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
I04-1-Macromolecular Crystallography (fixed wavelength)
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Eleanor
Williams
,
Jana
Bagarova
,
Georgina
Kerr
,
Dong-Dong
Xia
,
Elsie S.
Place
,
Devaveena
Dey
,
Yue
Shen
,
Geoffrey A.
Bocobo
,
Agustin H.
Mohedas
,
Xiuli
Huang
,
Philip E.
Sanderson
,
Arthur
Lee
,
Wei
Zheng
,
Aris N.
Economides
,
James C.
Smith
,
Paul B.
Yu
,
Alex N.
Bullock
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-β signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.
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Mar 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: The immunophilin FKBP12 is a known inhibitor of type I BMP and TGF-β receptors that competes for binding with their substrate SMADs. FKBP12 and the close paralog FKBP12.6 additionally assemble with ryanodine receptors to control Ca2+ release. Binding of FKBP12.6 to BMP/TGF-β receptors has yet to be investigated, but appears plausible given its high sequence similarity to FKBP12. Here, we found that FKBP12.6 can assemble with BMP and TGF-β-family type I receptors, but not with type II receptors. Cellular immunoprecipitation confirmed similar binding of FKBP12 and FKBP12.6 to the BMP receptor ALK2 (ACVR1), a known target of mutations in the congenital syndrome fibrodysplasia ossificans progressiva (FOP), as well as the pediatric brain tumor diffuse intrinsic pontine glioma (DIPG). SEC-MALS analyses using purified proteins indicated a direct 1:1 interaction between FKBP12.6 and the receptor’s cytoplasmic domains. The 2.17 Å structure of this ALK2-FKBP12.6 complex bound to the inhibitor dorsomorphin showed FKBP12.6 binding to the GS domain of ALK2 in a manner equivalent to the FKBP12 complex, with ALK2 residues Phe198 and Leu199 extending into the FK506-binding pocket of FKBP12.6. These findings suggest a level of redundancy in FKBP-family regulation of BMP and TGF-β signaling.
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Jan 2021
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I03-Macromolecular Crystallography
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David
Smil
,
Jong Fu
Wong
,
Eleanor
Williams
,
Roslin J.
Adamson
,
Alison
Howarth
,
David
Mcleod
,
Ahmed
Mamai
,
Soyoung
Kim
,
Brian
Wilson
,
Taira
Kiyota
,
Ahmed
Aman
,
Julie
Owen
,
Gennady
Poda
,
Kurumi
Horiuchi
,
Ekaterina
Kuznetsova
,
Haiching
Ma
,
J. Nicole
Hamblin
,
Sue
Cramp
,
Owen
Roberts
,
Aled M.
Edwards
,
David
Uehling
,
Rima
Al-Awar
,
Alex N.
Bullock
,
Jeff
O'Meara
,
Methvin
Isaac
Open Access
Abstract: There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain penetrant ALK2 inhibitors based on lead compound LDN-214117. Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009, M4K2117, and M4K2163, each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced pre-clinical compounds suitable for further development and evaluation in orthotopic models of DIPG.
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Aug 2020
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I03-Macromolecular Crystallography
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Deeba
Ensan
,
David
Smil
,
Carlos A.
Zepeda-Velázquez
,
Dimitrios
Panagopoulos
,
Jong Fu
Wong
,
Eleanor P.
Williams
,
Roslin
Adamson
,
Alex N.
Bullock
,
Taira
Kiyota
,
Ahmed
Aman
,
Owen G.
Roberts
,
Aled M.
Edwards
,
Jeff A.
O’meara
,
Methvin B.
Isaac
,
Rima
Al-Awar
Diamond Proposal Number(s):
[19301]
Open Access
Abstract: Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009, an analogue of the previously reported ALK2 inhibitor LDN-214117. Although highly selective for ALK2 over the TGF-βR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives (26a–c), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.
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May 2020
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I03-Macromolecular Crystallography
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Jerome
Fortin
,
Ruxiao
Tian
,
Ida
Zarrabi
,
Graham
Hill
,
Eleanor
Williams
,
Gonzalo
Sanchez-Duffhues
,
Midory
Thorikay
,
Parameswaran
Ramachandran
,
Robert
Siddaway
,
Jong Fu
Wong
,
Annette
Wu
,
Lorraine N.
