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A meeting organized by
Laurent MEIJER, Conrad KUNICK & Yann HERAULT
March 28 – April 1st, 2017
Saint Malo, Bretagne, France
Information: www.dyrk-conference.com
Conference coordinator: Pauline DE LAFFOREST
dyrk-conference@manros-therapeutics.com
DYRK1A, related kinases and
human disease
“DYRK1A, related kinases & human disease” 1
“DYRK1A, related kinases & human disease”
Saint Malo, France
March 28 – April 1st, 2017
Phosphorylation by protein kinases is the most universally used mechanism by cells to
control their structural proteins and enzymes. All major physiological phenomena are regulated by
phosphorylation and many diseases are associated with abnormal phosphorylation. Therefore, the
last four decades have seen considerable efforts in the study of functions and regulations of
kinases in essentially all aspects of biology. In parallel, the search for pharmacological inhibitors of
kinases has become a major area of research in the pharmaceutical industry for the discovery and
development of new therapies.
DYRK1A (‘dual specificity, tyrosine phosphorylation regulated kinase 1A’) phosphorylates
many substrates involved in signaling pathways. It plays key roles in mRNA splicing, gene
transcription, cell survival, differentiation, endocytosis, neuronal development and functions.
Abnormalities in DYRK1A dosage are associated with cognitive disorders observed in Down
syndrome, Mental Retardation Disease 7 (MRD7) and Alzheimer’s disease. DYRK1A plays key
functions in pancreatic cells, cancer, inflammation, megakaryoblastic leukemia. Close orthologs are
found in plants, yeast, algae and unicellular parasites.
The conference will focus on DYRK1A, DYRK1B, 2, 3, 4 and the closely related CLKs (‘cdc2-
like kinases). Presentations will cover all structural (crystal structures), regulatory and functional
aspects of these kinases, their substrate specificity, functions at the cell and organism levels, the
development of potent and selective pharmacological inhibitors and their potential therapeutic use
as treatment of various human diseases.
Welcome to Saint Malo! We hope you will enjoy the conference program,
the interactions between participants, the local food, the scenery and
… the (unpredictable) weather!
Laurent MEIJER, Conrad KUNICK & Yann HERAULT
& Pauline DE LAFFOREST
“DYRK1A, related kinases & human disease” 2
PROGRAM
TUESDAY MARCH 28 – afternoon & evening
17.00 Opening of Registration
Le Palais du Grand Large
1 quai Duguay-Trouin
35 400 SAINT MALO
19.00 Welcome address
Le Palais du Grand Large
Salle Rotonde
19.10 Welcoming drinks
WEDNESDAY MARCH 29 – morning
8.00 – 9:00 Registration
SESSION 1. DYRK1A & Down syndrome Chair: Jean-Maurice DELABAR
9.00 – 9.05 WELCOME!
9.05 – 9.30 Yann HERAULT
Challenging the role of DYRK1A in Down syndrome by studying the genotype and phenotype
relationships in animal models
9.30 – 9.55 Michel J. ROUX
Retinal phenotypes in murine models of Down syndrome
9.55 – 10.20 Sungchan CHO
A chemical with proven clinical safety rescues Down-syndrome-related phenotypes through DYRK1A
inhibition
10.20 – 10.45 Akiko KOBAYASHI
Repression of DYRK1A restores impaired cortical development and abnormal learning behavior of
Down syndrome mice
10.45 – 11.05 COFFEE AND TEA BREAK
11.05 – 11.30 Thu Lan NGUYEN
Deciphering the molecular mechanisms of action of pharmacological DYRK1A kinase inhibitors for
therapeutic use in Down Syndrome preclinical models
11.30 – 11.55 Cécile CIEUTA-WALTI
Preclinical study of safety and biomarkers of efficacy of Epigallocatechin-3-gallate at three different
doses in Wild-type and transgenic DYRK1A mice
“DYRK1A, related kinases & human disease” 3
SESSION 2. DYRK1A & CBS Chair: Mariona L. ARBONES
11.55 – 12.20 Jean-Louis GUEANT
The influence of the metabolism of monocarbons on epigenomic mechanisms: the interacting role of
the remethylation and transsulfuration pathways
12.20 – 12.45 Gaëlle FRIOCOURT
Identification of cystathione -synthase pharmacological inhibitors to alleviate the intellectual deficiency
of patients with Down syndrome
12.45 – 12.50 Alice LEON
Identification of a new function of Cystathionine -Synthase (CBS) and characterization of its link with
DYRK1A using S. cerevisiae
12.50 - 14.30 LUNCH
WEDNESDAY MARCH 29 – afternoon
SESSION 3. DYRK1A & Alzheimer’s disease Chair: Yann HERAULT
14.30 – 14.55 Jérôme BRAUDEAU
APP
GSK3 and DYRK1A levels during human-like AD progression
14.55 – 15.20 Benoît SOUCHET
Preventing DYRK1A catabolism in reactive astrocytes as a novel therapeutic approach to treat
15.20 – 15.45 Jean-Maurice DELABAR
DYRK1A, biomarker or
SESSION 4. DYRK1A & Neurogenesis Chair: Susana DE LA LUNA
15.45 – 16.10 Mariona L. ARBONES
Control of neuron numbers by DYRK1A: lessons from mouse models
16.10 - 16.30 COFFEE AND TEA BREAK
16.30 – 16.55 Nobuhiro KURABAYASHI
DYRK1A overexpression contributes to enhanced astrogliogenesis in a Down syndrome mouse model
16.55 – 17.20 Aline DUBOS
Role of Dyrk1a in GABAergic neurons during development
SESSION 5. DYRK1A mutations, dosage & disease Chair: Masatoshi HAGIWARA
17.20 – 17.45 Bregje WM VAN BON
De novo mutations of DYRK1A lead to a syndromic form of ID
“DYRK1A, related kinases & human disease” 4
17.45 – 18.10 Amélie PITON
DYRK1A haploinsuficiency is a frequent cause of intellectual disability: How to better diagnose it?
18.10 – 18.35 Véronique BRAULT
Dyrk1a gene dosage in glutamatergic neurons has a key effect in cognitive deficits observed in mouse
models of MRD7 and Down syndrome
THURSDAY MARCH 30 – morning
SESSION 6. DYRK & CLK inhibitors Chair: Stefan KNAPP
9.00 – 9.25 Masatoshi HAGIWARA
Development of inhibitors of CDK9, CLK1, DYRK1a, and their clinical application
9.25 – 9.50 Thierry BESSON
New DYRK1A inhibitors
9.50 – 10.15 Franz BRACHER
Novel DYRK1 inhibitors derived from -carboline alkaloids
10.15 – 10.40 Robin KETTELER
Novel Scaffold Kinase Inhibitors of DYRK/CLK Family Members
10.40 - 11.00 COFFEE AND TEA BREAK
11.00 – 11.25 Stefan KNAPP
Rational design strategies for improving selectivity of inhibitors targeting splicing regulating kinases
11.25 – 11.50 Conrad KUNICK
DYRK and CLK inhibitors: Is selectivity feasible?
11.50 – 12.15 Rosanna MEINE
2-Substituted indole-3-carbonitriles as new DYRK inhibitors
12.15 – 12.40 Laurent MEIJER
Leucettines, a family of DYRK1A inhibitors: from marine sponge to drug candidate
12.40 -14.30 LUNCH
THURSDAY MARCH 30 – afternoon
14.30 – 14.55 Jonathan C. MORRIS
Developing inhibitors of the kinases that regulate alternative splicing
14.55 – 15.20 Scott HENDERSON
Making the most of public domain data with Knime® : renovating GSK3 and CDK2 scaffolds
15.20 – 15.30 Marie-Louise JUNG
Smart screening libraries and successful medicinal chemistry: kinase inhibitors search
“DYRK1A, related kinases & human disease” 5
SESSION 7. DYRK1A & Diabetes Chair: Conrad KUNICK
15.30 – 15.55 Bridget K. WAGNER
Inhibition of DYRK1A in the pancreatic beta cell
15.55 – 16.20 Robert J. DE VITA
DYRK1A as a small molecule target for human β-cell proliferation for the treatment of diabetes
16.20 – 16.40 COFFEE AND TEA BREAK
SESSION 8. DYRK1B Chair: Simon COOK
16.40 – 17.05 Walter BECKER
Impaired maturation of DYRK1B mutants that are associated with a form of the metabolic syndrome
17.05 – 17.30 Maria GAITANOU
Mirk/Dyrk1B kinase is a novel dual function molecule inducing cell cycle exit and neuronal
differentiation in the embryonic chick spinal cord
17.30 – 17.55 Edward P. GELMANN
Blocking DYRK1B Phosphorylation of NKX3.1 Prostate Tumor Suppressor Inhibits Prostate Growth and
Increases Apoptosis A Potential Therapeutic Strategy.
17.55 – 18.20 Rachael HUNTLY
Analysis of DYRK signalling by phospho-SILAC mass spectrometry
FRIDAY MARCH 31 – morning
SESSION 9. DYRKs, DNA repair and cancer Chair: Marc BLONDEL
9.15 – 9.25 Martin MEHNERT
Multilayered proteomic analysis of cancer mutations in the Dyrk2 kinase complex
9.25 – 9.50 Kiyotsugu YOSHIDA
Tumor suppressive function of DYRK2
9.50 – 10.15 Julia ROEWENSTRUNK
Interaction of the protein kinase DYRK1A with RNF169 suggests a role for this kinase in DNA repair
10.15 – 10.40 Larisa LITOVCHICK
The role of DYRK1A in DNA repair
10.40 – 11.00 COFFEE AND TEA BREAK
11.00 – 11.25 Athena F. PHOA
Drug-target engagement and efficacy of DYRK1A inhibitors in glioblastoma cells
“DYRK1A, related kinases & human disease” 6
11.25 – 11.50 Rahul BHANSALI
The multi-faceted regulatory role of DYRK1A in normal and malignant lymphopoiesis
11.50 – 12.15 Susana DE LA LUNA
The DYRK1A kinase positively regulates angiogenic responses in endothelial cells
12.15 – 12.40 Rajeev SINGH
DYRK1B and Hedgehog signaling: a complex crosstalk
12.50 - 14.30 LUNCH
FRIDAY MARCH 31 – afternoon
SESSION 10. DYRK1A & T cell regulation Chair: Laurent MEIJER
14.30 – 14.55 Bernard KHOR
From kinome to DYRK1A in T cell regulation and inflammation
SESSION 11. DYRKs substrates & regulators Chair: Walter BECKER
14.55 – 15.20 Kassandra M. ORI-MCKENNEY
Multimodal regulation of the microtubule cytoskeleton by DYRK1a
15.20 – 15.45 Reinhard W. KOSTER
Genetic modeling of neurodegenerative diseases in zebrafish for bioimaging studies and compound
evaluation
15.45 – 16.10 Maribel LARA-CHICA
Identification of new DYRK2 substrates and their implications in carcinogenesis
16.10 – 16.30 COFFEE AND TEA BREAK
16.30 – 16.55 Simon COOK
Analysis of DYRK signalling by RNAseq profiling
16.55 – 17.20 Yoshihiko MIYATA
Functional regulation of different DYRK family protein kinases by distinctive cellular binding partners
17.20 – 17.45 Chiara DI VONA
DYRK1A as a gene-specific RNA polymerase II CTD kinase
17.45 – 18.10 Despina SMIRLIS
Leishmania infantum DYRK1: a negative regulator of the G1 to S cell-cycle transition, essential for the
development of infective stationary phase promastigotes
SESSION 12. General conclusions Chairs: L. MEIJER, C. KUNICK & Y. HERAULT
“DYRK1A, related kinases & human disease” 7
SATURDAY APRIL 1st - morning
Saturday morning we are meeting with parents of children with DYRK1A mutations (we had a large
number of requests, it was rather unexpected, but we thought we had to do something!). This session
will be in French. Anyone is welcome, just indicate to Pauline that you would like to join this extra
session.
SESSION 13. Session grand public: Présentation de la protéine DYRK1A, ses fonctions, ses
régulateurs et son implication dans diverses pathologies humaines : retour sur les résultats
présentés lors du congrès Chair: Amélie PITON
10.00 – 10.30 Yann HERAULT & Laurent MEIJER
Résumé de la conférence, points forts des avancées sur la connaissance de DYRK1A
10.30 – 11.00 PAUSE CAFÉ & THÉ
11.00 – 11.30 Amélie PITON, Marie VINCENT, Marjolaine WILLEMS
Le syndrome MRD7
11.30 – 11.45 Yann HERAULT
Le modèle souris pour mieux comprendre le syndrome MRD7
11.45 – 12.30
Paroles aux familles, discussion
12.30 LUNCH
“DYRK1A, related kinases & human disease” 8
ABSTRACTS
(by alphabetical order of speaker)
Control of neuron numbers by DYRK1A: lessons from mouse models
Sònia NAJAS, María JOSE BARALLORE, Mariona L. ARBONES
Instituto de Biología Molecular de Barcelona (IBMB-CSIC), c/ Baldiri i Reixac 15, 08028 Barcelona, Spain
Neurons in the mammalian brain are generated prenatally from a heterogeneous population of
progenitors that divide producing more progenitors (expansion divisions) or producing neurons
(differentiative divisions). Neurons are usually generated in excess and a fraction of them die during
development by physiological apoptosis. Therefore, alterations in the neurogenic potential of embryonic
neural stem cells or in the activity of apoptotic cell death pathways may have a significant impact in the
number of the different neuron types that integrate into functional circuits. Studies in mouse models
carrying 1 or 3 functional copies of Dyrk1a have shown that DYRK1A controls brain size and neuron
numbers in a dosage-dependent and region-specific manner (1). In both haploinsufficient Dyrk1a+/-
embryos and transgenic embryos carrying 3 copies of mouse Dyrk1a (TgBACDyrk1a), neurogenesis is
preserved in regions of the ventral mesencephalon where dopaminergic neurons involved in the control
of voluntary movement and regulation of emotion are generated. However, at postnatal stages the
number of these neurons were decreased in Dyrk1a+/- mice and increased in TgBACDyrk1a mice due to
a dysregulation of Caspase 9-mediated cell death pathway (2). In contrast, neuron counts in the
postnatal neocortex of Dyrk1a mutant mice, the region involved in higher-order brain functions,
inversely correlate with DYRK1A protein levels. Examination of this structure indicated that
neurogenesis is increased in the Dyrk1a+/- model and reduced in the TgBACDyrk1a model, and that
variations in the division mode (proliferative vs. differentiative divisions) of the stem cells (radial glial
progenitors) that give rise to cortical excitatory neurons contribute to the neurogenic defects observed
in these two models. Reduced neurogenesis in TgBACDyrk1a embryos correlates with a longer cell
cycle G1 phase and decreased nuclear levels of the cell cycle activator Cyclin D1 in radial glial
progenitors. These defects are consistent with the ability of DYRK1A to phosphorylate T286 in Cyclin
D1, which promotes its nuclear export and subsequent degradation via the ubiquitin-proteasome
pathway (3). During the talk, I will present new data showing the effect of DYRK1A overexpression in
the production of cortical inhibitory neurons and discuss the pathogenic effects of DYRK1A gene-
dosage variations in neocortical development.
(1) Guedj et al., 2012. Neurobiol. Dis. 46, 190-203.
(2) Barallobre et al., 2014. Cell Death Dis. 5, e1289.
(3) Najas et al., 2015. EBioMedicine 2, 120-134.
Financial support: this work was supported by the Spanish Ministry of Economy, Innovation and Competitiveness (MINECO),
the Spanish network on Rare Diseases (CIBERER) and the Jérôme Lejeune Foundation.
“DYRK1A, related kinases & human disease” 9
Impaired maturation of DYRK1B mutants that are associated with a form of the
metabolic syndrome
Samira ABU JHAISHA, Esti W. WIDOWATI, Stefan KNAPP1, Walter BECKER2
1Institute for Pharmaceutical Chemistry and Buchmann Institute for Molecular Life Sciences (BMLS), Johann Wolfgang Goethe
University, Frankfurt am Main, Germany. 2Institute of Pharmacology and Toxicology, RWTH Aachen University, Aachen,
Germany
DYRK family protein kinases depend on the autophosphorylation of a conserved tyrosine
residue in the catalytic domain to acquire full catalytic activity (1). In addition to the sequence similarity
of their kinase domains, all DYRK kinases share a structural element named DYRK homology (DH) box
in the N-terminal domain (2). Recently, two missense mutations affecting the DH box of DYRK1B have
been found to co-segregate with a rare autosomal-dominant form of the metabolic syndrome (3). The
present study aims to elucidate the consequences of these amino acid substitutions (H90P and R102C)
on the molecular function of DYRK1B.
In vitro kinase assays showed that the mutations did not alter the specific activity of mature
kinase molecules. However, cell culture experiments showed that a significant part of the mutant
DYRK1B protein accumulated in detergent-insoluble cytoplasmic aggregates and was
underphosphorylated on tyrosine. The mutant DYRK1B variants were more vulnerable to the HSP90
inhibitor ganetespib and showed enhanced binding to the co-chaperone CDC37 as compared to wild
type DYRK1B. These results support the hypothesis that the mutations in the DH box interfere with the
maturation of DYRK1B by tyrosine autophosphorylation and compromise the conformational stability of
the catalytic domain, which renders the kinase susceptible to misfolding and aggregation.
(1) Becker W & Sippl W, 2011. Activation, regulation, and inhibition of DYRK1A. FEBS J. 278, 246-56.
(2) Becker W & Joost HG, 1999. Structural and functional characteristics of Dyrk, a novel subfamily of
protein kinases with dual specificity. Progr. Nucleic Acids Res. Mol. Biol. 62, 1-17.
(3) Keramati AR et al., 2014. A form of the metabolic syndrome associated with mutations in DYRK1B.
N. Engl. J. Med. 370, 1909-1919.
“DYRK1A, related kinases & human disease” 10
New DYRK1A inhibitors
Thierry BESSON
Normandie University, UNIROUEN, INSA Rouen, CNRS, COBRA UMR 6014, 76000 Rouen, France.
Our research group is mainly invested in the synthesis of C,N,S- or C,N,O-containing
heterocyclic precursors of bioactive molecules able to modulate the activity of kinases in signal
transduction, and especially Ser/Thr kinases (CDK5, GSK3, CLK1 and CK1) and dual-specificity
kinases (DYRK family). DYRK1A is certainly the most studied and is a novel, high-potential therapeutic
target for pharmacological interventions seeking to modify the course of Alzheimer disease. In our
continuous effort aiming at preparing novel heterocyclic scaffolds able to modulate the activity of
kinases synthetic routes to functionalized thiazolo[5,4-f]quinazolines were particularly studied The
chemical highlight of this work was the use of a special reagent (4,5-dichloro-1,2,3-dithiazolium
chloride) and a 6-amino-2-cyanobenzo[d] thiazole-7-carbonitrile derivatives as a versatile molecular
platform. Introduction of various aliphatic, aromatic or amino substituents and transformation of
carbonitrile group into various chemical functions (e.g. imidate, ester, amidine...) allowed the efficient
preparation of a library of novel thiazoloquinazoline derivatives. The most active compounds are five
methyl carbimidate derivatives exhibiting significant and selective inhibition against DYRK1A and
DYRK1B (subnanomolar range).
Figures. (Left) Chemical structures of methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidates 1-5 and
DYRK1A and DYRK1B IC50 (nM) values. (Right) Modelling of 1 and 2 with DYRK1A/1B, calculated
from structural modelling and docking studies.
