I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure–activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
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Dec 2020
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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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I02-Macromolecular Crystallography
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Alfredo
Picado
,
Apirat
Chaikuad
,
Carrow I.
Wells
,
Safal
Shrestha
,
William J.
Zuercher
,
Julie E.
Pickett
,
Frank E.
Kwarcinski
,
Parvathi
Sinha
,
Chandi S.
De Silva
,
Reena
Zutshi
,
Shubin
Liu
,
Natarajan
Kannan
,
Stefan
Knapp
,
David H.
Drewry
,
Timothy M.
Willson
Diamond Proposal Number(s):
[442]
Abstract: STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.
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Nov 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[105433]
Open Access
Abstract: Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
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Nov 2020
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[442]
Abstract: Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, non-canonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK and CLK. Using the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a non-canonical binding mode in CLK1, providing a rational for selectivity over the closely-related CLK3 which harbors a smaller DFG-1 alanine. Our data suggests that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.
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Aug 2020
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I03-Macromolecular Crystallography
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Jonathan
Elie
,
Omid
Feizbakhsh
,
Nathalie
Desban
,
Béatrice
Josselin
,
Blandine
Baratte
,
Amandine
Bescond
,
Julien
Duez
,
Xavier
Fant
,
Stéphane
Bach
,
Dominique
Marie
,
Matthieu
Place
,
Sami
Ben Salah
,
Agnes
Chartier
,
Sabine
Berteina-raboin
,
Apirat
Chaikuad
,
Stefan
Knapp
,
Fabrice
Carles
,
Pascal
Bonnet
,
Frédéric
Buron
,
Sylvain
Routier
,
Sandrine
Ruchaud
Diamond Proposal Number(s):
[10619]
Open Access
Abstract: Haspin is a mitotic protein kinase required for proper cell division by modulating Aurora B kinase localisation and activity as well as histone phosphorylation. Here a series of imidazopyridazines based on the CHR-6494 and Structure Activity Relationship was established. An assessment of the inhibitory activity of the lead structures on human Haspin and several other protein kinases is presented. The lead structure was rapidly optimised using a combination of crystal structures and effective docking models, with the best inhibitors exhibiting potent inhibitory activity on Haspin with IC50 between 6 and 100 nM in vitro. The developed inhibitors displayed anti-proliferative properties against various human cancer cell lines in 2D and spheroid cultures and significantly inhibited the migration ability of osteosarcoma U-2 OS cells. Notably, we show that our lead compounds are powerful Haspin inhibitors in human cells, and did not block G2/M cell cycle transition due to improved selectivity against CDK1/CyclinB.
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Jan 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8421]
Open Access
Abstract: Since hyperactivity of the protein kinase DYRK1A is linked to several neurodegenerative disorders, DYRK1A inhibitors have been suggested as potential therapeutics for Down syndrome and Alzheimer’s disease. Most published inhibitors to date suffer from low selectivity against related kinases or from unfavorable physicochemical properties. In order to identify DYRK1A inhibitors with improved properties, a series of new chemicals based on [b]-annulated halogenated indoles were designed, synthesized, and evaluated for biological activity. Analysis of crystal structures revealed a typical type-I binding mode of the new inhibitor 4-chlorocyclohepta[b]indol-10(5H)-one in DYRK1A, exploiting mainly shape complementarity for tight binding. Conversion of the DYRK1A inhibitor 8-chloro-1,2,3,9-tetrahydro-4H-carbazol-4-one into a corresponding Mannich base hydrochloride improved the aqueous solubility but abrogated kinase inhibitory activity.
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Nov 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Eidarus
Salah
,
Deep
Chatterjee
,
Alessandra
Beltrami
,
Anthony
Tumber
,
Franziska
Preuss
,
Peter
Canning
,
Apirat
Chaikuad
,
Petra
Knaus
,
Stefan
Knapp
,
Alex N.
Bullock
,
Sebastian
Mathea
Diamond Proposal Number(s):
[6391, 10619]
Abstract: LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique 'rock-and-poke' mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.
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Oct 2019
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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
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Jan S.
Kramer
,
Stefano
Woltersdorf
,
Thomas
Duflot
,
Kerstin
Hiesinger
,
Felix F.
Lillich
,
Felix
Knöll
,
Sandra K.
Wittmann
,
Franca M.
Klingler
,
Steffen
Brunst
,
Apirat
Chaikuad
,
Christophe
Morisseau
,
Bruce D.
Hammock
,
Carola
Buccellati
,
Angelo
Sala
,
G. Enrico
Rovati
,
Matthieu
Leuillier
,
Sylvain
Fraineau
,
Julie
Rondeaux
,
Victor
Hernandez Olmos
,
Jan
Heering
,
Daniel
Merk
,
Denys
Pogoryelov
,
Dieter
Steinhilber
,
Stefan
Knapp
,
Jeremy
Bellien
,
Ewgenij
Proschak
Abstract: The emerging pharmacological target soluble epoxide hydrolase (sEH) is a bifunctional enzyme exhibiting two different catalytic activities, which are located in two distinct domains. Although the physiological role of the C-terminal hydrolase domain is well-investigated, little is known about its phosphatase activity located in the N-terminal domain of the sEH (sEH-P). Herein, we report the discovery and optimization of the first inhibitor of human and rat sEH-P, applicable in vivo. X-ray structure analysis of the sEH phosphatase domain complexed with an inhibitor provides insights in the molecular basis of small-molecule sEH-P inhibition and helps to rationalize the structure-activity relationships. 4-(4-(3,4-Dichlorophenyl)-5-phenyloxazol-2-yl)butanoic acid (22b, SWE101) has an excellent pharmacokinetic and pharmacodynamic profile in rats and enables the investigation of the physiological and pathophysiological role of sEH-P in vivo.
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Aug 2019
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