drápela
,
Karel
Souček
,
Herbert
Waldmann
,
Stefan
Knapp
,
Kamil
Paruch
Diamond Proposal Number(s):
[10619]
Abstract: Reported is the identification of the furo[3,2‐b]pyridine core as a novel scaffold for potent and highly selective inhibitors of cdc‐like kinases (CLKs) and efficient modulators of the Hedgehog signaling pathway. Initially, a diverse target compound set was prepared by synthetic sequences based on chemoselective metal‐mediated couplings, including assembly of the furo[3,2‐b]pyridine scaffold by copper‐mediated oxidative cyclization. Optimization of the subseries containing 3,5‐disubstituted furo[3,2‐b]pyridines afforded potent, cell‐active, and highly selective inhibitors of CLKs. Profiling of the kinase‐inactive subset of 3,5,7‐trisubstituted furo[3,2‐b]pyridines revealed sub‐micromolar modulators of the Hedgehog pathway.
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Dec 2018
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I03-Macromolecular Crystallography
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Michael
Forster
,
Apirat
Chaikuad
,
Teodor
Dimitrov
,
Eva
Döring
,
Julia
Holstein
,
Benedict-Tilman
Berger
,
Matthias
Gehringer
,
Kamran
Ghoreschi
,
Susanne
Müller
,
Stefan
Knapp
,
Stefan A.
Laufer
Diamond Proposal Number(s):
[10619]
Abstract: Janus kinases are major drivers of immune signaling and have been the focus of anti-inflammatory drug discovery for more than a decade. Because of the invariable co-localization of JAK1 and JAK3 at cytokine receptors, the question if selective JAK3 inhibition is sufficient to effectively block downstream signaling has been highly controversial. Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381 (23) featuring high isoform and kinome selectivity. Crystallography revealed that this inhibitor induces an unprecedented binding pocket by interactions of a nitrile substituent with arginine residues in JAK3. Herein we describe detailed structure activity relationships necessary for induction of the arginine pocket and the impact of this structural change on potency, isoform selectivity and efficacy in cellular models. Furthermore, we evaluated the stability of this novel inhibitor class in in vitro metabolic assays and were able to demonstrate an adequate stability of key compound 23 for in vivo use.
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May 2018
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