The crystal structures of the complex revealed a non-canonical binding mode of compound 1
and 2 in DYRK2 explaining the remarkable selectivity and potency of these inhibitors. The structural
data and comparison presented here provide therefore a template for further improvement of this
inhibitor class and for the development of novel inhibitors selectively targeting DYRK kinases.
(1) Leblond,B. et al., 2013. WO 2013026806. Chem. Abstr. 158, 390018.
(2) Foucourt A., et al., 2014. Molecules 19, 15546.
(3) Foucourt A., et al., 2014. Molecules 19, 15411.
(4) Thompson, B., et al., 2015. J. Exp. Med. 212, 723.
(5) Courtadeur, S., et al., 2015. J. Neurochem. 133, 440.
(6) Besson, T. et al., 2016. J. Med. Chem. 59, 10315.
Financial support : f
acknowledged. We also thank the LABEX SynOrg (ANR-11-LABX-0029) for financial support.
“DYRK1A, related kinases & human disease” 11
The multi-faceted regulatory role of DYRK1A in normal and malignant
lymphopoiesis
Rahul BHANSALI1, Paul LEE2, Malini RAMMOHAN1, John CRISPINO1
1Division of Hematology/Oncology Northwestern University, Chicago, IL, USA; 2Division of Pediatric Hematology/Oncology,
Chicago, IL, USA
The chromosome 21 gene DYRK1A encodes a versatile kinase implicated in various roles
integral to cellular function. Our group found a tumorigenic role for DYRK1A in Down syndrome-Acute
Megakaryoblastic Leukemia (1), and we subsequently studied its role in normal hematopoiesis using a
conditional knockout mouse model. Ultimately, we uncovered that DYRK1A is required for normal
lymphoid, but not myeloid, development through its phosphorylation and destabilization of cyclinD3
leading to quiescence and cellular maturation. While loss of cell cycle exit primarily stems from
stabilization of cyclinD3, these cells do not proliferate uncontrollably; rather, they show premature
exhaustion (2). This finding prompted our proteomics-based investigation into the various roles of
DYRK1A in lymphopoiesis and its possible involvement in the pathogenesis of Acute Lymphoblastic
Leukemia (ALL).
In order to elucidate which pathways were affected by DYRK1A inhibition, we treated primary
murine pre-B cells with a DYRK1-specific inhibitor, followed by tandem mass tagging and analysis by
mass spectrometry. This generated several thousand peptides that were differentially phosphorylated
between the treated and untreated samples. With an inclusion criterion of 1.5-fold decrease in
phosphorylation upon DYRK1A inhibition, we refined our list to 36 proteins, including cyclin D3, which
are strong candidates for DYRK1A substrates or members of proximally involved pathways. Using
bioinformatics analysis of GO Biological Processes, we found that these proteins showed enrichment of
pathways vital for cell development such as cell cycle, cell division and mitosis, RNA metabolism, and
JAK-STAT signaling. Further investigation of these 36 proteins has provided an excellent opportunity to
develop a more profound understanding of several lymphopoietic pathways that may involve DYRK1A
and how these relate to the development of ALL when dysregulated.
Clinically, these results, in combination with data showing that DYRK1A expression is increased
in ALL relative to other tumor types, implicate DYRK1A as a potential target in ALL. But this study also
provides an interesting perspective in cancer biology because this versatile kinase may have both
oncogene and tumor suppressor qualities in the same cell type, elaborating upon the traditional models
of carcinogenesis.
(1) Malinge, S., et al., 2012. Increased dosage of the chromosome 21 ortholog Dyrk1a promotes
megakaryoblastic leukemia in a murine model of Down syndrome. J. Clin. Invest. 122, 948-962.
(2) Thompson, B.J., et al., 2015. DYRK1A controls the transition from proliferation to quiescence during
lymphoid development by destabilizing Cyclin D3. J. Exp. Med. 212, 953-970.
Financial support: we would like to thank the Rally Foundation for Childhood Cancer Research and the American Society of
Hematology for their generous support of this project.
“DYRK1A, related kinases & human disease” 12
Novel DYRK1 inhibitors derived from β-carboline alkaloids
Franz BRACHER
Department of Pharmacy Center for Drug Research, Ludwig-Maximilians University, Butenandtstr. 5-13, 81377 Munich,
Germany
Natural products are a rich source of lead structures for drug development. In the last decade
we performed extensive investigations on the potential of -carboline alkaloids and synthetic analogues
thereof as lead structures for the development of selective kinase inhibitors. Accompanied by
comprehensive research on novel synthetic approaches towards the -carboline scaffold, we
developed new and selective kinase inhibitors derived from the alkaloids bauerine C (an 1-oxo--
carboline from a blue-green alga), annomontine (a aminopyrimidyl--carboline alkaloid from tropical
plants), and the plant alkaloid harmine.
Highly selective inhibitors of DYRK1 were obtained by systematic structure variation of harmine,
and the most advanced new inhibitors, e.g. AnnH75, were free from undesired MAO A inhibitory
activity. Concise structure-activity relationships could be elaborated (in cooperation with Prof. Dr.
Walter Becker, Aachen), and a co-crystal structure (PDB: 4YU2; provided by the group of Prof. Dr.
Stefan Knapp, Oxford) gave further insight into the binding mode.
(1) K. Rüben, A. Wurzlbauer, A. Walte, W. Sippl, F. Bracher, W. Becker, 2015. Selectivity profiling and
biological activity of novel -carbolines as potent and selective DYRK1 kinase inhibitors. PLOS One
10, e0132453.
(2) A. Walte, K. Rüben, R. Birner-Grünberger, C. Preisinger, S. Bamberg-Lemper, N. Hilz, F. Bracher,
W. Becker, 2013. Mechanism of dual specificity kinase activity of DYRK1A. FEBS J. 280, 4495-
4511.
(3) O. Fedorov, K. Huber, A. Eisenreich, P. Filippakopoulos, O. King, A. N. Bullock, D. Fabro, J.
Trappe, U. Rauch, F. Bracher, S. Knapp, 2011. Specific CLK inhibitors from a novel chemotype for
regulation of alternative splicing. Chem. & Biol. 18, 67.
“DYRK1A, related kinases & human disease” 13
APPβ processing initiates full Tau pathology in a novel age dependent
Alzheimer’s disease rat model: GSK3β and DYRK1A levels during human-like AD
progression
Mickael AUDRAIN, Benoit SOUCHET, Sandro ALVES, Romain FOL, Alexis HADDJERI, Satoru TADA,
Charlène JOSEPHINE, Alexis-Pierre BEMELMANS, Nicole DEGLON, Philippe HANTRAYE, Yvette
AKWA, Jean-Marie BILLARD, Brigitte POTIER, Patrick DUTAR, Nathalie CARTIER, Jérôme
BRAUDEAU1
1AgenT, CEA FAR, 18 route du panorama, 92265 Fontenay aux Roses, France
progressive accumulation of -amyloid peptide (A), a gradual Tau hyperphosphorylation, and displays
a decline in cognitive functions followed by senile plaques and tangles formation. Despite billions of
dollars invested in R&D to find an effective treatment, AD clinical trials still have one of the highest
failure rate of any disease area over 99% compared with 81% for cancer (1). This high failures rate
could be attributed in part to current animal models, which do not fully recapitulate the human AD
course. Especially, the link between APP processing and Tau pathology remains challenging in
transgenic animals.
Indeed, growing evidences suggests that APP processing and soluble amyloid- (A) release
are upstream of Tau pathology. However, the lack of animal models mimicking these both cerebral
pathologies as observed in human AD raises questions regarding both amyloid cascade hypothesis
validity and underlying mechanism.
In order to decipher relationship between amyloid and tau pathologies, we developed the first
inducible and progressive AD rat model, named AAV-AD rat. Here, we induced rat model thanks AAV
gene transfer of mutated form of human APP and PS1. This modeling strategy, already described in
mouse (2), led to produce amyloid derivatives while avoiding significant transgene overexpression.
Soluble A42 levels and the A42/40 ratio gradually increased in rat hippocampus and CSF to levels
close to those found in human AD. Senile plaque formation progressively occurred late in the life of the
animals, i.e. 2.5 years after induction. More importantly, endogenous Tau was progressively
hyperphosphorylated over time and associated with increased levels of GSK3 and DYRK1A kinases,
resulting finally in tangle-like aggregation (AT8 positive cells) in old rats.
Detailed analysis of biochemical, histological, electrophysiological, and behavioural critical
preclinical steps of AD progression in the AAV-AD rat allowed us to decipher the early links between
APP processing and Tau pathology and propose a sequential AD progression hypothesis during
infraclinical stages.
(1) Cummings et al., 2014. Alzheimer's disease drug-development pipeline: few candidates, frequent
failures. Alzheimer's Res. Ther. 6, 37.
(2) Audrain M, Fol R, Dutar P, Potier B, Billard JM, Flament J, Alves S, Burlot MA, Dufayet-Chaffaud G,
Bemelmans AP, Valette J, Hantraye P, Déglon N, Cartier N, Braudeau J., 2016. Alzheimer's
disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease
progression. Mol. Neurodegener. 11, 5.
“DYRK1A, related kinases & human disease” 14
Dyrk1a gene dosage in glutamatergic neurons has a key effect in cognitive
deficits observed in mouse models of MRD7 and Down syndrome.
Véronique BRAULT1,2,3,4, Javier FLORES-GUTIERREZ1,2,3,4, Marie-Christine BIRLING5, Guillaume
PAVLOVIC5, Loïc LINDNER5, Mohammed SELLOUM5, Tania SORG5, Valérie LALANNE5, Hamid
MEZIANE5, Giovanni IACONO6, Doulaye DEMBELE1,2,3,4, Yann HERAULT1,2,3,4,5
1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France, 2 Centre National de la Recherche Scientifique,
UMR7104, Illkirch, France, 3Institut National de la Santé et de la Recherche Médicale, U964, Illkirch, France, 4 Université de
Strasbourg, Illkirch, France, 5 Institut Clinique de la Souris, PHENOMIN, GIE CERBM, Illkirch, France
Perturbation of the excitation/inhibition (E/I) balance leads to neurodevelopmental diseases
associated to autism spectrum disorders, intellectual disability and epilepsy. The DYRK1A gene
present on human chromosome 21 (Hsa21) codes for a kinase with numerous substrates. Mutations in
this gene leads to an intellectual disability syndrome associated with microcephaly, epilepsy and
autistic troubles (MRD7) (1). In mice, haploinsufficiency of Dyrk1a leads to decreased brain size with
altered microarchitecture of pyramidal cells (2). Overexpression of DYRK1A, on the other hand, has
been linked with learning and memory defects observed in people with Down syndrome (DS). Analysis
of DS trisomic mouse models revealed that Dyrk1a plays a crucial role in the perturbation of the E/I
balance that leads to the deficit in synaptic transmission and memory observed in those models
(3).Those findings led to preclinical programs using either GABA antagonists or DYRK1A inhibitors to
rescue learning and memory deficits in DS mouse models. Dyrk1a is expressed in both glutamatergic
and GABAergic neurons, but its impact on each neuronal population has not yet been elucidated. We
started to investigate the impact of Dyrk1a gene copy number variation in glutamatergic neurons using
a genetic approach. We used a conditional knockout allele of Dyrk1a mated with a transgenic mouse
expressing the Cre recombinase in hippocampal and cortical glutamatergic neurons (Tg(Camk1a-Cre)).
We combined those mice with the trisomic mouse model Ts1Yey to return to two copies of Dyrk1a in
glutamatergic neurons. We also produced Dyrk1aCamk2a/+ and Dyrk1aCamk2a/Camk2a mice in order to look at
the impact of the deficit of Dyrk1a in glutamatergic neurons. Dyrk1a gene dosage change in
glutamatergic neurons did not impact working memory deficits or susceptibility to epilepsy as tested
with the pro-convulsivant PTZ. However, object recognition memory was impacted by gene copy
number indicating a major effect of Dyrk1a trisomy on the glutamatergic pathway in declarative
memory.
(1) Van Bon, B.W. et al., 2016. Disruptive de novo mutations of DYRK1A lead to a syndromic form of
autism and ID. Mol. Psychiatry. 21, 126-132.
(2) Benavides-Piccione, R. et al., 2005. Alterations in the phenotype of neocortical pyramidal cells in
the Dyrk1A+/- mouse. Neurobiol. Dis. 20, 115-122.
(3) Souchet, B. et al., 2015. Pharmacological correction of excitation/inhibition imbalance in Down
syndrome mouse models. Front. Behav. Neurosci. 9, 267.
Financial support: the project is supported by the French National Centre for Scientific Research (CNRS), the French
National Institute of Health and Medical Research (INSERM), the University of ôme
“DYRK1A, related kinases & human disease” 15
A chemical with proven clinical safety rescues Down-syndrome-related
phenotypes through DYRK1A inhibition
Miri CHOI1,8, Hyeongki KIM1,2, Kyu-Sun LEE3,4, Ae-Kyeong KIM3, Kwangman CHOI1, Mingu KANG1,8,
Seung-Wook CHI5, Min-Sung LEE5, Jeong-Soo LEE3,4, So-Young LEE6, Woo-Joo SONG7, Kweon
YU3,4, and Sungchan CHO1,2
1Anticancer Agent Research Center, KRIBB, Ochang. 2Department of Biomolecular Science, University of Science and
Technology (UST) 3Neurophysiology Research Group, Hazard Monitoring BioNano Research Center, KRIBB 4Department of
Functional Genomics, University of Science and Technology (UST) 5Disease Target Structure Research Center, KRIBB
6International Cooperation Office, Ministry of Food & Drug Safety 7Department of Biochemistry and Molecular Biology,
Neurodegeneration Control Research Center, School of Medicine, Kyung Hee University 8School of Pharmacy, Graduate
School of Chungbuk National University, , Republic of Korea.
DYRK1A is important in neuronal development and function, and its excessive activity is
considered a significant pathogenic factor in Down syndrome and Alzheimer's disease. Although the
inhibition of DYRK1A is a new strategy to modify the disease, very few inhibitors have been reported
yet, and their potential clinical uses require further evaluation. Here, we newly identify CX-4945, the
safety of which has been already proven in the clinical setting, as a potent inhibitor of DYRK1A that
acts in an ATP-competitive manner. The inhibitory potency of CX-4945 on DYRK1A (IC50=6.8 nM) in
vitro was higher than that of harmine, INDY or proINDY, which are well-known potent inhibitors of
DYRK1A. CX-4945 effectively reverses the aberrant phosphorylation of Tau, amyloid precursor protein
(APP) and presenilin 1 (PS1) in mammalian cells. To our surprise, feeding with CX-4945 significantly
restored the neurological and phenotypic defects induced by the overexpression of minibrain, an
ortholog of human DYRK1A, in the Drosophila model. Moreover, oral administration of CX-4945 acutely
suppressed Tau hyperphosphorylation in the hippocampus of DYRK1A-overexpressing mice. Our
research results demonstrate that CX-4945 is a potent DYRK1A inhibitor and also suggest that it has
therapeutic potential for DYRK1A-associated diseases. Currently, to determine whether CX-4945
rescues the cognitive deficits in DS and AD, the behavioral tests in DYRK1A-overexpressing mouse
model is on the way.
(1) Kim et al., 2016. A chemical with proven clinical safety rescues Down-syndrome-related phenotypes
through DYRK1A inhibition. Dis. Model Mech. 9, 839-848.
“DYRK1A, related kinases & human disease” 16
Preclinical study of safety and biomarkers of efficacy of Epigallocatechin-3-
gallate at three different doses in wild-type and transgenic DYRK1A mice.
Cecile CIEUTA-WALTI1,2, A.-S. REBILLAT1, J.M. DELABAR3, J.L. PAUL4, J. DAIROU5, N. JANEL6.
1Institut Lejeune, 37 rue des volontaires 75015 Paris. 2Université de Sherbrooke, QC, Canada. 3INSERM U 1127, CNRS UMR
7225, Sorbonne Universités, UPMC Univ Paris 06 UMR S 1127, Institut du Cerveau et la Moelle épinière, CNRS UMR7225,
INSERM UMRS 975, Paris. 4AP-HP, Hôpital Européen Georges Pompidou, Service de Biochimie, 75015 Paris, France.
5Laboratoire de Chimie et Biochimie Pharmacologique et Toxicologiques, Unive
- , 6Unité de Biologie Fonctionnelle & Adaptative,
Université Paris Diderot-Paris 7, CNRS UMR 8251, Equipe processus dégénératifs, stress et vieillissement.
Objective : To study the safety and biomarkers of efficacy of three doses of Epigallocatechin-3-gallate
(EGCG) in wild-type and TgDYRK1A mice for the purpose of conducting a clinical study in children with
Down Syndrome.
Methods: The group 1 consisting of 4 subgroups of 20 wild-type (WT) mice who were fed with EGCG
(Fontup) for 3 times at dose 1 (25 mg/kg), 2 (50 mg/kg), 3 (75 mg/kg) and 4 (placebo). The group 2 of
WT and transgenic (Tg)DYRK1A mice were fed with dose 3 twice a day (150 mg/kg/d) for one week.
Blood analyses of plasma efficacy (plasma EGCG dosage, homocysteine, GSH, fibrinogen, ApoD)
have been down in the group 1 and safety and neural markers of efficacy (plasma DYRK1A, ALT,
galectin-3, brain BDNF, P-ERK) in groups 1 and 2.
Results: blood analyses of efficacy show in the group 1: plasma level of EGCG is proportional to the
administered dose (p<0.0001). Plasma homocysteine level is only significantly increased (p<0.01) with
the dose 2 with a non-pathologic rate. Plasma GSH level is increased for dose 2 (p<0.03) and 3
(p<0.001). Plasma level of fibrinogen is decreased (p<0.0001) at doses 1, 2 and 3. Plasma ApoD level
is decreased (p<0.02) for dose 2 and 3 and for dose 1 (p<0.005); blood analyses of safety show in the
group1: plasma DYRK1A level is unchanged. Plasma ALT level decreased with dose 2 (p<0.07) and
appears to have a protective effect on the liver. Plasma level of galectin-3 (a marker of cardiac function)
is unchanged; blood analyses of safety show in the group 2 (150 mg/kg/d for one week): plasma ALT
level is not significantly increased in TgDYRK1A mice and unchanged in WT mice. Plasma level of
Galectin-3 is decreased in the basal condition in TgDYRK1A mice (p<0.01) and is not significantly
increased with dose 3. Plasma level of galectin-3 decreased in WT mice (p<0.001); blood analyses of
neural markers of efficacy show for the group 1: brain BDNF level is increased respectively for dose 1
and 3 (p<0.0001) and for dose 2 (p<0.05). Brain PERK/ERK level is decreased for the dose 1
(p<0.0001), 2 (p<0.06) and 3 (p<0.03). Additional analyses were done with dose 2 in brain TgDYRK1A
mice and showed that two neural markers of efficacy, PERK/ERK (p<0.01) and DDB1 (p<0.03) levels
are decreased.
Conclusion: This preclinical study shows that EGCG is safe for hepatic and cardiac function at these 3
doses in WT mice, which correspond in humans approximately to 2.5, 5 and 7.5 mg/kg and with the
dose of 15 mg/kg/d during one week. Blood analyses show that plasma level of EGCG, homocysteine,
GSH, ApoD are good biomarkers of EGCG efficacy and that dose 2 is probably the best dose, which
corresponds in humans to 5 mg/kg per dose, i.e. 10 mg/kg/d. These results confirm the dose to be used
for safety and potential efficacy of EGCG in our future clinical study in children with Down Syndrome.
(1) Janel N, et al., 2014. Translational
Psychiatry 4, e425.