Apuzzo
,
Jillian
Haight
,
Annick
You-Ten
,
Bryan E.
Snow
,
Andrew
Wakeham
,
David J.
Goldhamer
,
Daniel
Schramek
,
Alex N.
Bullock
,
Peter Ten
Dijke
,
Cynthia
Hawkins
,
Tak W.
Mak
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1 G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3b K27M and Pik3ca H1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1 G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.
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Mar 2020
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I04-Macromolecular Crystallography
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Gonzalo
Sánchez-Duffhues
,
Eleanor
Williams
,
Pascal
Benderitter
,
Valeria
Orlova
,
Michiel
Van Wijhe
,
Amaya
Garcia De Vinuesa
,
Georgina
Kerr
,
Josselin
Caradec
,
Kirsten
Lodder
,
Hetty C.
De Boer
,
Marie-José
Goumans
,
Elisabeth M. W.
Eekhoff
,
Antonio
Morales-Piga
,
Javier
Bachiller-Corral
,
Pieter
Koolwijk
,
Alex N.
Bullock
,
Jan
Hoflack
,
Peter
Ten Dijke
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Fibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the Activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to Activins, thereby turning them into bone inducing agents. To date there is no cure for FOP. The further development of FOP patient‐derived models may contribute to discover novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO.
We have characterized peripheral blood‐derived endothelial colony forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo Endothelial‐to‐mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with Activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP.
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Aug 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Structure–activity relationship and crystallographic data revealed that quinazolinone-containing fragments flip between two distinct modes of binding to activin receptor-like kinase-2 (ALK2). We explored both binding modes to discover potent inhibitors and characterized the chemical modifications that triggered the flip in binding mode. We report kinase selectivity and demonstrate that compounds of this series modulate ALK2 in cancer cells. These inhibitors are attractive starting points for the discovery of more advanced ALK2 inhibitors.
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Aug 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Individuals with the rare developmental disorder fibrodysplasia ossificans progressiva (FOP) experience disabling heterotopic ossification caused by a gain of function mutation in the intracellular region of the BMP type I receptor kinase ALK2, encoded by the gene ACVR1. Small molecule BMP type I receptor inhibitors that block this ossification in FOP mouse models have been derived from the pyrazolo[1,5-a]pyrimidine scaffold of dorsomorphin. While the first derivative LDN-193189 exhibited pan inhibition of BMP receptors, the more recent compound LDN-212854 has shown increased selectivity for ALK2. Here we solved the crystal structure of ALK2 in complex with LDN-212854 to define how its binding interactions compare to previously reported BMP and TGFβ receptor inhibitors. LDN-212854 bound to the kinase hinge region as a typical type I ATP-competitive inhibitor with a single hydrogen bond to ALK2 His286. Specificity arising from the 5-quinoline moiety was associated with a distinct pattern of water-mediated hydrogen bonds involving Lys235 and Glu248 in the inactive conformation favoured by ALK2. The structure of this complex provides a template for the design of future ALK2 inhibitors under development for the treatment of FOP and other related conditions of heterotopic ossification.
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Sep 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[442]
Open Access
Abstract: GDF8, or myostatin, is a member of the TGF-β superfamily of secreted polypeptide growth factors. GDF8 is a potent negative regulator of myogenesis both in vivo and in vitro. We found that GDF8 signaling was inhibited by the small molecule ATP competitive inhibitors Dorsomorphin and LDN-193189. These compounds were previously shown to be potent inhibitors of BMP signaling by binding to the type BMP type I receptors ALK1/2/3/6. We present the crystal structure of the type II receptor ActRIIA with dorsomorphin, and demonstrate that dorsomorphin or LDN-193189 target GDF8 induced Smad2/3 signaling and repression of myogenic transcription factors. As a result, both inhibitors rescue myogenesis in myoblasts treated with GDF8. As revealed by quantitative live cell microscopy, treatment with dorsomorphin or LDN-193189 promoted the contractile activity of myotubular networks in vitro. We therefore suggest these inhibitors as suitable tools to promote functional myogenesis.
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Nov 2014
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