I03-Macromolecular Crystallography
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Abstract: The mutation V600E in B-Raf leads to mitogen activated protein kinase (MAPK) pathway activation, uncontrolled cell proliferation, and tumorigenesis. ATP competitive type I B-Raf inhibitors, such as vemurafenib (1) and PLX4720 (4) efficiently block the MAPK pathways in B-Raf mutant cells, however these inhibitors induce conformational changes in the wild type B-Raf (wtB-Raf) kinase domain leading to heterodimerization with C-Raf, causing paradoxical hyperactivation of the MAPK pathway. This unwanted activation may be avoided by another class of inhibitors (type II) which bind the kinase in the DFG-out conformation, such as AZ628 (3) preventing heterodimerization. Here we present a new B-Raf kinase domain inhibitor, based on a phenyl(1H-pyrrolo [2,3-b]pyridin-3-yl)methanone template, that represents a hybrid between 4 and 3. This novel inhibitor borrows the hinge binding region from 4 and the back pocket binding moiety from 3. We determined its binding mode, performed activity/selectivity studies, and molecular dynamics simulations in order to study the conformational effects induced by this inhibitor on wt and V600E mutant B-Raf kinase. We discovered that the inhibitor was active and selective for B-Raf, binds in a DFG-out/αC-helix-in conformation, and did not induce the aforementioned paradoxical hyperactivation in the MAPK pathway. We propose that this merging approach can be used to design a novel class of B-Raf inhibitors for translational studies.
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Feb 2023
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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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I03-Macromolecular Crystallography
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Angelina R.
Sekirnik
,
Jessica K.
Reynolds
,
Larissa
See
,
Joseph P.
Bluck
,
Amy R.
Scorah
,
Cynthia
Tallant
,
Bernadette
Lee
,
Katarzyna B.
Leszczynska
,
Rachel L.
Grimley
,
R. Ian
Storer
,
Marta
Malattia
,
Sara
Crespillo
,
Sofia
Caria
,
Stephanie
Duclos
,
Ester M.
Hammond
,
Stefan
Knapp
,
Garrett M.
Morris
,
Fernanda
Duarte
,
Philip C.
Biggin
,
Stuart J.
Conway
Open Access
Abstract: TRIM33 is a member of the tripartite motif (TRIM) family of proteins, some of which possess E3 ligase activity and are involved in the ubiquitin-dependent degradation of proteins. Four of the TRIM family proteins, TRIM24 (TIF1α), TRIM28 (TIF1β), TRIM33 (TIF1γ) and TRIM66, contain C-terminal plant homeodomain (PHD) and bromodomain (BRD) modules, which bind to methylated lysine (KMen) and acetylated lysine (KAc), respectively. Here we investigate the differences between the two isoforms of TRIM33, TRIM33α and TRIM33β, using structural and biophysical approaches. We show that the N1039 residue, which is equivalent to N140 in BRD4(1) and which is conserved in most BRDs, has a different orientation in each isoform. In TRIM33β, this residue coordinates KAc, but this is not the case in TRIM33α. Despite these differences, both isoforms show similar affinities for H31–27K18Ac, and bind preferentially to H31–27K9Me3K18Ac. We used this information to develop an AlphaScreen assay, with which we have identified four new ligands for the TRIM33 PHD-BRD cassette. These findings provide fundamental new information regarding which histone marks are recognized by both isoforms of TRIM33 and suggest starting points for the development of chemical probes to investigate the cellular function of TRIM33.
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Sep 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Nadilly
Bonagas
,
Nina M. S.
Gustafsson
,
Martin
Henriksson
,
Petra
Marttila
,
Robert
Gustafsson
,
Elisée
Wiita
,
Sanjay
Borhade
,
Alanna C.
Green
,
Karl S. A.
Vallin
,
Antonio
Sarno
,
Richard
Svensson
,
Camilla
Göktürk
,
Therese
Pham
,
Ann-Sofie
Jemth
,
Olga
Loseva
,
Victoria
Cookson
,
Nicole
Kiweler
,
Lars
Sandberg
,
Azita
Rasti
,
Judith E.
Unterlass
,
Martin
Haraldsson
,
Yasmin
Andersson
,
Emma R.
Scaletti
,
Christoffer
Bengtsson
,
Cynthia B. J.
Paulin
,
Kumar
Sanjiv
,
Eldar
Abdurakhmanov
,
Linda
Pudelko
,
Ben
Kunz
,
Matthieu
Desroses
,
Petar
Iliev
,
Katarina
Färnegårdh
,
Andreas
Krämer
,
Neeraj
Garg
,
Maurice
Michel
,
Sara
Häggblad
,
Malin
Jarvius
,
Christina
Kalderén
,
Amanda Bögedahl
Jensen
,
Ingrid
Almlöf
,
Stella
Karsten
,
Si Min
Zhang
,
Maria
Häggblad
,
Anders
Eriksson
,
Jianping
Liu
,
Björn
Glinghammar
,
Natalia
Nekhotiaeva
,
Fredrik
Klingegård
,
Tobias
Koolmeister
,
Ulf
Martens
,
Sabin
Llona-Minguez
,
Ruth
Moulson
,
Helena
Nordström
,
Vendela
Parrow
,
Leif
Dahllund
,
Birger
Sjöberg
,
Irene L.
Vargas
,
Duy Duc
Vo
,
Johan
Wannberg
,
Stefan
Knapp
,
Hans E.
Krokan
,
Per I.
Arvidsson
,
Martin
Scobie
,
Johannes
Meiser
,
Pal
Stenmark
,
Ulrika Warpman
Berglund
,
Evert J.
Homan
,
Thomas
Helleday
Open Access
Abstract: The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.
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Feb 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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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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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Olivier E.
Nonga
,
Darja
Lavogina
,
Erki
Enkvist
,
Katrin
Kestav
,
Apirat
Chaikuad
,
Sarah E.
Dixon-Clarke
,
Alex N.
Bullock
,
Sergei
Kopanchuk
,
Taavi
Ivan
,
Ramesh
Ekambaram
,
Kaido
Viht
,
Stefan
Knapp
,
Asko
Uri
Diamond Proposal Number(s):
[8421]
Open Access
Abstract: We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
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Jul 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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