(2) Noll C. et al., 2009. DYRK1A, a novel determinant of the methionine-homocysteine cycle in different
mouse models overexpressing this Down-syndrome-associated kinase. PLoS One e7540.
(3) De la Torre R et al., 2014. Epigallocatechin-3-gallate, a DYRK1A inhibitor, rescues cognitive deficits
in Down syndrome mouse models and in humans. Mol. Nutr. Food Res. 58, 278288.
“DYRK1A, related kinases & human disease” 17
Analysis of DYRK signalling by phospho-SILAC mass spectrometry
Rachael HUNTLY1, Anne ASHFORD1, David OXLEY1, Kathryn S. LILLEY2 and Simon J. COOK1.
1The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK. 2Department of Biochemistry, University
of Cambridge, Cambridge CB2 1QW, UK.
The DYRKs are proline-directed protein kinases that sit in the CMGC arm of the human kinome,
being distantly related to the ERKs and CDKs. Hundreds of substrates/interacting partners are known
for the ERKs and CDKs and we have a good conceptual understanding of how these protein kinases
function. In contrast, relatively few DYRK substrates have been defined that might provide a biological
context for DYRK functions. Since the DYRKs undergo co-translational cis-autoactivation we have
utilised tetracycline-inducible expression systems to drive DYRK1B or DYRK2 expression in HEK293
cells coupled with stable isotope labelling of amino acids in cell culture (SILAC), phosphopeptide
enrichment and LC-MS/MS to identify DYRK-inducible phosphoproteins. Mascot and Proteome
DiscovererTM software packages allowed identification and quantification of phosphopeptides whilst
Motif-x revealed that the majority of DYRK-induced phosphorylation events were at pS-P or pT-P
motifs, consistent with them potentially being direct DYRK substrates. Gene Ontology (GO) analysis for
both DYRK1B- and DYRK2-induced changes in phosphorylation suggested RNA
processing/metabolism, protein turnover, translation, signal transduction and mitosis as DYRK-
regulated processes. Follow-up validation of the hits from these screens is ongoing but we have
already confirmed the identification of new substrates of DYRK1B and DYRK2 involved in RNA
processing and protein turnover/proteostasis.
Financial support: work funded by a Biotechnology and Biological Sciences Research Council (BBSRC) PhD studentship
(University of Cambridge), a BBSRC Project Grant and a BBSRC Institute Strategic Programme Grant.
“DYRK1A, related kinases & human disease” 18
DYRK1A, biomarker or target for Alzheimer’s disease
N JANEL, P ALEXOPOULOS, A BADEL, F LAMARI, A-C CAMPROUX, J LAGARDE, S SIMON, C
FERAUDET-TARISSE, P LAMOURETTE, M ARBONES, JL PAUL, B DUBOIS, MC POTIER, M
SARAZIN, Jean-Maurice DELABAR
Institut
Cedex, France.
interventions to delay the onset of dementia, but current biomarkers are invasive and/or costly to
assess. Validated plasma biomarkers would circumvent these challenges. We previously identified the
kinase DYRK1A in plasma. To validate DYRK1A as biomarker for AD risk, we assessed levels of
DYRK1A and the related markers BDNF and homocysteine in two unrelated AD patient cohorts with
age-matched controls.
Receiver-operating-characteristic curves and logistic regression analyses showed that
combined assessment of DYRK1A, BDNF, and homocysteine has a sensitivity of 0.952, a specificity of
0.889, and an accuracy of 0.933 in testing for AD. The blood levels of these markers provide a risk
assessment profile. Combined assessment of these three markers outperforms most of the previous
markers and could become a useful substitute to the current panel of AD biomarkers. These results
associate a decreased level of DYRK1A with AD and challenge the use of DYRK1A inhibitors in
peripheral tissues as treatment. It may also help to predict future cognitive decline in cognitively normal
individuals.
“DYRK1A, related kinases & human disease” 19
The DYRK1A kinase positively regulates angiogenic responses in endothelial
cells
Esteban J. ROZEN1, Julia ROEWENSTRUNK1, María J. BARALLOBRE2, Chiara DI VONA1, Carole
JUNQ3, Ana F. FIGUEIREDO4, Jeroni LUNA5, Cristina FILLAT5, Mariona L. ARBONES2, Mariona
GRAUPERA4, Miguel A. VALVERDE3, Susana DE LA LUNA1
1Gene Regulation, Stem Cells and Cancer Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of
Science and Technology, Barcelona, Spain. 2Institut de Biologia Molecular de Barcelona (IBMB), Barcelona, Spain. 3Molecular
Physiology Laboratory, Universitat Pompeu Fabra (UPF), Barcelona, Spain. 4Vascular Signalling Laboratory, Institut
d´Investigació Biomèdica de Bellvitge (IDIBELL), Barcelona, Spain. 5Institut D'Investigacions Biomèdiques August Pi i Sunyer
(IDIBAPS), Barcelona, Spain.
Angiogenesis is a highly regulated process essential for correct organ development and
maintenance, and its deregulation contributes to inflammation, cardiac disorders and cancer. The
Ca2+/calcineurin/Nuclear Factor of Activated T-cells (NFAT) signaling pathway is central to endothelial
cell angiogenic responses, and it is activated by stimuli like the vascular endothelial growth factor
(VEGF). NFAT activity is regulated by phosphorylation/dephosphorylation, and phosphorylation by
dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) is thought to be an inactivating
event. Contrary to the expectations, we show here that the DYRK family member DYRK1A positively
regulates NFAT transcriptional responses in primary endothelial cells following VEGF stimulation. Down
regulation of DYRK1A in endothelial cells reduces the release of Ca2+ from intracellular stores in
response to VEGF, which results in increased NFAT phosphorylation, increased cytoplasmic NFAT
accumulation and consequently, reduced NFAT-dependent transcriptional responses. The DYRK1A
effect appears to be exerted at the level of VEGF receptor accumulation leading to diminished PLC1
activation. DYRK1A knockdown markedly impairs VEGF-mediated endothelial cell proliferation and
tube formation. Concurring with the in vitro results, Dyrk1a heterozygous mice show defects in
developmental retinal vascularization and in VEGF-mediated vascular outgrowth in aortic ring assays.
Therefore, our data establish a novel regulatory circuit, DYRK1A/ Ca2+/NFAT, that is critical to fine-tune
endothelial cell proliferation and angiogenesis.
“DYRK1A, related kinases & human disease” 20
DYRK1A as a Small Molecule Target for Human β-Cell Proliferation for the
Treatment of Diabetes
K. KUMAR; P. WANG; H. WANG; H. LI; P.UNG; A. SCHLESSINGER; DP. FELSENFELD; H. LIU;
S.SIVENDRAN; A. BENDER; A. KUMAR; J.C. ALVAREZ-PEREZ; A. GARCIA-OCANA; R. SANCHEZ;
D.K. SCOTT; A.F. STEWART; R.J. DE VITA
Icahn School of Medicine at Mt. Sinai, New York, USA
Dual Specificity Tyrosine-Regulated Kinase 1a (DYRK1A) is a kinase that has been implicated
Syndrome. Recently, we have reported that small molecule kinase inhibitors, harmine and INDY, are
able to induce human -cells to proliferate in vitro and in vivo (1). We have also defined one key target
relevant to human -cell replication to be DYRK1A, a result confirmed by two other labs (2, 3). This
exciting discovery that small molecule compounds with DYRK1A inhibitory activity promote human
pancreatic -cell proliferation provides a unique opportunity for potential translation to human clinical
studies to treat the -cell deficiency that characterizes all types of diabetes.
We will present details on the validation of DYRK1A as one target important for -cell
proliferation. With that target in hand, the translational effort shifts to questions regarding the optimal
kinase selectivity for -cell proliferation, removing non-kinase off-target activities of known DYRK1a
inhibitors and de novo design of new DYRK1a inhibitor scaffolds. We will present our efforts to address
these important translational challenges to optimize small molecule DYRK1a inhibitor compounds to
treat diabetes and opportunities to deliver them specifically to the beta cell.
(1) Wang P, Felsenfeld DP, Liu H, Sivendran S, Bender A, Kumar A, Alvarez-Perez JC, Garcia-Ocana
A, Sanchez R, Scott DK, Stewart AF, 2015. A high-throughput chemical screen reveals that
harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication. Nature
Med. 21, 383-388.
(2)
DE, Clemons PA, Kulkarni RN, Wagner B, 2016. Inhibition of DYRK1A stimulates human -cell
proliferation. Diabetes 65, 1660-1671.
(3) Shen W, Taylor B, Jin Q, Nguyen-Tran V, Meeusen S, Zhang YQ, Kamireddy A, Swafford A,
Powers AF, Walker J, Lamb J, Bursalaya B, DiDonato M, Harb G, Qiu M, Filippi CM, Deaton L, Turk
CN, Suarez-Pinzon WL, Liu Y, Hao X, Mo T, Yan S, Li J, Herman AE, Hering BJ, Wu T, Martin
Seidel H, McNamara P, Glynne R, Laffitte B, 2015. Inhibition of DYRK1A and GSK3B induces
human -cell proliferation. Nat. Commun. 6, 8372.
Financial support: Mt Sinai Seed Fund; NIH NIDDK R01DK015015-01-A.
“DYRK1A, related kinases & human disease” 21
DYRK1A as a gene-specific RNA polymerase II CTD kinase
Chiara DI VONA1,2, Laura BARBA1,2 and Susana DE LA LUNA1,2,3
1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona
08003, Spain, 2Universitat Pompeu Fabra (UPF), Barcelona, Spain. 2Institució Catalana de Recerca i Estudis Avançats
(ICREA), Passeig Lluís Companys, 23, Barcelona, Spain
Our perception of how kinases regulate gene expression has recently been expanded to
consider not only their influence on transcription factors and co-regulators but also that on histones,
chromatin remodelers or components of the basal transcription machinery, all of which may be directly
modified by kinases at specific genomic loci. DYRK1A (dual-specificity tyrosine-regulated kinase)
belongs to a highly conserved family of kinases represented in all eukaryotes and it is known to fulfil
key roles during brain development. We have mapped the genome-wide profile of DYRK1A interactions
with chromatin and found that the kinase is recruited to RNA polymerase II (RNAPII)-dependent
promoters. DYRK1A binds chromatin regions displaying a highly conserved palindromic sequence that
lies close to the transcription start site of target genes, a sequence that appears to be necessary for
DYRK1A-mediated transcriptional activation. The recruitment of RNAPII at the promoters of target
genes is reduced in cells depleted of DYRK1A, indicating that the reduction in DYRK1A levels could
impact negatively on the association of the preinitiation complex with promoters. Moreover, DYRK1A
phosphorylates the carboxy-terminal domain (CTD) of the RNAPII both at serine 2 and serine 5 in vitro.
Consistently, silencing of DYRK1A leads to a reduction in RNAPII phosphorylation in these two
residues along the body of targets genes, and to a decrease in the expression of downstream target
genes. Interestingly, a subset of DYRK1A targets comprises ribosomal protein genes, and indeed,
downregulation of DYRK1A diminishes cell growth. We thus propose a role for DYRK1A as a gene
specific CTD kinase and as chromatin-associated transcriptional regulator that is part of the cellular
machinery controlling cell growth.
“DYRK1A, related kinases & human disease” 22
Role of Dyrk1a in GABAergic neurons during development
Aline DUBOS1, 2, 3, Camille SALEMBIER1, 2, 3, Yann HERAULT1, 2, 3, 4
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France. 2Centre National de la
Recherche Scientifique, UMR7104, Illkirch, France. 3Institut National de la Santé et de la Recherche Médicale, U964, Illkirch,
France. 4CELPHEDIA, PHENOMIN, Institut Clinique de la Souris, 1 rue Laurent Fries, 67404 Illkirch, France.
Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is considered to be
the major candidate gene for cognitive deficit in Down syndrome (DS) and its mutations is responsible
for the autosomal dominant mental retardation 7 syndrome (MRD7), a syndromic form of intellectual
disabilities with microcephaly, epilepsy and autistic troubles. In mice, Dyrk1a over-expression
(TgDyrk1a) leads to increased brain size (1) and an increase of GABAergic markers and neurons (2).
Strikingly, TgDyrk1a mice present an Excitation/Inhibition (E/I) imbalance toward inhibition which
confers them resistance to pentylenetetrazol (PTZ)-induced seizures (2). On the other hand, mice
heterozygous for Dyrk1a present a microcephaly (1, 3), smaller glutamatergic pyramidal cells and
decreased GAD67 GABAergic marker (2-4). The overall converge toward increased excitation which
lead to increased susceptibility to PTZ-induced seizures in Dyrk1a+/- mice (2). This suggest an essential
role of Dyrk1a gene dosage in neuronal development and in the establishment of a functional balance
between excitatory and inhibitory systems. To determine the pathophysiology mechanisms associated
with Dyrk1a dosage variations, we analysed the role of Dyrk1a during neuronal development by initially
focusing on its role on GABAergic neurons development using a Dyrk1a conditional KO mouse line
crossed with the interneuron specific Dlx6a-Cre mouse line.
First, we analysed GABAergic neurons migration at E15.5, period corresponding to a peak in
interneuron migration, in Dyrk1aDlx6a-Cre/wt mice crossed with the GAD65-GFP reported mouse line in
order to visualise interneurons. Preliminary results showed a decrease in the total number of GFP+
cells in the neocortex of Dyrk1aDlx6a-Cre/wt x GAD65-GFP embryos, due to a significant decreased of
GFP+ cells in the subventricular zone (SVZ)/intermediate zone (IZ) and the subplate (SP)/cortical plate
(CP) migratory streams, but not in the marginal zone (MZ). To better characterise the migration defect
observed in Dyrk1aDlx6a-Cre/wt x GAD65-GFP embryos, we analysed the SVZ/IZ migratory stream at
E15.5 and showed a delay of migration of the GFP+ cells in this stream. In addition, the number of
GFP+ cells was reduced all along the SVZ/IZ migratory stream. To exclude that the reduction of GFP+
cells was due to defect in interneuron progenitors proliferation, we counted the number of dividing
progenitors and did not observed any difference between Dyrk1aDlx6a-Cre/wt and Dyrk1awt/wt embryos,
suggesting that it may be due to event occurring after proliferation. Our preliminary results suggest a
role of Dyrk1a in interneuron development by affecting their migration in the neocortex.
(1) Guedj F, Pereira PL, Najas S, Barallobre MJ, Chabert C, Souchet B, et al., 2012. DYRK1A: a
master regulatory protein controlling brain growth. Neurobiol. Dis. 46,190-203.
(2) Souchet B, Guedj F, Sahun I, Duchon A, Daubigney F, Badel A, et al., 2014. Excitation/inhibition
balance and learning are modified by Dyrk1a gene dosage. Neurobiol. Dis. 69, 65-75.
(3) Fotaki V, Dierssen M, Alcantara S, Martinez S, Marti E, Casas C, et al., 2002. Dyrk1A
haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology
in mice. Mol. Cell Biol. 22, 6636-6647.
(4) Benavides-Piccione R, Dierssen M, Ballesteros-Yanez I, Martinez de Lagran M, Arbones ML, Fotaki
V, et al., 2005. Alterations in the phenotype of neocortical pyramidal cells in the Dyrk1A+/- mouse.
Neurobiol. Dis. 20, 115-122.
“DYRK1A, related kinases & human disease” 23
Identification of cystathione β-synthase pharmacological inhibitors to alleviate
the intellectual deficiency of patients with Down syndrome
Gaëlle FRIOCOURT1, Damien MARECHAL2,3, Nadège LOAËC1, Alice LEON1, Yann HERAULT2,3*,
Marc BLONDEL1*
1Inserm UMR1078, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Etablissement
Français du Sang (EFS) Bretagne, CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, Brest, France.
2IGBMC, CNRS, Illkirch, France. 3Institut Clinique de la Souris, Illkirch, France.
* Both authors contributed equally
Down syndrome (DS) is the most common genetic cause of intellectual disability (ID), affecting
1/700 to 1/1000 live births. Like DYRK1A, CBS (Cystathionine -synthase) gene is located on
chromosome 21 and both are suspected to be involved in the ID observed in DS. CBS catalyses the
condensation of serine and homocysteine to form cystathionine. It plays a critical role by linking the
folate and the methionine cycles and by regulating homocysteine levels. In addition, CBS converts
cysteine to hydrogen sulde, an important neuromodulator in the brain. Loss-of-function mutations in
CBS lead to homocystinuria, characterised by elevated level of homocysteine in urine, skeletal and
cardiovascular problems and severe ID. On the contrary, several studies have shown that CBS protein
level and enzyme activity are increased in DS patients, suggesting that the level of expression/activity
of this enzyme may be critical for proper cerebral function. Accordingly, the group of Y. Hérault has
recently observed that Cbs-overexpressing transgenic mice present defects in short term memory,
suggesting that a partial inhibition of CBS activity may represent a good strategy to improve memory
and learning in patients with Down syndrome.
The search for pharmacological inhibitors of CBS has so far been limited to in vitro approaches
and has not led to the identification of any compounds active in vivo. Moreover, no method of chemical
library screenings in a eukaryotic cellular context has ever been described. We thus recently developed
a yeast model overexpressing CYS4, the orthologue of CBS, and based on the knowledge of the
metabolic functions of this enzyme, we set-up a three-steps screening method allowing the
identification of molecules specifically inhibiting CYS4/CBS. Using this simple and convenient assay,
we screened 2000 FDA-approved drugs. Four different molecules have been found active on both
CYS4 and CBS. Interestingly, three of them appear to have common chemical properties. One of them
has recently been shown to improve cognitive defects in Cbs-overexpressing transgenic mice. As two
of these molecules are already in trials or clinics and pass the blood-brain barrier, therapeutic
repositioning may be considered at relatively short-term.
Financial support: This work is funded by the Fondation Jérôme Lejeune and has benefited from a seeding grant from the
ITMO BCDE (Biologie Cellulaire, Développement et Evolution).
“DYRK1A, related kinases & human disease” 24
Mirk/Dyrk1B kinase is a novel dual function molecule inducing cell cycle exit and
neuronal differentiation in the embryonic chick spinal cord
Nikos KOKKORAKIS1, Panagiotis K POLITIS2, Rebecca MATSAS1 and Maria GAITANOU1
1Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, 2Center of Basic Research, Biomedical
Research Foundation of the Academy of Athens, Greece.
Regulation of cell cycle progression/exit of neuronal precursors is essential for proper
generation of the nervous system. Mirk/Dyrk1B has long been studied as a cell cycle regulator in
skeletal muscle differentiation controlling cyclin D1 levels by inducing its proteosomal degradation. We
have previously demonstrated that Dyrk1B is also expressed in the brain and in primary cortical
neurons in culture, while it promotes cell cycle exit and neuronal differentiation in Neuro 2a cells. Here
we cloned the chick Dyrk1B ortholog and demonstrated that it is expressed by cycling neuronal
progenitors as well as by differentiated motor neurons in the embryonic spinal cord.
Furthermore, we used a gain-of-function approach to investigate the role of Dyrk1B in vivo in the
chick spinal cord. Expression of Dyrk1B/GFP or control GFP protein was achieved by unilateral in ovo
electroporation in the neural tube of E2 chick embryos, which were then analyzed at E4. We found that
overexpression of Dyrk1B induces cell cycle exit. GFP+ cells in the Dyrk1B/GFP electroporated
embryos showed a reduction by 2.3-fold in BrdU incorporation, by 10-fold in the expression of the
mitotic marker PH3, by 4.9-fold in Prox-1 and by 1.5-fold in the number of Sox2+ neural progenitors as
compared with GFP+ cells in control embryos. In addition Dyrk1B/GFP electroporated cells showed an
increased expression of Doublecortin by 1.4 fold in comparison with GFP electroporated control cells.
In conclusion, we identified Mirk/Dyrk1B as a novel dual function molecule inducing cell cycle
exit and neuronal differentiation in the developing chick spinal cord.
(1) Deng, X., Ewton, D. Z., and Friedman, E., 2009. Mirk/Dyrk1B maintains the viability of quiescent
pancreatic cancer cells by reducing levels of reactive oxygen species. Cancer Res. 69, 33173324.
(2) Friedman E., 2013. Mirk/Dyrk1B Kinase in Ovarian Cancer. Int. J. Mol. Sci. 14, 5560-5575.
(3) Tsioras K., Papastefanaki F., Politis P.K., Matsas R., Gaitanou M., 2013. Functional Interactions
between BM88/Cend1, Ran- Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell
Cycle Progression/Exit in Mouse Neuroblastoma Cells. PLOS ONE 8, 11, e82172.
“DYRK1A, related kinases & human disease” 25
Blocking DYRK1B phosphorylation of NKX3.1 prostate tumor suppressor inhibits
prostate growth and increases apoptosis – A potential therapeutic strategy.
Bowen CAI, Lian-Niang SONG, Maria VILENCHIK, Edward P. GELMANN
Columbia University, New York, NY, and Felicitex Therapeutics, Cambridge, MA, USA.
NKX3.1 is a prostate-specific homeodomain protein that is the gatekeeper tumor suppressor of
the majority of prostate cancers (1, 2). NKX3.1 is haploinsufficient and mere decrease in protein levels
is sufficient to attenuate tumor suppression (3). Moreover, NKX3.1 expression, though reduced in most
prostate cancers, is virtually never lost completely, even at sites of advanced, metastatic, castration-
resistant prostate cancer (4). As a result, therapeutic intervention designed to increase levels of
NKX3.1 protein has the potential to attenuate growth of prostate cancer at any phase of clinical
progression. We have shown that DYRK1B phosphorylates NKX3.1 on serine 185 to trigger
ubiquitination and mediate steady state protein turnover. Either DYRK1B knock down or its
pharmacologic inhibition prolongs NKX3.1 half-life in cultured cells. A third generation DYRK inhibitor,
FX9847 that has a very narrow spectrum of kinase inhibition and a high level of activity against
DYRK1A, HASPIN/GSG2, and DYRK1B has favorable pharmacokinetics in mice and has been shown
to have antitumor activity against pancreatic, lung, and colon cancer xenograft models. In anticipation
of testing the effect of FX9847 on the murine prostate, we have shown that FX9847 blocks DYRK1B
phosphorylation of NKX3.1 in cultured cells at concentrations that have no adverse effects on cell
proliferation. To determine whether Nkx3.1 gene targeting in the mouse, that causes prostate
hyperplasia and dysplasia, is a good model for the effects on the prostate of Dyrk1b inhibition, we
engineered mice with mutation of the Dyrk1b target amino acid Nkx3.1(S186), changing the substrate
serine to alanine. Mice with only a single copy of this mutant gene, Nkx3.1S186A/-, had reduced prostate
size, increase Nkx3.1 protein expression, and regions of anoikis and apoptosis in the prostate gland.
Next we will cross mice with the Nkx3.1S186A allele with mice engineered for Pten loss in the prostate to
determine whether Nxk3.1 with prolonged half-life can suppress prostate tumorigenesis activated by
Pten I loss. These experiments will determine the target phenotype for testing FX9847 in mouse
models of prostate cancer.
(1) Asatiani E, Huang WX, Wang A, Rodriguez OE, Cavalli LR, Haddad BR, et al., 2005. Deletion,
methylation, and expression of the NKX3.1 suppressor gene in primary human prostate cancer.
Cancer Res 65, 1164-1173.
(2) Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, et al., 2013. Punctuated
evolution of prostate cancer genomes. Cell 153, 666-677.
(3) Bhatia-Gaur R, Donjacour AA, Sciavolino PJ, Kim M, Desai N, Norton CR, et al., 1999. Roles for
Nkx3.1 in prostate development and cancer. Genes & Dev. 13, 966-977.
(4) Chuang AY, DeMarzo AM, Veltri RW, Sharma RB, Bieberich CJ, Epstein JI, 2007.
Immunohistochemical differentiation of high-grade prostate carcinoma from urothelial carcinoma.
Am. J. Surg. Pathol. 31, 1246-1255.
“DYRK1A, related kinases & human disease” 26
The influence of the metabolism of monocarbons on epigenomic mechanisms:
the interacting role of the remethylation and transsulfuration pathways
Jean-Louis GUEANT
Inserm U954, Nutrition-Genetics and Environmental Exposure, Faculty of Medicine, University of Lorraine, 54500 Nancy,
France.
The methionine cycle plays a central role in the metabolism of monocarbons. The remethylation
of homocysteine catalyzed by methionine synthase in the presence of vitamin B12 and folate allows the
synthesis of methionine, which is the immediate precursor of the universal methyl group donor S-
adenosylmethionine (SAM). SAM is the substrate of transmethylations reactions involved in epigenomic
mechanisms, including the methylation of DNA, RNA and histones, as well as the modulation of the
activity of co-regulators of nuclear receptors. Methylation is also involved in the synthesis of
intermediate metabolites. The transsulfuration pathway of homocysteine is the cataplerotic pathway of
the methionine cycle, which regulates its flux by adapting the concentration of homocysteine.
Cystathionine -synthase (CBS) is the key enzyme of this cataplerotic pathway. The CBS gene is
localized on human chromosome 21. CBS removes homocysteine from the methionine cycle and
directs the flux of sulfur to the biosynthesis of cysteine. It binds three cofactors, heme, whose function
-phosphate (PLP), part of the catalytic site where homocysteine and
serine are condensed to cystathionine, and S-adenosylmethionine (SAM), which upon binding greatly
stimulates its activity. The deficiency of CBS results in recessively inherited metabolic disease,
homocystinuria, characterized by high concentrations of homocysteine, methionine and S-
adenosylhomocysteine and greatly decreased cysteine and cystathionine. As a consequence, deficient
CBS activity results in decreased H2S production, which is compensated by enhanced synthesis of
hydrogen sulfide. The regulation of the expression and activity of CBS depends on complex regulatory
mechanisms that involve the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase (Dyrk1a).
The gene of Dyrk1A is also localized on human chromosome 21. Recent data by the group of N. Janel
showed that Dyrk1a protein expression is correlated to CBS activity in modified and non-modified
genetic mice models, in brain and liver.
“DYRK1A, related kinases & human disease” 27
Development of inhibitors of CDK9, CLK1, DYRK1a, and their clinical application.
Masatoshi HAGIWARA
Department of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan
Kinase families of CDKs, CLKs and DYRKs are involved in the regulation of gene expression.
Therefore, we have developed specific inhibitors of these kinases to clarify their physiological functions.
Through the development of these chemicals, we eventually succeeded to find candidate compounds
available for therapeutic drugs to cure diseases such as viral infections, Duchenne muscular dystrophy,
and Down syndrome. The preparation of the clinical trial of the ant-virus drug is under the way in our
uni
academic drug discovery with open innovation.
(1) Yamamoto et al., 2014. CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses. J. Clin.
Invest. 124, 34793488.
(2) Nishida A et al., 2011. Chemical treatment enhances skipping of a mutated exon in the dystrophin
gene. Nature Comm. 2, 308.
(3) Ogawa Y et al., 2010. Development of a novel selective inhibitor of the Down syndrome-related
kinase Dyrk1A. Nature Comm. 1, 86.
(4) Kii I et al., 2016. Selective inhibition of the kinase DYRK1A by targeting its folding process. Nat.
Commun. 7, 11391.
“DYRK1A, related kinases & human disease” 28
Making the most of public domain data with Knime® : renovating GSK3β and
CDK2 scaffolds
Scott HENDERSON1, James BENNETT2, Fiona SORRELL2, Jonathan ELKINS2, Simon WARD1
1Sussex Drug Discovery Centre, School of Life Sciences, University of Sussex, Falmer BN1 9QJ, U.K. 2Nuffield Department of
Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute (TDI), University of Oxford, Old Road
Campus, Roosevelt Drive, Oxford OX3 7DQ, UK.
A recent publication describes the extensive screening of a set of diverse, drug like kinase
inhibitors (Published Kinase Inhibitor Set) and the identification of several new DYRK1A inhibitor
templates (1).
Hit-validation and small-scale expansion of a number of these templates has led to the
synthesis of inhibitors with nanomolar kinase inhibitory activities against the primary target, DYRK1A.
The physicochemical properties of the molecules are close to those required for predicted CNS
penetration and drug-likeness (Lipinski Ro5). Encouragingly whilst the compounds have off-target
activity (GSK3 and CDK2), making a small change to the structure changes the selectivity profile
significantly (2).
The open source Knime® Analytics Platform is being used to predict off-target selectivity,
druglikeness central nervous system multiparameter optimization (CNS MPO) score and overall drug
likeness in an effort to produce a selective, CNS penetrant tool from which further inhibitors can be
derived.
(1) Elkins, J. M., Fedele, V., Szklarz, M., Abdul Azeez, K. R., Salah, E., Mikolajczyk, J., Romanov, S.,
Sepetov, N., Huang, X.-P., Roth, B. L., et al., 2016. Nat. Biotechnol. 34, 95-103.
(2) F.X. Tavares, J.A. Boucheron, S.H. Dickerson, R.J. Griffin, F. Preugschat, S.A. Thomson, T.Y.
Wang and H.-Q.Zhou, 2004. J. Med. Chem. 47, 47164730.
“DYRK1A, related kinases & human disease” 29
Challenging the role of DYRK1A in Down syndrome by studying the genotype
and phenotype relationships in animal models
Yann HERAULT1,2, Maria DEL MAR MUNIZ MORENO1, Guillaume PANI1, Thu Lan NGUYEN1,3,
Damien MARECHAL1, Claire CHEVALIER1, Valérie NALESSO1, Aline DUBOS1, Michel ROUX1,
Véronique BRAULT1, Arnaud DUCHON1
1Institut de Génétique Biologie Moléculaire et Cellulaire, IGBMC, CNRS, INSERM, Université de Strasbourg, UMR7104,
UMR964, 1 rue Laurent Fries, 67404 Illkirch, France. 2CELPHEDIA-PHENOMIN-ICS, 1 rue Laurent Fries, 67404 Illkirch,
France.
Genetic diseases with intellectual disability (ID) involved impairment of mental abilities that
impacts adaptive functioning in the conceptual, the social or the practical domain with or without other
features. ID can occur during the developmental period and is defined by an intellectual quotient below
70. Several genetic causes, including trisomy 21 (Down syndrome, DS), deletion or duplication of
with ID. To better understand the genotype-phenotype relationship in DS, we generated several models
in different organisms. Here we will report the characterization of several DS mouse models using
standardized behavioral and cognitive paradigms, completed with transcriptomic approaches in brain
region that are affected. Based on the new series of models, several pathways have been identified,
challenging the main role of Dyrk1a in DS and highlighting the complexity of genetic interactions. The
data generated are challenging our current knowledge on DS and its impact on brain and cognitive
functions. Such studies will lead to a better understanding of mechanisms controlling cognition and
behavior in model organism and human and how to define new preclinical treatments.
“DYRK1A, related kinases & human disease” 30
Analysis of DYRK signalling by RNAseq profiling
Rachael HUNTLY, Anne ASHFORD, Simon COOK.
The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.
how their deregulation may contribute to disease phenotypes (i.e. cancer and metabolic syndrome).
DYRK1B, highly expressed in muscle cells, functions in myoblast differentiation1, but less is known
about normal functions of DYRK2. Whilst transcriptional responses to DYRK1B and DYRK2 depletion
have previously been explored by microarray analysis (1-3), transcriptional changes that result from
their elevated expression are unclear. To investigate DYRK-driven changes on global gene expression,
we have utilised tetracycline-inducible expression systems to drive DYRK1B or DYRK2 expression in
HEK293 cells followed by cDNA library generation for RNAseq and subsequent data analysis using
SeqMonk bioinformatics software. Gene Ontology (GO) analysis for both DYRK1B and DYRK2-induced
changes were consistent with our own observations and published work. For example, DYRK1B
induction led to differential expression of genes implicated in skeletal muscle differentiation whilst
DYRK2 was linked to pro-apoptotic signals. Additionally, GO terms suggested novel functions for
DYRK1B in mediating the transcriptional regulation of other major developmental pathways. Finally, our
data may provide insights into how the DYRK proteins interact with other known signalling cascades
(i.e. MAPK pathways). This data provides novel insights into an understudied area of DYRK biology
and highlights clear functional differences at the transcriptional level between class I and class II
DYRKs.
(1) Deng X et al., 2005. J. Biol. Chem. 280, 4894-4905.
(2) Mimoto et al., 2016. Oncogene doi:10.1038/onc.2016.349.
(3) Mimoto et al., 2017. Cancer Lett. 384, 27-38.
Financial support: work funded by BBSRC DTP PhD studentship (University of Cambridge).
“DYRK1A, related kinases & human disease” 31
Smart screening libraries and successful medicinal chemistry: kinase inhibitors
search
Marie-Louise JUNG
Prestwick Chemical, -Illkirch, France.
Prestwick Chemical (1), created 1999, has a worldwide recognition for providing smart
screening libraries. The Prestwick Drug-Fragment Library is a collection of compounds arising from
smart fragmentation of 1565 off-patent approved drugs. The Prestwick CNS Drug Library is a unique
collection of 320 structurally diverse approved and marketed central nervous system drugs. The
Prestwick Phytochemical Library is a collection of 320 natural products, mostly derived from plants. The
C. Elegans Library Of Drugs Designed For Caenorhabditis Elegans Research Programs is a collection
of 240 small molecules that have been carefully selected for chemical structural diversity as well as for
good tolerability in C. elegans. The Prestwick Pyridazine Library comprises 400 innovative pyridazine
and pyridazone derivatives. Finally the most famous chemical library is the Prestwick Chemical Library,
a unique collection of 1280 small molecules, mostly approved drugs (FDA, EMA and other agencies)
selected by a team of medicinal chemists and pharmacists for their high chemical and pharmacological
-
-ies have been substantially reported by users
in more than 350 publications. Examples of repositioned drugs into the kinase inhibitors field will be
presented. Moreover, an impressive track record of compounds designed and prepared at Prestwick
and being in clinical phase can be claimed. Currently 10 molecules are developed in the clinic, coming
out of our lead optimization work, and one is on the market. An example for discovering DYRK
inhibitors will be emphasized.
(1) http://www.prestwickchemical.com/
“DYRK1A, related kinases & human disease” 32
Novel scaffold kinase inhibitors of DYRK/CLK family members
Krisna PRAK1, Janos KRISTON-VIZI1, A.W. Edith CHAN2, Christin LUFT1, Joana R COSTA1, Niccolo
PENGO1, and Robin KETTELER1
1MRC Laboratory for Molecular Cell Biology, University College London, London, UK. 2Wolfson Institute for Biomedical
Research, UCL, UK.
Protein kinases are essential regulators of most cellular processes and are involved in the
etiology and progression of multiple diseases. The cdc2-like kinases (CLKs) have been linked to
various neuro-degenerative disorders, metabolic regulation, virus infection and autism. Recently, it has
been shown that inhibition of CLK2 can restore sociability in a mouse model (1), suggesting that CLK2
is a promising drug target.
Here, we have developed a screening workflow for the identification of potent CLK2 inhibitors
and identified compounds with a novel chemical scaffold structure, the benzobisthiazoles that has not
been previously reported for kinase inhibitors (2). We propose binding models of these compounds to
CLK family proteins and key residues in CLK2 that are important for the compound interactions and the
kinase activity. We identified structural elements within the benzobisthiazole that determines CLK2 and
CLK3 inhibition, thus providing a rationale for selectivity assays. In summary, our results will inform
structure-based design of CLK family inhibitors based on the novel benzobisthiazole scaffold.
(1) Bidinosti et al., 2016. CLK2 inhibition ameliorates autistic feature associated with SHANK3
deficiency. Science 351, 1199-1203.
(2) Prak K, Kriston-Vizi J, Chan AW, Luft C, Costa JR, Pengo N, and Ketteler R., 2016.
Benzobisthiazoles represent a novel scaffold for kinase inhibitors of CLK family members.
Biochemistry 55, 608-617.
“DYRK1A, related kinases & human disease” 33
From kinome to DYRK1A in T cell regulation and inflammation
Bernard KHOR, John D GAGNON, Gautam GOEL, Marly I ROCHE,Kara L CONWAY, Leslie N
SHAMJI, Stuart L SCHREIBER, Arlene H SHARPE, Stanley Y SHAW, Ramnik J XAVIER
1201 9th Ave, Seattle, WA 98122, USA
The immune system balances dueling needs to respond vigorously against pathogens while
remaining tolerant to self by engaging pro- and anti-inflammatory forces in precise and dynamic
equilibrium. The CD4+ T cell is a central regulator of this equilibrium, acting via its ability to differentiate
into either pro-inflammatory effector subsets (e.g. Th1, Th17) or anti-inflammatory subsets, largely
represented by regulatory T cells (Tregs). While several key genetic factors regulating these lineage
decisions have been identified, therapeutic tools to modulate this balance remain relatively lacking and
represent a continuing unmet need.
To this end, we undertook a small molecule discovery approach to identify new, druggable
proteins that regulate T cell differentiation. We were particularly interested in kinase targets not only
because many facets of T cell biology are known to be regulated by kinases, but also because of the
relative advanced state of kinase inhibitor efforts as reflected by their clinical availability, especially in
oncology.
In order to capture targets of maximal physiologic relevance, we developed a pipeline to
interrogate primary CD4+ T cells in an unbiased high-throughput fashion to identify enhancers of Treg
differentiation. We identified DYRK1A as a novel reciprocal regulator of Treg/Th17 differentiation (1).
Accordingly, DYRK1A inhibitors exert significant anti-inflammatory effect, promoting Treg while inhibiting
Th17 differentiation. These inhibitor-enhanced Tregs appear fully functional in vitro and in animal models
of inflammation, and treatment with the DYRK1A inhibitor harmine attenuates inflammation in a murine
model of asthma.
These results highlight a potential novel clinical role for DYRK1A inhibition in treating inflammatory
disorders. Studies are underway to investigate whether converse DYRK1A hyperactivity provides a
unifying explanation for the increased autoimmunity and impaired Treg function in patients with Down
syndrome and whether these patients may especially benefit from DYRK1A inhibitor therapies.
(1) Khor, B. et al., 2015. The kinase DYRK1A reciprocally regulates the differentiation of Th17 and
regulatory T cells. Elife 4, doi: 10.7554/eLife.05920.
Financial support: funding by NIH T32CA009216, T32HL066987, K08DK104021, S10OD012027, P30DK043351,
U01DK062432,
Research Program and Sir Henry Dale Fellowship Grant Number 105663/Z/14/Z.
“DYRK1A, related kinases & human disease” 34
Rational design strategies for improving selectivity of inhibitors targeting splicing
regulating kinases
Stefan KNAPP1,2, Apirat CHAIKUAD1,2, Martin SCHRODER1, Clara REDONDO1, Franz BRACHER6,
Jon ELKINS1, Jonathan C. MORRIS3, David BATES4 , Laurent MEIJER5 Thierry BESSON7
1Johann Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt am
Main, Germany. 2Structural Genomic Consortium, University of Oxford. Old Road Campus, Oxford OX3 7DQ, UK. 3School of
Chemistry, UNSW Australia, Sydney, Australia. 4Biology, Division of Cancer and Stem Cells, School of Medicine, University of
Nottingham, Queen's Medical Centre, Nottingham NG2 7UH, UK. 5ManRos Therapeutics, Roscoff, France. 6Department of
Pharmacy, Center for Drug Research, Butenandtstraße 5-13, D - 81377 Munich, Germany. 7Normandie Univ, UNIROUEN,
INSA Rouen, CNRS, COBRA UMR 6014, 76000 Rouen, France.
The CLK, SRPK and DYRK family of kinases have been tightly associated with the regulation of
mRNA splicing. Our group is interested in the development of highly selective inhibitors, so called
chemical probes, using rational, structure based design approaches targeting each of these kinase
families. As a basis for this research we have solved high resolution crystal structures of all CLK family
members, DRYK1A as well as SRPK1 and SRPK2 in complex with ATP competitive inhibitors.
Structural comparisons and kinome wide inhibitor profiles identified a number of unique structural
features that can be used for designing more selective inhibitors. For instance, we recently targeted the
unique insertion domain in SRPK1 resulting in the development of inhibitors with exclusive target
selectivity. The most selective inhibitor of this series (SPINX31) inhibited SRPK1 at low nM potency in
vitro as well as in cell based assay systems. In mouse models of macular degeneration, we were able
to demonstrate that SRPK1 inhibition leads to a shift in splice form expression from pro-angiogenic to
anti-angiogenic VEGF, resulting in potent inhibition of neovascularization in the eye. Some CLK family
members harbours unique residue combinations in the ATP site comprising for instance large
hydrophobic residues N-terminal to the DFG motif. The presence of these residues result in unique
binding modes and a set of typical CLK inhibitor off-targets that share similar residue combination in the
active site. However, sequence variations within these kinases allowed us to develop inhibitors with
good CLK selectivity. In some cases we were able to obtain also selectivity against the closely related
DYRK kinase family.
In this talk I will summarize insights from ligand complexes and how we use them for the
development of a selective chemical tool set to study kinase function in mRNA splicing.
(1) Batson, J., Toop, H.D., Redondo, C., Babaei-Jadidi, R., Chaikuad, A., Wearmouth, S.F., Gibbons,
B., Allen, C., Tallant, C., Zhang, J., Du, C., Hancox, J.C., Hawtrey, T., Da Rocha, J., Griffith, R.,
Knapp, S., Bates, D.O., and Morris, J.C., 2017. Development of Potent, Selective SRPK1 Inhibitors
as Potential Topical Therapeutics for Neovascular Eye Disease. ACS Chem. Biol.
(2) Bullock, A.N., Das, S., Debreczeni, J.E., Rellos, P., Fedorov, O., Niesen, F.H., Guo, K.,
Papagrigoriou, E., Amos, A.L., Cho, S., Turk, B.E., Ghosh, G., and Knapp, S., 2009. Kinase domain
insertions define distinct roles of CLK kinases in SR protein phosphorylation. Structure 17, 352-362.
(3) Chaikuad, A., Diharce, J., Schroder, M., Foucourt, A., Leblond, B., Casagrande, A.S., Desire, L.,
Bonnet, P., Knapp, S., and Besson, T., 2016. An Unusual Binding Model of the Methyl 9-
Anilinothiazolo[5,4-f] quinazoline-2-carbimidates (EHT 1610 and EHT 5372) Confers High Selectivity
for Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases. J. Med. Chem. 59, 10315-10321.
(4) Fedorov, O., Huber, K., Eisenreich, A., Filippakopoulos, P., King, O., Bullock, A.N., Szklarczyk, D.,
Jensen, L.J., Fabbro, D., Trappe, J., Rauch, U., Bracher, F., and Knapp, S., 2011. Specific CLK
inhibitors from a novel chemotype for regulation of alternative splicing. Chem. Biol 18, 67-76.
(5) Tahtouh, T., Elkins, J.M., Filippakopoulos, P., Soundararajan, M., Burgy, G., Durieu, E., Cochet, C.,
Schmid, R.S., Lo, D.C., Delhommel, F., Oberholzer, A.E., Pearl, L.H., Carreaux, F., Bazureau, J.P.,
Knapp, S., and Meijer, L. (2012). Selectivity, cocrystal structures, and neuroprotective properties of
leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid
leucettamine B. J. Med. Chem. 55, 9312-9330.
“DYRK1A, related kinases & human disease” 35
Repression of DYRK1A restores impaired cortical development and abnormal
learning behavior of Down syndrome mice
Akiko KOBAYASHI, Isao KII, Yuto SUMIDA, Yukiko OKUNO, Suguru YOSHIDA, Tomonari AWAYA,
Takamitsu HOSOYA, Masatoshi HAGIWARA
Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-
ku, Kyoto, 606-8501, Japan
Impaired neurogenesis has been observed in psychiatric/neurodevelopmental diseases such as
Down syndrome (DS). Trisomy of chromosome 21, DS, is the most common genetic cause of
intellectual disability. Although prenatal diagnosis has become prevalent, no therapies currently exist for
the rescue of neurocognitive impairment of DS. Here, we tried a new screen to find out compounds
which promote the proliferation of neural stem cells (NSCs). Newly identified growth inducer, which has
potent inhibitory activity against dual-specificity tyrosine phosphorylation-regulated kinase 1A
(DYRK1A), rescued proliferative deficits in Ts65Dn-derived neurospheres and human fibroblasts
derived from DS individuals. The oral administration of the compound, named ALGERNON, restored
the reduced proliferation of NSCs in murine DS models and increased the number of newborn neurons.
Moreover, administration of ALGERNON to pregnant dams rescued the abnormal cortical development
in DS mouse embryos and abnormal behavior in DS-offspring. These data suggest that DYRK1A
represents a potential therapeutic target for individuals with DS, and possibly other disorders involving
aberrant neurogenesis.
“DYRK1A, related kinases & human disease” 36
Genetic modeling of neurodegenerative diseases in zebrafish for bioimaging
studies and compound evaluation.
Astrid BUCHBERGER, Kazuhiko NAMIKAWA, Reinhard W. KOSTER
TU Braunschweig, Zoological Institute, Cellular and Molecular Neurobiology, Spielmannstrasse 7, 38106 Braunschweig,
Germany.
Zebrafish larvae are small, develop outside their mother, and are produced in large quantities.
Moreover, this model organism is genetically tractable with embryos that are nearly transparent and
therefore ideally suited for bioimaging studies and compound analysis. We have used these
advantages for modelling spinocerebellar Ataxia Type 1 (SCA1), which is caused by a gain of function
of nuclear polyglutamine-containing Ataxin 1 (Atx1) resulting in the progressive degeneration of
cerebellar Purkinje neurons (PCs) (1). After isolation of a PC-specific regulatory element we have used
combinatorial Gal4-genetics to express pathogenic human Atx1 in differentiating zebrafish PCs (2).
Aggregate formation and signs of neuronal degeneration can be observed with a few days with Gal4-
expression allowing for targeting additional reporters or disease-modulating factors into affected PCs.
In addition, these larvae can be easily used to interfere with PC degeneration by compound treatment.
Interestingly, the zebrafish atx1 homolog together with its evolutionary conserved interaction
partners was found to be expressed in sensory hair cells, which become functional already early during
development, mediate vibration-induced swimming and are in direct contact with the aqueous medium.
Sensory hair cells expressing the human pathogenic Atx1-mutant in transgenic larvae degenerate
rapidly resulting in striking behavioural deficits. Furthermore, we show that hair cell degeneration can
be rescued either genetically or by small compound treatment providing proof of principle for identifying
SCA1-interfering substances that can be tested subsequently for blood-brain-barrier passage.
Zebrafish contain three dyrk1 homologs. While dyrk1aa and dyrk1ab have not been studied so
far, dyrk1b was shown to be expressed during early development and is crucially involved in endoderm
formation and craniofacial patterning (3). We have isolated the zebrafish dyrk1aa homolog, which is
highly conserved to human dyrk1A. Expression analysis revealed a nearly pan-neuronal expression
during embryonic development. In the adult brain dyrk1aa is weakly expressed in the telencephalon,
optic tectum and hypothalamus but displays strong expression in the granule cell layer of the
cerebellum. A key to genetic modelling of human diseases in zebrafish is the availability of cell type
specific regulatory elements. We have set out to establish a bidirectional pan-neuronal enhancer that
allows to simultaneously identify transgene expressing cells by fluorescent protein expression. In
parallel, a cerebellar granule cell specific regulatory element was identified. This will be used to express
human dyrk1a throughout the brain or selected neuronal cell types. As proof of principle, we have
targeted cerebellar Purkinje cells with human dyrk1a overexpression. Transgenic zebrafish do not show
gross cerebellar malformations, but appear to display hypoactive behaviour a phenotype that can be
subjected to small compound analysis.
(1) Orr, H.T., 2012. Cell biology of spinocerebellar ataxia. J. Cell Biol. 197, 167-177.
(2) Distel, M., Wullimann M.F., and R.W. Köster, 2009. Optimized Gal4 genetics for permanent gene
expression mapping in zebrafish. PNAS 106, 13365-13370.
(3) Mazmanian, G., Kovshilovsky, M., Yen, D., Mohanty, A., Mohanty, S., Nee, A., and R.M. Nissen,
2010. The zebrafish dyrk1b gene is important for endoderm formation. Genesis 48, 20-30.
“DYRK1A, related kinases & human disease” 37
DYRK and CLK inhibitors: Is selectivity feasible?
Conrad KUNICK1,2
1Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig, Beethovenstraße 55, 38106
Braunschweig, Germany. 2Center of Pharmaceutical Engeneering (PVZ), Technische Universität Braunschweig, Franz-Liszt-
Straße 35A, 38106 Braunschweig, Germany.
Protein kinases belonging to the DYRK and the CLK families share high structural similarities of
their ATP binding pockets. Thus, chemical inhibitors of DYRK and CLK kinases are typically not very
selective for a distinct DYRK or CLK family member. While this could be a favorable property of a drug
against neurodegenerative diseases in which hyperactivities of both CLK and DYRK kinases are
involved, high selectivity of an agent is desirable when it is used as a chemical probe or as a tool in
biological assays. In the course of our search for novel kinase inhibitory chemotypes we have identified
the following two molecules with orthogonal DYRK and CLK inhibition selectivity. KuFal194 (1) is a
highly selective DYRK1A inhibitor with single-digit nanomolar potency on the isolated enzyme. This
molecule also exhibited activity in cellular assays, albeit in considerable higher (micromolar)
concentrations (1). KuWal151 (2) was discovered as member of a novel compound class designated
- -3
inhibitors, most mini-indirubines inhibit non-selectively CLK and DYRK kinases. Among the mini-
indirubines, 2 was selective for CLK1/4 without interfering with DYRK kinases (2). In the presentation,
syntheses of the new inhibitors as well as models of their orientation in the ATP binding pockets of host
kinases will be reported.
KuFal194 KuWal151
(1) Falke, H., et al., 2015. J. Med. Chem. 58, 3131-3143.
(2) Walter, A., 2015. Dissertation Technische Universität Braunschweig.
“DYRK1A, related kinases & human disease” 38
DYRK1A overexpression contributes to enhanced astrogliogenesis in a Down
syndrome mouse model
Nobuhiro KURABAYASHI and Kamon SANADA
Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku,
Tokyo 113-0033, Japan
Down syndrome (DS) is caused by trisomy for human chromosome 21. Individuals with DS commonly
exhibit mental retardation, which is associated with anomalies in brain development. In the neocortex of
DS brain, the density of neurons is remarkably reduced, whereas that of astrocytes is increased.
Similar to abnormalities seen in DS brain, mouse models of DS show deficits in brain development, and
neural progenitor cells that give rise to neurons and glia show mis-regulation in their differentiation.
These suggest that the mis-regulation of progenitor differentiation contributes to alteration in numbers
of neurons and astrocytes in DS brain. Nevertheless, the molecular basis underlying these defects
remains largely unknown. We demonstrated that increased dosage of DYRK1A contributes to
enhanced astrocytic differentiation of progenitors in the Ts1Cje mouse model of DS. Further, we link
the increased dosage of DYRK1A to elevated activity of STAT, a transcription factor critical for
astrogliogenesis. Together, our findings indicate that potentiation of the DYRK1A-STAT pathway in
progenitors contributes to aberrant astrogliogenesis in DS.
(1) Kurabayashi N et al. (2015) EMBO Rep. 16, 15481562
(2) Kurabayashi N and Sanada K. (2013) Genes Dev. 27, 2708-2721
(3) Kurabayashi N et al. (2010) Mol. Cell. Biol. 30, 1757-1768.
Funding: Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of
Japan, and research grants from the Mitsubishi Foundation and Takeda Science Foundation
“DYRK1A, related kinases & human disease” 39
Identification of new DYRK2 substrates and their implications in carcinogenesis.
Maribel LARA-CHICA, Rosario MORRUGARES, Carla JIMENEZ-JIMENEZ, Eduardo MUNOS, Marco
A. CALZADO CANALE
Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC). Avda. Menéndez Pidal, s/n. 14004. Córdoba. Spain
Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a relevant Ser/Thr
kinase involved in the regulation of cell processes such as cytokinesis, cell proliferation and
differentiation (1). Several studies have shown its important role in carcinogenesis, due to the ability to
phosphorylate and regulate some relevant proteins implicated in this process, such as p53, c-Jun/c-
Myc, SIAH2, Snail or Rpt3 (2, 3). These data have centred the attention of the scientific community on
a key candidate as a target for anticancer therapy. Nevertheless, there are few known substrates able
to be regulated by DYRK2. In this sense, it is highly important to further study how this kinase exerts its
function during tumour development and progression, through the identification of new substrates that
take an active part in these processes.
To assess this point, we performed several technical approaches (kinase array and MS/MS) in order
to identify new potential DYRK2 substrates. We obtained a list of relevant proteins involved in the
different steps of carcinogenesis, including CDC25A among them. Given the essential role of this
phosphatase as a key regulator of the cell-cycle progression, we focused on studying the effect of
DYRK2 on CDC25A. Our study demonstrates that DYRK2 down-regulates CDC25A expression,
facilitating its proteasomal degradation. This degradation depends on DYRK2 kinase activity, being
this kinase the most effective in degrading CDC25A among the rest of the members of its family.
This effect is independent of HIPK2 and ATM/ATR activities. An important inverse correlation of the
expression of DYRK2/CDC25A was detected under different stimuli of the kinase and its specific
silencing by siRNA. Furthermore, this inverse correlation was confirmed in lung cancer cells, which
occurs specially in a cellular model system of bronchial epithelial cell squamous differentiation.
Taken together, our findings show DYRK2 as a new regulator of relevant proteins involved in
tumour development and progression, such as CDC25A, what helps to improve our knowledge of the
tumorigenic process and could open a road to the development of new therapeutic strategies against
cancer.
(1) Aranda et al., 2011. FASEB J. 25, 449-62.
(2) Nihira and Yoshida, 2015. Cell Cycle 14, 802-807.
(3) Guo et al., 2016. Nat. Cell Biol. 18, 202-212.
Financial support: this work was supported by MINECO (SAF2016-75228-R). MLC was supported by an FPU fellowship
(FPU13/03393) from MECD.
“DYRK1A, related kinases & human disease” 40
Identification of a new function of Cystathionine β-Synthase (CBS) and
characterization of its link with DYRK1A using S. cerevisiae
Alice LEON1, Nadège LOAEC1, Yann HERAULT2,3, Marc BLONDEL1, Gaëlle FRIOCOURT1
1Inserm UMR1078, Faculté de Médecine et des Sciences de la Santé, Université de Bretagne Occidentale, Brest, France.
2IGBMC, CNRS, Illkirch, France.3Institut Clinique de la Souris, Illkirch, France.
Cystathionine -Synthase (CBS) is an enzyme misregulated in two inherited intellectual
deficiencies (ID): homocystinuria, which is caused by CBS loss-of-function mutations, and Down
syndrome (DS), where CBS overexpression, due to its localization on the chromosome 21, would
therefore be responsible for the cognitive phenotype of patients. In this latter disease, CBS along with
DYRK1A are genes thought to contribute to ID in DS, and several studies in mouse suggest a genetic
and probably functional link between these two genes.
Modulating the activity of CBS is therefore a promising therapeutic strategy to reduce cognitive
disorders of patients. Yet, to date efforts have failed to identify drugs that efficiently and specifically
inhibit CBS activity in vivo
In order to identify cellular partners of CBS, as well as some of its potential modifier genes, we
set up a genetic screen based on a yeast model overexpressing CYS4, the yeast homolog of CBS.
Indeed, the identification of genes related to CBS, and particularly to the consequences of its
overexpression, may help to define new therapeutic targets for CBS-related ID, in particular in DS
patients.
Our genetic screens led to the identification of several genes that suggest a previously
undescribed role for Cys4p/CBS in vesicular trafficking, which is a central pathway for synaptic
transmission. In addition, we found that both MCK1 and YAK1, the yeast homologs of human GSK3
genes and DYRK1A respectively, modulate the phenotypic consequences of CYS4 overexpression in
yeast. These results suggest that the functional link between these genes, which has been highlighted
in several studies, is conserved in S. cerevisiae.
A more extensive analysis of these pathways, first in yeast, then in mammalian cells, will help us
to better understand how a misregulation of CBS activity can cause ID, and thus to propose new
therapeutic strategies.
Financial support :
Recherche ». This work is funded by the Fondation Jérôme Lejeune and ITMO BCDE (Biologie Cellulaire, Développement et
Evolution).
“DYRK1A, related kinases & human disease” 41
The role of DYRK1A in DNA repair
Vijay MENON, Varsha ANANTHAPADMANABHAN, Larisa LITOVCHICK
Department of Internal Medicine and Massey Cancer Center, 401 College Street, Richmond, Virginia, 23298, USA.
The function of DYRK1A protein kinase is regulated by its gene dosage whereby both gains and
losses of one copy of DYRK1A gene on chromosome 21 result in developmental abnormalities. In order
to better understand the function and regulation of DYRK1A, we applied MudPIT proteomic approach to
identify DYRK1A-interacting proteins in human cells. Four biological replicate experiments were
performed to identify proteins reproducibly detected in the DYRK1A immunoprecipitates but not in the
controls. Six proteins detected in all four biological replicate experiments were also most highly
enriched in the DYRK1A immunoprecipitates, suggesting that these proteins form stable and abundant
complexes with DYRK1A. One of these proteins, RNF169, has been recently characterized as a
component of ubiquitin-mediated cascade involved in the repair of DNA double-strand breaks (DSBs).
Presence of specific chromatin marks including ubiquitination regulates the choice between two major
DSB repair pathways: homologous recombination repair (HRR) and non-homologous end joining
(NHEJ), mediated by recruitment of chromatin-binding DNA damage response proteins including
53BP1 and RNF169. Binding of 53BP1 could prevent the resection of the DNA strands near the
damage site necessary for the HRR while RNF169 is thought to promote the HRR by limiting 53BP1
accumulation. To determine whether DYRK1A plays a role in these processes, we knocked out its
expression in human and mouse cell lines using CRISPR-Cas9 approach. We found that both the
number of the 53BP1 IRIFs and their persistence over time were significantly reduced in the cell lines
that lacked DYRK1A. This effect was dependent on the presence of RNF169, suggesting that DYRK1A
at the DSBs. Next, we sought to
determine the mechanism of this regulation and found that RNF169 is phosphorylated by DYRK1A at
two sites located in a highly conserved domain with no known function. Interestingly, the phospho
mimetic mutant of RNF169 displayed a decreased ability to inhibit 53BP1 IRIF formation when
compared to the wild type or the non-phosphorylatable RNF169 alleles. Since loss of DYRK1A could be
relevant to cancer due to its widespread gene copy number losses, we determined the effect of
DYRK1A loss on the ability of the cells to repair their DNA. Using the DR-GFP reporter of HRR and the
neutral comet assays, we found that loss of DYRK1A results in an increased efficiency of the DNA DSB
repair. Our findings implicate DYRK1A in the critical processes of DNA damage response and reveal a
novel function of this important protein kinase.
Financial support: NIH R01CA188571
“DYRK1A, related kinases & human disease” 42
Multilayered proteomic analysis of cancer mutations in the Dyrk2 kinase complex
Martin MEHNERT, Matthias GSTAIGER, Ruedi AEBERSOLD
Institute of Molecular Systems Biology, Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland
Dual-specificity tyrosine-phosphorylation-regulated kinase 2 (Dyrk2) belongs to the class II
(Dyrk2, Dyrk3, Dyrk4) kinases of the Dyrk family and is suggested to be involved in regulating key
cellular processes such as cell proliferation, cytokinesis and cellular differentiation. Furthermore, Dyrk2
functions in the DNA damage response by phosphorylating p53 thereby promoting cellular apoptosis
upon genotoxic stress (1). A dysregulation and mutations of Dyrk2 were found in various cancer types
classifying Dyrk2 as potential oncogene. In addition to its kinase function Dyrk2 acts as a scaffold for
the assembly of a multifunctional protein complex encompassing the ubiquitin ligase Ubr5 and the
substrate receptor subunit DDB1-VprBP (2). The Dyrk2-dependent phosphorylation of recruited
substrates is required for the subsequent ubiquitylation by the E3 ligase and its proteasomal
degradation. Substrates of the Dyrk2 kinase complex are the AAA-ATPase katanin p60 acting in the
reorganization of spindle microtubules during mitosis and the catalytic subunit of the telomerase
(TERT) whose dysregulation is reported in HIV disease and cancer (3).
We performed a proteomic interaction analysis of the Dyrk2 kinase network using affinity
purification mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID-MS) and
identified about 80 high confident interactors, in particular factors of cell cycle regulation, apoptosis and
nuclear transport. The integration of cancer associated point mutations into Dyrk2 affected the
interaction network and caused a disassembly of the Dyrk2 kinase complex. Proteomic and
phosphoproteomic profiling of wild-type and CRISPR/Cas9 engineered Dyrk2 knockout cells by label-
free quantitative mass spectrometry (SWATH-MS) revealed 189 differentially regulated proteins and
about 60 altered phospho
the various cancer related Dyrk2 mutants followed by a topological and structural analysis of the cancer
perturbed kinase complex by cross-linking coupled mass spectrometry.
(1) Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K, 2007. Dyrk2 is targeted to the nucleus and
controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol. Cell. 25,
725-738.
(2) Maddika S, Chen J, 2009. Protein kinase Dyrk2 is a scaffold that facilitates assembly of an E3
ligase. Nat. Cell Biol. 11, 409-419.
(3) Jung HY, Wang X, Jun S, Park JI, 2013. Dyrk2-associated EDD-DDB1-VprBP E3 ligase inhibits
telomerase by TERT degradation. J. Biol. Chem. 288, 7252-7262.
“DYRK1A, related kinases & human disease” 43
Leucettines, a family of DYRK1A inhibitors: from marine sponge to drug
candidate
Laurent MEIJER1, Thu Lan NGUYEN1,2, Morgane CAM1, Tania TAHTOUH1, Emilie DURIEU1,
Emmanuelle LIMANTON3, Benoît VILLIERS1, Céline BRUYERE1, Xavier FANT4, Stefan KNAPP5,
Benoît SOUCHET6, Jérôme BRAUDEAU6,7, Antigoni MANOUSOPOULOU8, Spiros D. GARBIS8,
Arnaud DUCHON2, Yann HERAULT2,9, François CARREAUX3, Jean-Pierre BAZUREAU3
1ManRos Therapeutics, Roscoff, France. 2Institut de Génétique Biologie Moléculaire et Cellulaire, Illkirch, France. 3Université
de Rennes 1, Laboratoire Sciences Chimiques, Rennes, France. 4Station Biologique de Roscoff, France. 5Institute for
Pharmaceutical Chemistry, Goethe-University & Buchmann Institute for Molecular Life Sciences, Frankfurt am Main, Germany.
6MIRCen, CEA & Université Paris Saclay, France. 7AgenT, Fontenay-aux-Roses, France. 8Cancer Sciences & Clinical &
Experimental Medicine, University of Southampton, UK. 9Institut Clinique de la Souris, Illkirch, France.
There is growing evidence implicating DYRK1A in the onset and development of
Leucettines, an archetype of DYRKs inhibitors, will be reviewed in this presentation (1-3).
A random screen of natural products allowed us to identify the marine sponge Leucettamine B
as an inhibitor of DYRKs. Synthesis of over 500 analogues (collectively referred to as Leucettines) led
to a first optimized product, Leucettine L41. Leucettines were co-crystallized with DYRK1A, DYRK2,
CLK3, PIM1 and GSK-3. The selectivity of Leucettine L41 was extensively studied using 4 different
methods. A SAR study was carried out with 78 Leucettines which were tested for their inhibitory action
on 11 recombinant kinases and in five cellular assays (modulation of CLK1 pre-mRNA splicing,
protection towards glutamate -induced cell death, induction of autophagy, phosphorylation of Tau
Thr212, phosphorylation of cyclin D1 Thr286). Optimization of Leucettines towards a clinical drug
candidate implies the development of an orally available drug able to cross the blood brain barrier while
maintaining selectivity, potency and patentability. Recent progress in this direction will be presented.
Leucettine L41 was tested in two DS mouse models and 2 AD mouse models as will be
presented by our collaborators. Both tgBACDyrk1a mice (a model expressing three DYRK1A gene
copies) and Ts65Dn mice (a partial trisomy model) models overexpress DYRK1A and display cognitive
impairment related to DS and AD. Leucettine L41 treatment led to normalization of the DYRK1A activity
and fully corrected the deficits seen in these DS models. Leucettine L41 also prevented cognitive
deficits triggered by icv injection of amyloid peptide A25-35 as well as those observed in the
APP/PS1E9 transgenic mice model of AD. Extensive proteomic and phosphoproteomic studies are
carried out to elucidate the molecular actions of Leucettine L41 in the brains of the mouse models as
well as in cellular models (cells expressing human DYRK1A or kinase-dead DYRK1A). Altogether these
results confirm that Leucettines are able to cross the blood brain barrier, to normalize DYRK1A activity,
to modify specific phosphorylation patterns and to restore normal cognitive functions in 2 DS and 2 AD
animal models.
Leucettines deserve further development as potential therapeutics against neurodegenerative
diseases, and possibly other diseases,
(1) Debdab, M. et al., 2011. Leucettines, a class of potent inhibitors of cdc2-like kinases and dual
specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine
B. Modulation of alternative pre-RNA splicing. J. Med. Chem. 54, 4172-4186.
(2) Tahtouh, T. et al., 2012. Selectivity, co-crystal structures and neuroprotective properties of
Leucettines, a family of protein kinase inhibitors derived from the marine sponge alkaloid
Leucettamine B. J. Med. Chem. 55, 9312-9330.
(3) Fant, X. et al., 2014. Cdc-like/dual-specificity tyrosine phosphorylation-regulated kinases Leucettine
L41 induces mTOR-Mol. Pharmacol. 85,
441-450.
Financial support : CRITT-Santé Bretagne, Fondation Jérôme Lejeune, France Alzheimer 29, Fonds Unique Interministériel
projets PHARMASEA & TRIAD, Pôle Mer Bretagne, FP 7-PEOPLE-2011---
01 BLUEGENICS project.
“DYRK1A, related kinases & human disease” 44
2-Substituted indole-3-carbonitriles as new DYRK inhibitors
Rosanna MEINE1,2, Nadège LOAEC3, Laurent MEIJER3, Conrad KUNICK1,2
1Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig, Beethovenstraße 55, 38106
Braunschweig, Germany. 2Center of Pharmaceutical Engeneering (PVZ), Technische Universität Braunschweig, Franz-Liszt-
Straße 35A, 38106 Braunschweig, Germany. 3ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, France.
In our group the DYRK1A inhibitor 10-iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acid was
developed. It exhibits good inhibitory activity against DYRK1A (IC50 = 6 nM) and is highly selective
against other DYRKs and CLK kinases. However, in cellular assays the DYRK1A inhibition was
considerably lower because of poor physicochemical properties (1).
Here we report a fragment-based drug design starting from 7-chloro-1H-indole-3-carbonitrile as
a small analogue of the 10-halogen substituted 11H-indolo[3,2-c]quinolone-6-carboxylic acids. During
the development also water solubility and other physicochemical properties were taken into account.
Analogous to the 11H-indolo[3,2-c]quinolone-6-carboxylic acids a 7-iodo substituent was favorable.
Furthermore, various residues at the 1 and 2 position of the indole scaffold were introduced. A phenyl
substituent in position 2 led to potent DYRK1A inhibitors. An additional methyl substitution at the indole
nitrogen led to a very potent and selective DYRK1B inhibitor. The synthesis of the compound series,
docking experiments and the results of enzyme assays will be presented.
(1) Falke, H. et al., 2015. J. Med. Chem. 58, 3131-3143.
Financial support: this work was supported by the state of Lower Saxony, Germany, by a Georg-Christoph-Lichtenberg-
“DYRK1A, related kinases & human disease” 45
Functional regulation of different DYRK family protein kinases by distinctive
cellular binding partners
Yoshihiko MIYATA, Eisuke NISHIDA
Department of Cell & Developmental Biology, Graduate School of Biostudies, Kyoto University. Kitashirakawa Oiwake-cho,
Sakyo-ku, Kyoto 606-8502, Japan.
The human DYRK family consists of mutually-related five protein kinases, DYRK1A, DYRK1B,
DYRK2, DYRK3, and DYRK4.
chromosome 21, and plays an important role in the functional and developmental regulation of many
types of cells, including neuronal cells. Physiological roles of other members of DYRK family remain
less evident. Here we report identification of cellular proteins that associate with specific members of
DYRKs. We identified WDR68 (DCAF7), an evolutionarily conserved WD40-repeat protein, as a cellular
binding partner of DYRK1A. WDR68 was originally identified in petunia as a factor controlling the
transcription of flower anthocyanin biosynthetic genes. WDR68 was indispensable for the proliferation
and survival of mammalian cells. DYRK1A and DYRK1B, but not other DYRKs, bound to WDR68 via
the N-terminal domains. Importantly, DYRK1A-binding induced nuclear accumulation of WDR68. We
then identified the molecular chaperone TRiC/CCT as a major WDR68-binding protein. Knockdown of
cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its
DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the
knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the
TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT
is essential for correct protein folding, DYRK1A-binding, and nuclear accumulation of WDR68.
We also found that molecular chaperones Hsp90, Cdc37, and Hsp70 associated with DYRK1B
and DYRK4, but not with other DYRKs. Treatment of cells with an Hsp90 inhibitor geldanamycin
induced dissociation of Hsp90 and Cdc37, but not Hsp70, from DYRK1B and DYRK4. DYRK1B and
DYRK4 underwent formation of cytoplasmic aggregation and degradation by the Hsp90 inhibitor,
suggesting that the chaperone function of Hsp90/Cdc37 is required for solubility and stability of these
kinases. Altogether, we suggest that DYRK family protein kinases are distinctively regulated by
respective binding partners in cells.
“DYRK1A, related kinases & human disease” 46
Developing inhibitors of the kinases that regulate alternative splicing
Jonathan C. MORRIS
School of Chemistry, University of New South Wales, Sydney, Australia
The splicing of pre-mRNA is one of the main processes that influence protein diversity in
humans and recent studies have estimated that up to 88% of multi-exon protein coding genes are
alternatively spliced. This diversity highlights the importance that alternative splicing plays in regulatory
affairs, such as cell growth, differentiation and apoptosis. Alternative splicing is carried out by cellular
machinery known as the spliceosome and this machinery is regulated by the phosphorylation of key
splicing factors. By controlling the phosphorylation events, it is possible to regulate the production of
protein isoforms.
To achieve this, we have been developing small molecule inhibitors of the kinases (such as the
DYRKs, CLKs and SRPK1) responsible for the phosphorylation events. The pyrrolopyrimidine scaffold,
as seen in variolin B and 1, has been found to yield potent inhibitors of the CLKs and DYRKs. We have
also identified other scaffolds that are potent SRPK1 inhibitors.
Preliminary biological data will be presented that demonstrates how some of these inhibitors
can be used in the regulation of Treg differentiation and function, as well as in the regulation of the
alternative splicing of VEGFA, one of the critical proteins involved in angiogenesis.
“DYRK1A, related kinases & human disease” 47
Deciphering the molecular mechanisms of action of pharmacological DYRK1A
kinase inhibitors for therapeutic use in Down Syndrome preclinical models.
Thu Lan NGUYEN1,2, Arnaud DUCHON1, Antigoni MANOUSOPOULOU4, Spiros D. GARBIS4,
Emmanuelle LIMANTON5, François CARREAUX5, Jean-Pierre BAZUREAU5, Laurent MEIJER2, Yann
HERAULT1,3
1Institut de Génétique Biologie Moléculaire et Cellulaire, IGBMC, CNRS, INSERM, Université de Strasbourg, UMR7104,
UMR964, 1 rue Laurent Fries, 67404 Illkirch, France 2ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff,
France 3Institut Clinique de la Souris, CELPHEDIA/PHENOMIN, CNRS, INSERM, Université de Strasbourg, 1 rue Laurent
Fries, 67404 Illkirch, France 4Cancer Sciences & Clinical and Experimental Medicine, University of Southampton, Highfield,
Southampton, UK 5Université de Rennes 1, Laboratoire Sciences Chimiques, 35042 Rennes, France.
Down Syndrome or Trisomy 21 (DS), is due to the presence of an extra copy of chromosome
21. As the most frequent mental retardation it affects about 1 new born per 700 births.
Among the candidates implicated in DS intellectual disabilities, the Dual Specificity Tyrosine
Phosphorylation Regulated Kinase, DYRK1A, found in the DS critical region of chromosome 21, is one
of the most relevant (1). Indeed, several studies have shown a correlation between an increase of its
kinase activity and the intellectual defects observed in DS models. EGCG, known to inhibit DYRK1A,
was successfully used to rescue cognitive traits in mice and human (2, 3).
In order to understand the mechanisms underlying the impact of DYRK1A dosage on cognitive
alterations, we used several trisomic mouse models expressing DYRK1A alone or with additional
Hsa21 homologous genes and specific DYRK1A inhibitors from ManRos Therapeutics.
We will present here the consequence of treatments with Leucettine 41, a synthetic DYRK1A
inhibitor, following repetitive administration to several DS mouse models on the behaviour and cognition
and on several activities of DYRK1A. Further analysis of the phosphoproteome of DS mouse models
treated or not with L41 unravels a few targets and pathways which are involved in the restoration of
cognitive capacities of DS models, highlighting molecular synaptic mechanisms. These results support
the potential of more selective DYRK1A inhibitor as a therapeutic approach to improve cognitive
functions in DS patients.
(1) Duchon, A. & Herault, Y., 2016. DYRK1A, a Dosage-Sensitive Gene Involved in
Neurodevelopmental Disorders, Is a Target for Drug Development in Down Syndrome. Front.
Behav. Neurosci. 10, 104.
(2) De la Torre, R., De Sola, S., Pons, M., Duchon, A., de Lagran, M. M., Farré, M., Fitó, M., Benejam,
B., Langohr, K., Rodriguez, J., Pujadas, M., Bizot, J. C., Cuenca, A., Janel, N., Catuara, S., Covas,
M. I., Blehaut, H., Herault, Y., Delabar, J. M. & Dierssen, M., 2014. Epigallocatechin-3-gallate, a
DYRK1A inhibitor, rescues cognitive deficits in Down syndrome mouse models and in humans. Mol.
Nutr. Food Res. 58, 278-288.
(3) De la Torre, R., de Sola, S., Hernandez, G., Farré, M., Pujol, J., Rodriguez, J., Espadaler, J. M.,
Langohr, K., Cuenca-Royo, A., Principe, A., Xicota, L., Janel, N., Catuara-Solarz, S., Sanchez-
Benavides, G., Bléhaut, H., Dueñas-Espín, I., Del Hoyo, L., Benejam, B., Blanco-Hinojo, L., Videla,
S., Fitó, M., Delabar, J. M., Dierssen, M. & group, T.S., 2016. Safety and efficacy of cognitive
training plus epigallocatechin-3-gallate in young adults with Down's syndrome (TESDAD): a double-
blind, randomised, placebo-controlled, phase 2 trial. Lancet Neurol. 15, 801-810.
Financial support : CIFRE, FUI TRIAD, Fondation Jérôme Lejeune.
“DYRK1A, related kinases & human disease” 48
Multimodal regulation of the microtubule cytoskeleton by DYRK1a
Brigette JONG, Tracy TAN, Melissa BORDEN, Kassandra M. ORI-MCKENNEY
One Shields Avenue, 145 Briggs, University of California, Davis, Davis, CA 95616, USA
Cellular architecture is governed by the organization of cytoskeletal networks and determines
the functional output of a cell. It is therefore essential to understand the regulatory mechanisms of
cytoskeleton organization as a cell develops, changes, or maintains its internal structure, because
altering these processes can disrupt cell function and ultimately lead to pathological conditions. We and
others have shown that DYRK1a regulates the microtubule cytoskeleton directly by phosphorylating
beta-tubulin, and indirectly through phosphorylation of the microtubule associated protein, Tau. We
have utilized a chemical genetic approach to identify direct downstream substrates of Drosophila MNB
in larval lysates, as well as mammalian DYRK1a in brain lysates, and discovered additional conserved
proteins that act upon the microtubule cytoskeleton. Using in vitro kinase assays, single molecule TIRF
microscopy, and in vivo neuronal imaging, we have begun to characterize the role of DYRK1a in
shaping the microtubule landscape through differential regulation of the microtubule associated
proteins, Tau and MAP7. By analyzing neurons from mitosis to neuronal differentiation and pruning, we
hope to provide a complete map of the DYRK1a kinase network during neuronal development, and
elucidate the multiple ways in which this kinase modulates the microtubule cytoskeleton during different
cellular processes.
Financial support: this project is supported by NIH grant R00HD080981 to KMOM.
“DYRK1A, related kinases & human disease” 49
Drug-target engagement and efficacy of DYRK1A inhibitors in glioblastoma cells
Athena F. PHOA1, Qingqing ZHOU2, Ramzi H. ABBASSI1, Monira HOQUE1, Brett W. Stringer3, Bryan
W. DAY3, Terrance G. JOHNS4, Michael KASSIOU2, Lenka MUNOZ1
1School of Medical Sciences, The University of Sydney, NSW 2006. 2School of Chemistry, The University of Sydney, NSW
2006. 3QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, QLD 4006. 4Oncogenic Signaling Laboratory,
Centre for Cancer Research, Hudson Institute of Medical Research, 27 Wright Street, Clayton, VIC 3168, Australia.
Glioblastoma is an aggressive brain tumour resistant to conventional chemo- and radiotherapy.
All patients succumb to the disease within two years, and glioblastoma remains a major unmet medical
need. Amplification and mutations of the epidermal growth factor receptor (EGFR) occur in over 60% of
glioblastoma cases. Recent evidence suggests that to effectively target EGFR, complete degradation
and removal of the receptor is necessary, as sole inhibition of the kinase catalytic function is insufficient
to achieve anti-cancer effects (1, 2). Dual-specificity tyrosine phosphorylation-regulated kinase 1A
(DYRK1A) is up-regulated in glioblastomas and has been shown to prevent endocytotic degradation of
EGFR (3). DYRK1A activity results in enhanced EGFR signalling and tumour growth. Therefore,
inhibition of DYRK1A is a potential therapeutic intervention for EGFR-dependent glioblastomas. A
series of novel DYRK1A inhibitors was designed and synthesized. The library was screened for their
anti-cancer efficacy in established and patient-derived glioblastoma cell lines with varying EGFR
expression. The most potent DYRK1A inhibitors in the series (IC50
numerous cell lines (EC50 M) and promoted EGFR degradation by decreasing its half-life by 3-fold.
In addition to their anti-proliferative properties, the most potent inhibitors also reduced migration and
invasion of glioblastoma cells. Target engagement was confirmed with genetic knockdown and the
cellular thermal shift assay (CETSA) (4). We demonstrate that DYRK1A knockdown phenocopies
bility in cells is
increased upon drug treatment, confirming that these drugs bind to DYRK1A in cells. In summary, we
present detailed pharmacology investigation of novel DYRK1A inhibitors and identification of lead
compounds with anti-cancer properties necessary to combat glioblastoma.
(1) Z Weihua, et al., 2008. Cancer Cell 13, 385-393.
(2) X Tan, et al., 2015. Cell 160, 145-160.
(3) N Pozo, et al., 2013. J. Clin. Invest. 123, 2475-2478.
(4) S Gourdain, et al., 2014. Nat. Protocols 9, 2100-2122.
“DYRK1A, related kinases & human disease” 50
DYRK1A haploinsuficiency is a frequent cause of intellectual disability: How to
better diagnose it?
Angélique QUARTIER1, Marjolaine WILLEMS2, Marie VINCENT3, Cyril MIGNOT4, Salima EL
CHEHADEH5, Michèle MATHIEU-DRAMARD6, Vincent LAUGEL7,8, Nadège CAMELS9, Bénédicte
GERARD9, Jean-Louis MANDEL1,9, Amélie PITON1,9
1Département de Médicine translationnelle et Neurogénétique, IGBMC, CNRS UMR 7104/INSERM U964/Université de
Strasbourg, Illkirch, France Chaire de Génétique Humaine, Collège de France, Illkirch, France; 2Département de Génétique
Médicale, Centre de Référence Maladies Rares Anomalies du Développement et Syndromes Malformatifs Sud-Languedoc
Roussillon, Hôpital Arnaud de Villeneuve, Montpellier, France; 3Service de Génétique Médicale, CHU de Nantes, Nantes,
France; 4Assistance Publique-Hôpitaux de Paris, Département de Génétique and Centre de Référence Déficiences
Intellectuelles de Causes Rares and GRC UPMC "Déficiences Intellectuelles et Autisme", Groupe Hospitalier Pitié-Salpêtrière,
Paris, France; 5Département de Génétique, CHU de Hautepierre, Strasbourg, France; 6Unité de Génétique Clinique, CHU
d'Amiens, Amiens, France; 7Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg,
Strasbourg, France; Département de Neuropédiatrie, Hôpital de Hautepierre, CHU de Strasbourg, Strasbourg, France;
8Laboratoire de Génétique Médicale, INSERM U1112, Faculté de Médecine de Strasbourg, Hôpitaux Universitaires de
Strasbourg, Strasbourg, France; 9Laboratoire de diagnostic génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg,
France.
Heterozygous de novo loss-of-function mutations in DYRK1A cause a syndromic form of
Intellectual disability (ID) associated with growth retardation including microcephaly, specific
dysmorphy, stereotypy, ataxia and other variable clinical features (Mental Retardation, autosomal
dominant 7, MRD7). MRD7 is a frequent cause of ID as loss-of-function mutations are found in around
0.5% of ID patients. Since recently, several de novo missense variations have been identified,
sometimes in patients not highly evocative of MRD7 which raises the question of their true
pathogenicity. Therefore, functional tests need to be developed to establish if these missense variants
participate, fully or partially, to the cognitive impairments observed in these patients. DYRK1A encodes
a protein serine/threonine kinase expressed throughout life, involved in many cellular processes, and
particularly in the regulation of gene expression. We are performing transcriptomic studies to identify
test the effect on DYRK1A function of the different missense variants identified. In parallel, we have
undertaken to better define the clinical manifestations associated to DYRK1A mutations in French
patients. This better delineation of the clinical spectrum of MRD7 and the development of functional
tests will be useful to improve diagnosis of patients affected by this frequent form of ID. This will also
allow to better understand the normal and pathological functions of DYRK1A.
(1) Bronicki LM et al., 2015. Ten new cases further delineate the syndromic intellectual disability
phenotype caused by mutations in DYRK1A. Eur. J. Hum. Genet. 23, 1482-1487.
(2) Redin C et al., 2014. Efficient strategy for the molecular diagnosis of intellectual disability using
targeted high-throughput sequencing. J. Med. Genet. 51, 724-736.
“DYRK1A, related kinases & human disease” 51
Interaction of the protein kinase DYRK1A with RNF169 suggests a role for this
kinase in DNA repair
Julia ROEWENSTRUNK1,2, Chiara DI VONA1,2, Eva BORRAS1,2, Eduard SABIDO1,2, Susana DE LA
LUNA1,2,3
1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona
08003, Spain. 2Universitat Pompeu Fabra (UPF), Barcelona, Spain. 3Institució Catalana de Recerca i Estudis Avançats
(ICREA), Pg. Lluís Companys 23, Barcelona 08010, Spain.
DYRK1A (dual-specificity tyrosine-regulated kinase) is overexpressed in Down syndrome
individuals. This overexpression is proposed to be associated to risk of childhood leukemia and
neurodevelopmental abnormalities. On the other hand, reduced levels of DYRK1A due to truncating
mutations cause a rare human syndrome characterized by intrauterine growth retardation and
microcephaly, among other clinical traits. Although the list of functional activities associated to DYRK1A
has increased in recent years, a complete picture on DYRK1A physiological roles is still missing. To
better understand DYRK1A activities an interactome analysis based on affinity DYRK1A purification
coupled to mass spectrometry analysis was undertaken. RNF169 (Really interesting new gene finger
protein 169), an E3-ubiquitin ligase described as a key component of the cellular response to double-
strand breaks (DSBs), appeared as one of the top DYRK1A nuclear targets, and the functional
characterization of this interaction has uncovered a novel physiological role for DYRK1A. DYRK1A
interacts directly with RNF169 through a dedicated motif in the non-catalytic N-terminus, and this
interaction is essential for the recruitment of DYRK1A to DSB sites. RNF169 is a substrate of DYRK1A
in vitro and in vivo. Using a combination of mass spectrometry analysis, mutagenesis and in vitro
kinase assays, several DYRK1A-dependent phosphosites have been identified in RNF169.
Interestingly, DYRK1A knockdown leads to increased radiation sensitivity. Our results point to a
possible role for DYRK1A in the regulation of DNA-damage response through phosphorylation of the
E3 ubiquitin ligase RNF169.
“DYRK1A, related kinases & human disease” 52
Retinal phenotypes in murine models of Down syndrome
Michel J. ROUX, Charlotte AMIOT, Damien MARECHAL, Yann HERAULT
IGBMC, Université de Strasbourg - CNRS UMR 7104, INSERM U964, 1 rue Laurent Fries, 67404 ILLKIRCH, France
The retina of Down syndrome patients is thicker than the one from controls, an anomaly
reproduced in two murine models of the disease, Ts65Dn and Tg(Dyrk1a) and attributed to the
increased dosage of Dyrk1a (1). As HSA21 genes not triplicated in Ts65Dn may affect the
expression/function of DYRK1A, we have analyzed by optical coherence tomography (OCT) the retina
of Ts65Dn and Tg(Dyrk1a) mice, and compared the results to those obtained on various additional
models, notably Dp(16)1 Yeh mice, which have a triplicated segment of MMU16 longer than Ts65Dn,
and the crosses between Tg(Dyrk1a) and Dp1Yah, which carries a triplication of the MMU17 genes
orthologue to HSA21. As Down syndrome patients present anterior segment defects, as myopia and
keratoconus, at a higher frequency than controls, we also measured by OCT corneal thickness, as well
as anterior chamber and vitreous chamber depths. In parallel, we have started to analyze by
immunohistochemistry the retinal neuron populations in Tg(Dyrk1a) mice, showing that most of the
increase in retinal thickness is due to changes in the amacrine cell population, which is more affected
than bipolar cells. Mirror results were obtained on the retina of Dyrk1a+/- mice, in which the amacrine
cell population is strongly reduced, as the retinal thickness.
(1) Laguna et al., 2103. Triplication of DYRK1A causes retinal structural and functional alterations in
Down syndrome. Hum. Mol. Genet. 22, 2775-2784.
“DYRK1A, related kinases & human disease” 53
DYRK1B and Hedgehog signaling: a complex crosstalk
V Rajeev SINGH, Matthias LAUTH
Philipps University Marburg, Institute of Molecular Biology and Tumor Research (IMT), Center for Tumor- and Immunobiology,
35043 Marburg, Germany
Hedgehog (Hh) signaling plays important roles in embryonic development and in tumor
formation. Apart from the well-established stimulation of the GLI family of transcription factors, Hh
ligands promote the phosphorylation and activation of mTOR and AKT kinases, yet the molecular
mechanism underlying these processes are unknown. Here, we identify the DYRK1B kinase as a
mediator between Hh signaling and mTOR/AKT activation. In fibroblasts, Hh signaling induces
DYRK1B protein expression, resulting in activation of the mTOR/AKT kinase signaling arm.
Furthermore, DYRK1B exerts positive and negative feedback regulation on the Hh pathway itself: It
negatively interferes with SMO-elicited canonical Hh signaling, while at the same time it provides
positive feed-forward functions by promoting AKT-mediated GLI stability. Due to the fact that the
mTOR/AKT pathway is itself subject to strong negative feedback regulation, pharmacological inhibition
of DYRK1B results in initial upregulation followed by downregulation of AKT phosphorylation and GLI
stabilization. Addressing this issue therapeutically, we show that a pharmacological approach
combining a DYRK1B antagonist with an mTOR/AKT inhibitor results in strong GLI1 targeting and in
pronounced cytotoxicity in human pancreatic and ovarian cancer cells.
(1) Friedman E, 2010. The Kinase Mirk/dyrk1B: A Possible Therapeutic Target in Pancreatic Cancer.
Cancers (Basel) 2, 14921512.
(2) Schneider P, Miguel Bayo-Fina J, Singh R, Kumar Dhanyamraju P, Holz P, Baier A, Fendrich V,
Ramaswamy A, Baumeister S, Martinez ED, Lauth M, 2015. Identification of a novel actin-dependent
signal transducing module allows for the targeted degradation of GLI1. Nat. Commun. 6, 8023.
(3) Lauth M, Bergstrom A, Shimokawa T, Tostar U, Jin Q, Fendrich V, Guerra C, Barbacid M, Toftgard
R, 2010. DYRK1Bdependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS.
Nat. Struct. Mol. Biol. 17, 718725.
(4) Gruber W, Hutzinger M, Elmer DP, Parigger T, Sternberg C, Cegielkowski L, Zaja M, Leban J,
Michel S, Hamm S, Vitt D, Aberger F, 2016. DYRK1B as therapeutic target in Hedgehog/GLI-
dependent cancer cells with Smoothened inhibitor resistance. Oncotarget 7, 71347148.
Financial support: This work was supported by grants obtained from the von-Behring-Röntgen Foundation and the German
Research Society (DFG LA2829/6-1).
“DYRK1A, related kinases & human disease” 54
Leishmania infantum DYRK1: a negative regulator of the G1 to S cell-cycle
transition, essential for the development of infective stationary phase
promastigotes
Vinícius PINTO COSTA ROCHA1, Mariko DACHER2, Simon YOUNG3, Foteini KOLOKOUSI4, Antonia
EFSTATHIOU4, Gerald F. SPATH2, Milena B. P. SOARES1, Despina SMIRLIS2,4
1Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, Rua Waldemar Falcão 121, Centro de Biotecnologia e
Terapia Celular, Hospital São Rafael, Avenida São Rafael 2152,41253-190 Salvador, BA, Brazil; 2Unité de Parasitologie
moléculaire et Signalisation, Department of Parasites and Insect Vectors, Institut Pasteur and INSERM U1201, Paris, France;
3Biomedical Sciences Research Centre, School of Biology, The University of St. Andrews, The North Haugh, St. Andrews, Fife
Scotland KY16 9ST, UK; 4Molecular Parasitology Lab, Hellenic Pasteur Institute,127 Bas. Sofias Avenue, 115 21 Athens,
Greece.
Leishmaniases are devastating infectious diseases caused by protozoan parasites of the genus
Leishmania. Leishmania has a digenetic life cycle alternating between promastigote forms, which
develop in the sand-fly vector, and an amastigote form, which grows in mammals after being bitten by
an infected sand-fly. In the promastigote form cell cycle arrest in the G1 phase is an important process
whereby Leishmania transforms from poorly infective into highly infective parasites. Metacyclics are
metabolically and biochemically mimicked by stationary phase promastigotes. Dual-specificity tyrosine-
regulated kinases (DYRKs) are known to act as negative regulators of growth and as positive
regulators of cell differentiation (1). Interestingly, a Leishmania DYRK1 homologue (LinJ.15.0180)
contains both DYRK specific and Leishmania specific- domains. In this study, we used transgenic
L.infantum parasites that over-express LinDYRK1. LinDYRK1over-expression caused a significant
delay in proliferation, associated with a delayed cell-cycle G1/S transition. To knockout LinDYRK1, as
loss-of-function analyses are often lethal, we used an established facilitated LinDYRK1 knockout
approach that relies on the episomal expression of our gene from an episome that is susceptible to
drug induced negative selection. Persistence of the episome in logarithmic parasites during negative
selection was used as a readout of essentiality. Loss of the LinDYRK1 expressing episome in
logarithmic parasites, was observed albeit at a low rate, enabling us to conclude that LinDYRK1
deletion could be compensated. Phenotype analyses of LinDYRK1 knockout parasites showed that
°C temperature shift, a natural
environmental stress-factor occurring during stage differentiation. Moreover, LinDYRK1-/- parasites
displayed defects in stationary growth phase as they rounded up, showing an abnormal cell-cycle
distribution with a marked increase of G2-M/G1 ratio, and severe surface aberrations, accumulation of
vesicular structures and lipid body formation. Knockout mutants exhibited subtle but clear differences in
the lipid composition that could account for lipid body emulsion size and thermosensitivity. Finally, null
mutants showed a pronounced reduction in their ability to survive in mouse macrophages. Overall this
work highlights the role of LinDYRK1 in mediating the growth-stress response balance, to promote pro-
survival and fitness of infective stationary phase parasites.
(1) Aranda C, Laguna A, de la Luna, 2011. DYRK family of protein kinases: evolutionary relationships,
biochemical properties, and functional roles. FASEB J. 25, 449-462.
Financial support: ACIP, International Division Institute Pasteur.
“DYRK1A, related kinases & human disease” 55
Preventing DYRK1A catabolism in reactive astrocytes as a novel therapeutic
approach to treat Alzheimer’s Disease.
Benoît SOUCHET1,2, M. AUDRAIN1, JM BILLARD3, J. DAIROU4, R. FOL1, NS. OREFICE1, S. TADA1,
G. DUFAYET1, S. ALVES1, B. POTIER3, P. DUTAR3, L. MEIJER5, N. JANEL6, N. CARTIER1, J.
BRAUDEAU1,7
1 MIRCen, CEA Fontenay-aux-Roses / INSERM UMR 1169, France; 2Université Paris Saclay, Paris, France; 3INSERM
UMR894, Centre de Psychiatrie et Neurosciences, Paris, France; 4Université Paris Descartes, Paris, France; 5ManRos
Therapeutics, Roscoff, France; 6Université Paris Diderot, Paris, France; 7AgenT, Fontenay-aux-Roses, France.
DYRK1A, dual specificity tyrosine phosphorylation regulated kinase 1 A, is a broad spectrum
kinase involved in the phosphorylation of numerous proteins including TAU proteins, APP processing
and neuroinflammation-related proteins. Altogether DYRK1A plays a crucial role in the pathogenesis of
DYRK1A is a kinase suspected to be a potential target to treat AD. We thus, investigated DYRK1A in
AD. In human hippocampal biopsies, we first identified an original DYRK1A catabolism leading to
decrease DYRK1A full length protein levels in AD patients through Calpain II overactivation as
previously observed (1
AD mice model (APPswe, PSEN1dE9; APP/PS1), we then
confirmed this catabolism to be located in reactive astrocytes. Pharmacological targeting of DYRK1A
using the Leucettine L41 compound, previously described in vitro as a DYRK1A inhibitor (2), prevented
DYRK1A catabolism in reactive astrocytes. While astrogliosis was not modified, L41 treatment
promoted the recruitment of phagocytosis-specialized microglia releasing low levels of inflammatory
mediators. Of note, this tissue remodeling was associated with a decrease of amyloid-plaque load,
synaptic plasticity improvement and cognitive abilities rescue. Surprisingly, no difference in the total
endogenous DYRK1A activity was observed in AD patients or vehicle and L41 treated APP/PS1 mice
relative to matched controls. Therefore, our study validates a AD therapeutic strategy targeting
DYRK1A. However, we also provide the first evidence that it would be preferable to orient future
investigations towards the DYRK1A catabolism in reactive astrocytes to treat this devastating disease.
(1) Jin N, Yin X, Gu J, Zhang X, Shi J, Qian W, et al., 2015. Truncation and Activation of Dual
Specificity Tyrosine Phosphorylation-regulated Kinase 1A by Calpain I: a molecular mechanism
linked to Tau pathology in Alzheimer disease. J. Biol. Chem. 290, 15219-15237.
(2) Debdab M, Carreaux F, Renault S, Soundararajan M, Fedorov O, Filippakopoulos P, et al., 2011.
Leucettines, a class of potent inhibitors of cdc2-like kinases and dual specificity, tyrosine
phosphorylation regulated kinases derived from the marine sponge leucettamine B: modulation of
alternative pre-RNA splicing. J. Med. Chem. 54, 4172-4186.
“DYRK1A, related kinases & human disease” 56
De novo mutations of DYRK1A lead to a syndromic form of ID
Bregje WM VAN BON
Clinical geneticist, Radboud University Medical Center, Nijmegen, the Netherlands.
Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) maps to the
Down syndrome critical region; copy number increase of this gene are thought to play a major role in
the neurocognitive deficits associated with Trisomy 21. In 2011, we investigated whether smaller copy
number variations were present at the DYRK1A locus in 3000 individuals and could confirm one de
novo intragenic deletion of 52 kb in an individual who presented with intellectual disability (ID),
microcephaly, a broad based gait and specific behavior (1). We noted a striking similarity with features
previously observed in different animal models with mutated Dyrk1A.
Further studies of patients with ID and autism spectrum disorder (ASD) confirmed truncation of
DYRK1A is associated with loss-of-function mutations. To understand the phenotypic spectrum
associated with DYRK1A mutations, we resequenced the gene in 7,162 ASD/DD patients and 2,169
unaffected siblings (2). Comparison of our data and published cases with 8,696 controls identified a
significant enrichment of DYRK1A truncating mutations (p = 0.00851) and an excess of de novo
mutations (p = 2.53×10) among ASD/ID patients. Phenotypic comparison of all novel and literature
cases identified a syndromal disorder including ID, microcephaly, ASD, stereotypic behavior and
apparent feeding problems.
In general, the level of ID is variable (3). The majority of individuals function in the moderate to
severe range of ID and a few individuals presented with mild ID. All individuals experienced apparent
speech problems; notably, expressive language was more severely affected compared to receptive
language. The majority of individuals show typical behavior including autism spectrum disorder, anxious
and stereotypic behavior. Feeding and sleeping problems are common and may persist in adulthood.
In addition, intrauterine growth retardation, seizures and gait disturbances were frequently
noted. About half of affected individuals developed epilepsy including atonic attacks, absences and
generalized myoclonic seizures. Motor development is often impaired by gait disturbances and
hypertonia. Although some individuals achieve independent walking at the upper age limit of normal,
the majority achieve walking after age two to three years.
Finally, in almost all individuals a specific facial gestalt could be recognized, especially at an
older age. During infancy and childhood, the face is characterized by deep-set eyes, mild upslanting
palpebral fissures, a short nose with a broad tip, and retrognathia with a broad chin. In adulthood, the
nasal bridge becomes high and the alae nasi short, giving the nose a more prominent appearance.
During my presentation I will focus on the clinical findings associated with DYRK1A loss of
function mutations and discuss possible pitfalls in molecular diagnosis.
(1) Van Bon BW, Hoischen A, Hehir-Kwa J, de Brouwer AP, Ruivenkamp C, Gijsbers AC, Marcelis CL,
de Leeuw N, Veltman JA, Brunner HG, de Vries BB, 2011. Intragenic deletion in DYRK1A leads to
mental retardation and primary microcephaly. Clin. Genet. 79, 296-299.
(2) Van Bon BW, Coe BP, Bernier R, Green C, Gerdts J, Witherspoon K, Kleefstra T, Willemsen MH,
Kumar R, Bosco P, Fichera M, Li D, Amaral D, Cristofoli F, Peeters H, Haan E, Romano C, Mefford
HC, Scheffer I, Gecz J, de Vries BB, Eichler EE, 2016. Disruptive de novo mutations of DYRK1A
lead to a syndromic form of autism and ID. Mol. Psychiatry. 21, 126-132.
(3) Van Bon BWM, Coe BP, de Vries BBA, Eichler EE. In: Pagon RA, Adam MP, Ardinger HH, Wallace
SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K, editors.
DYRK1A Related Intellectual Disability Syndrome. GeneReviews® [Internet]. Seattle (WA):
University of Washington, Seattle; 1993-2017. 2015 Dec 17. PMID: 26677511
“DYRK1A, related kinases & human disease” 57
Inhibition of DYRK1A in the pancreatic beta cell
Ercument DIRICE, Deepika WALPITA, Amedeo VETERE, Vlado DANCIK, Paul A. CLEMONS, Rohit N.
KULKARNI, Bridget K. WAGNER
Broad Institute, Chemical Biology and Therapeutics Science Program, 415 Main Street, Cambridge, MA 02142, USA.
Restoring functional beta-cell mass is an important therapeutic goal for both type 1 and type 2
diabetes. While proliferation of existing beta cells is the primary means of beta-cell replacement in
rodents, it has been unclear whether a similar principle applies to humans, as human beta cells are
remarkably resistant to stimulation of division. In order to identify small molecules capable of inducing
beta-cell proliferation, we developed a human islet cell culture system suitable for high-throughput
screening. Using this system, we found that 5-iodotubercidin (5-IT), an annotated adenosine kinase
inhibitor, strongly and selectively increases human beta-cell proliferation in vitro and in vivo.
Remarkably, 5-IT also increased glucose-dependent insulin secretion after prolonged treatment.
Kinome profiling revealed 5-IT to be a potent and selective inhibitor of the dual-specificity tyrosine
phosphorylation-regulated kinase (DYRK) and cell division cycle (CDC)-like (CLK) kinase families.
Induction of beta-cell proliferation by either 5-IT or harmine, another natural-product DYRK1A inhibitor,
was suppressed by co-incubation with the calcineurin inhibitor FK506, suggesting involvement of
DYRK1A and NFAT signaling. Gene-expression profiling in whole islets treated with 5-IT revealed
induction of proliferation- and cell cycle-related genes, suggesting that true proliferation is induced by 5-
IT. Furthermore, 5-IT promotes beta-cell proliferation in human islets grafted under the kidney capsule
of NOD-scid IL2Rgnull mice. These effects are selective to the beta cell, pointing to DYRK1A inhibition
as a therapeutic strategy to increase human beta-cell proliferation.
(1)
DE, Clemons PA, Kulkarni RN, Wagner BK, 2016. Inhibition of DYRK1A stimulates human beta-cell
proliferation. Diabetes 65, 1660-1671.
Financial support: this work was supported by a JDRF Strategic Research Agreement (17-2011-642). E.D is supported by a
Senior JDRF Fellowship (JDRF-3-APF-2014-220-A-N) and R.N.K. acknowledges support from R01 DK67536, R01 DK103215
and JDRF Grant (2-SRA-2015-17).
“DYRK1A, related kinases & human disease” 58
Tumor suppressive function of DYRK2
Kiyotsugu YOSHIDA
Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan.
Escape from apoptotic induction, dysregulation of cell cycle machinery, and acquisition of cell
migration ability are major features on cancer development. These characteristics are brought by
dysregulation of cellular homeostasis. Since the intracellular signal pathways crosstalk each other to
maintain the homeostasis, molecular-based studies are required to better understand the mechanism
for tumorigenesis. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a Ser/Thr
kinase, and the intracellular functions had not been elucidated for decades; however, recent studies
have shown that DYRK2 physiologically engages in these signal transductions. I will discuss the tumor
suppressive functions of DYRK2 in several aspects such as apoptosis induction, cell cycle regulation,
metastasis, and cancer stemness.
(1) Mimoto R, Imawari Y, Hirooka S, Takeyama H, Yoshida K, 2016. Impairment of DYRK2 augments
stem-like traits by promoting KLF4 expression in breast cancer. Oncogene doi:
10.1038/onc.2016.349
(2) Taira N, Mimoto R, Kurata M, Yamaguchi T, Kitagawa M, Miki Y, Yoshida K, 2012. DYRK2 priming
phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells. J. Clin.
Invest. 122, 859872.
(3) Taira N, Nihira K, Yamaguchi T, Miki Y, Yoshida K, 2007. DYRK2 is targeted to the nucleus and
controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Mol. Cell 25,
725-738.
“DYRK1A, related kinases & human disease” 59
PARTICIPANTS ADDRESSES
Mariona ARBONES
Instituto de Biología Molecular de Barcelona (IBMB-CSIC) - Parc Científic de Barcelona. C/
Baldiri i Reixac 15, 08028 Barcelona
SPAIN
<marbmc@ibmb.csic.es>
Jean-Pierre BAZUREAU
Université Rennes 1 ISCR UMR 6226 CORINT group Campus Beaulieu CS 74205
35042 Rennes
FRANCE
<jean-pierre.bazureau@univ-rennes1.fr>
Walter BECKER
Institute of Pharmacology and Toxicology - Medical Faculty of the RWTH Aachen University -
Wendlingweg 2 52072 Aachen
GERMANY
<wbecker@ukaachen.de>
Thierry BESSON
Laboratoire COBRA UMR 6014 Batiment IRCOF 1 rue Tesnière 76821 Mont Saint
Aignan Cedex
FRANCE
<thierry.besson@univ.rouen.fr>
Rahul BHANSALI
600 N Lake Shone Dr. Apt. 2811 Chicago IL 60611
USA
<rbhansali91@gmail.com>
Baptiste BILLOIR
CEA FAR 18 route du Panorama 92 260 Fontenay aux Roses
FRANCE
<baptiste.billoir@agent-biotech.com>
Henri BLEHAUT
le 92200 Neuilly Sur Seine
FRANCE
<blehaulth@gmail.com>
Marc BLONDEL
Inserm UMR 1078 Faculté de Médecine de Brest 22 av Camille Desmoulins
29200 Brest
FRANCE
<marc.blondel@univ-brest.fr>
Franz BRACHER
Department of Pharmacy - Ludwig-Maximilian University - Butenandtstr. 5-13 Munich
GERMANY
<franz.Bracher@cup.uni-muenchen.de>
“DYRK1A, related kinases & human disease” 60
Jérôme BRAUDEAU
CEA FAR 18 route du Panorama 92260 Fontenay aux Roses
FRANCE
<jerome.braudeau@agent.biotech.com>
Véronique BRAULT
IGBMC 1 rue Laurent Fries 67404 Illkrich
FRANCE
<vbrault@igbmc.fr>
Frédéric BURON
Institut de Chimie Organique et Analytique Pôle de Chimie rue de
Chartres BP 6759 45067 Orléans CEDEX 2
FRANCE
<frederic.buron@univ-orleans.fr>
Marco Antonio CALZADO CANALE
IMIBIC Avda. Menendez Pidal . s/n 14004 Cordoba
SPAIN
<mcalzado@uco.es>
Morgane CAM
ManRos Therapeutics Hôtel de recherche Centre de Perharidy 29680 Roscoff
FRANCE
<cam@manros-therapeutics.com>
François CARREAUX
Université Rennes 1 ISCR UMR 6226 CORINT group Campus Beaulieu CS 74205
35042 Rennes
FRANCE
<françois-carreaux@univ-rennes1.fr>
Sungchan CHO
KRIBB - 685-2 Yangcheong-ri - Ochang-eup - Cheongju-si Chungbuk
SOUTH KOREA
<sungchan@kribb.re.kr>
Miri CHOI
30 Yeongudanji-ro - Ochang-eup - Cheongwon-gu - Cheongju-si - Chungcheongbuk-do
28116
SOUTH KOREA
<mchoi16585@gmail.com>
Cécile CIEUTA-WALTI
Institut Lejeune 37 rue des Volontaires 75015 Paris
FRANCE
<cecile.cieuta-walti@institutlejeune.org>
Simon COOK
Signalling Laboratory The Babraham Institute Babraham Research Campus
Cambridge CB 22 3AT
UK
<simon.cook@babraham.ac.uk>
“DYRK1A, related kinases & human disease” 61
Pauline DE LAFFOREST
75 Paris
FRANCE
<paulinedelafforest@gmail.com>
Susana DE LA LUNA
Centre for Genomic Regulation (CRG) - The Barcelona Institute of Science and Technology -
Dr. Aiguader 88 - Barcelona 08003
SPAIN
<susana.luna@crg.eu>
Laureano DE LA VEGA
School of Medicine Division of Cancer Research University of Dundee Jacqui Wood
Cancer Center Ninewells Hospital and Medical School James Arrott Drive
Dundee DD1 9SY
UK
<l.delavega@dundee.ac.uk>
Thierry DE LA VILLEJEGU
Fondation Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<tdelavillejegu@fondationlejeune.org>
Jean-Maurice DELABAR
Institut du cerveau et de la moelle CNRS UMR 7225 INSERM U1127 UPMC, 47 bd de
CS21414 75646 Paris Cedex
FRANCE
<jeanmaurice.delabar@icm-institute.org>
Robert DE VITA
Icahn School of Medicine at Mt. Sinai New York
USA
<robert.devita@mssm.edu>
Nadia DI FRANCO
INSERM 1215 146 rue Léo Saignat 33077 Bordeaux Cedex
FRANCE
<nadia.difranco@inserm.fr>
Chiara DI VONA
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology
Carrer - Dr. Aiguader 88 - Barcelona 08003
SPAIN
<chiara.divona@crg.eu>
Aline DUBOS
IGBMC 1 rue Laurent Fries 67404 Illkrich
FRANCE
<dubos@igbmc.fr>
“DYRK1A, related kinases & human disease” 62
Szymon DZIOMBA
21/5 Janki Bryla Street 81-577 Gdynia
POLAND
<szymon.dziomba@gumed.edu.pl>
Jon ELKINS
Structural Genomics Consortium (SGC) UNICAMP State University of Campinas Cidade
Universitária Zeferino Vaz Av. Dr. A. Tosello, 550 Barão Geraldo Campinas / SP 13083-886
BRASIL
<jon.elkins@sgc.ox.ac.uk>
Gaëlle FRIOCOURT
Inserm UMR 1078 Faculté de Médecine de Brest 22 av Camille Desmoulins
29200 Brest
FRANCE
<gaelle.friocourt@univ-brest.fr>
Maria GAITANOU
Hellenic Pasteur Institute - Vasilissis Sofias 127 - Athens 11521
GREECE
<mgaitanou@pasteur.gr>
Edward GELMANN
Columbia University 177 Ft Washington Ave MHB 6N435 New York NY 10032
USA
<gelmanne@columbia.edu>
Jean-Louis GUEANT
Inserm U954 - Nutrition-Genetics and Environmental Exposure - Faculty of Medicine -
University of Lorraine - 54500 Nancy
FRANCE
<jean-louis.gueant@univ-lorraine.fr>
Masatoshi HAGIWARA
Department of Anatomy & Developmental Biology, Kyoto University Graduate School of
Medicine, Yoshida-Konoe-Cho - Sakyo-ku,Kyoto 6068501
JAPAN
<hagiwara.masatoshi.8c@kyoto-u.ac.jp>
Scott HENDERSON
South Chailey Lewes BN8 4QQ
UK
<SH573@sussex.ac.uk>
Yann HERAULT
IGBMC 1 rue Laurent Fries 67404 Illkrich
FRANCE
<herault@igbmc.fr>
“DYRK1A, related kinases & human disease” 63
Rachael HUNTLY
Signalling Laboratory The Babraham Institute Babraham Research Campus Cambridge
CB 22 3AT
UK
<rachael.huntly@babraham.ac.uk>
Olga ISSAKOVA
Nanosyn, 3100 Central Expressway Santa Clara CA 95051
USA
<oissakova@nanosyn.com>
Marie-Louise JUNG
Prestwick Chemical - 220 bd -Illkirch
FRANCE
<marielouise.jung@prestwickchemical.fr>
Robin KETTELER
MRC LMCB University College London Gower Street London WC1E 6BT
UK
<r.ketteler@ucl.ac.ukl>
Bernard KHOR
1201 9th Ave Seattle WA 98122
USA
<bkhor@benaroyaresearch.org>
Stefan KNAPP
Goethe-University Frankfurt - Institute of Pharmaceutical Chemistry- Max-von-Laue-Str. 9
60438 - Frankfurt am Main
GERMANY
<knapp@pharmchem.uni-frankfurt.de>
Akiko KOBAYASHI
Yoshida-Konoe-Cho - Sakyo-ku, Kyoto 6068501
JAPAN
<kobayashi.akiko.5e@kyoto-u.ac.jp>
Reinhard KÖSTER
TU Braunschweig - Zoology Institute - Cellular and Molecular Neurobiology -
Spielmannstraße 7 - 38106 Braunschweig
GERMANY
<r.koester@tu-bs.de>
Conrad KUNICK
TU Braunschweig - Institut für Medizinische und Pharmazeutische Chemie - Bundesstrasse
55 - 38106 Braunschweig
GERMANY
<c.kunick@tu-bs.de>
“DYRK1A, related kinases & human disease” 64
Nobuhiro KURABAYASHI
Molecular Genetics Research Laboratory, Graduate School of Science, The University of
Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033
JAPAN
<n_kurabayashi@gen.s.u-tokyo.ac.jp>
Maribel LARA CHICA
IMIBIC Avda. Menendez Pidal . s/n 14004 Cordoba
SPAIN
<bc2lachm@uco.es>
Christian LECHNER
GERMANY
<c.lechner@tu-bs.de>
Jean-Marie LE MENE
Fondation Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<directionrecherche@fondationjeromelejeune.org>
Catherine LEMONNIER
Fondation Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<directionrecherche@fondationjeromelejeune.org>
Alice LEON
22 avenue Camille Desmoulins 29200 Brest
FRANCE
<alice.leon10@gmail.com>
Mattias LINDBERG
University of Gothenburg Medecinaregatan 1G 41390 Gothenburg
SWEDEN
<mattias.lindberg@gu.se>
Larisa LITOVCHICK
401 College Street Richmond VA 23298
USA
<larisa.litovchick@vcuhealth.org>
Fei LIU
Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration,
Nantong University, Nantong, Jiangsu 226001, P. R.
CHINA
<feiliu63@hotmail.com>
Astrid LUNN
Université de Technologie de Compiègne
60200 Compiègne
FRANCE
<astridlunn@gmail.com>
“DYRK1A, related kinases & human disease” 65
Glynn MARTIN
Horizon Discovery Group Plc 8100 Cambridge Research Park Cambridge CB25 9TL
UK
<glynn.martin@horizondiscovery.com>
Martin MEHNERT
ETH Zurich Auguste-Piccard-Hof 1- 8093 Zurich
SWITZERLAND
<mehnert@imsb.biol.ethz.ch>
Laurent MEIJER
ManRos Therapeutics Hôtel de recherche Centre de Perharidy 29680 Roscoff
FRANCE
<meijer@manros-therapeutics.com>
Rosanna MEINE
Institut für Medizinische und Pharmazeutische Chemie - Technische Universität
Braunschweig - Beethovenstraße 55 - 38106 Braunschweig
GERMANY
<r.meine@tu-bs.de>
Benoit MELCHIOR
9381 Judicial Drive Suite 160 San Diego CA 92121
USA
<benoit@samumed.com>
Clotilde MIRCHER
Institut Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<Clotilde.mircher@institutlejeune.org>
Gopi Kumar MITTAPALI
9381 Judicial Drive Suite 160 San Diego CA 92121
USA
<gopi@samumed.com>
Yoshihiko MIYATA
Department of Cell & Developmental Biology - Graduate School of Biostudies - Kyoto
University, Room 501 - Faculty of Science Building-2 - Kitashirakawa Oiwake-cho - Sakyo-ku
- Kyoto 606-8502
JAPAN
<ymiyata@lif.kyoto-u.ac.jp>
Jonathan MORRIS
School of Chemistry UNSW Australia NSW, 2052
AUSTRALIA
<jonathan.morris@unsw.edu.au>
Rosario MORRUGARES
IMIBIC Avda. Menendez Pidal . s/n 14004 Cordoba
SPAIN
<rosmorcar@gmail.com>
“DYRK1A, related kinases & human disease” 66
Thu Lan NGUYEN
ManRos Therapeutics Hôtel de recherche Centre de Perharidy 29680 Roscoff
FRANCE
Institut de Génétique Biologie Moléculaire et Cellulaire, Université de Strasbourg, UMR7104,
UMR964, 1 rue Laurent Fries, 67404 Illkirch,
FRANCE
<nguyen@manros-therapeutics.com>
Tyler NICHOLS
22 Coleman Street Brighton East Sussex BN2 9SQ
UK
<t.nichols@sussex.ac.uk>
Kassandra ORI-McKENNEY
One Shields Avenue, 145 Briggs, University of California Davis - Davis, CA 95616
USA
<kmorimckenney@ucdavis.edu>
Mireia ORTEGA
Centre for Genomic Regulation (CRG) - The Barcelona Institute of Science and Technology -
Dr. Aiguader 88 - Barcelona 08003
SPAIN
<mireia.ortega@crg.eu>
Nassima OUMATA
ManRos Therapeutics Hôtel de recherché Centre de Perharidy 29680 Roscoff
FRANCE
<oumata@manros-therapeutics.com>
Athena PHOA
School of Medical Sciences, The University of Sydney, NSW 2006
47 Flood St, Leichhardt NSW 2040
AUSTRALIA
<apho6944@uni.sydney.edu.au>
Amélie PITON
IGBMC 1 rue Laurent Fries 67404 Illkrich
FRANCE
<piton@igbmc.fr>
Julia ROEWENSTRUNK
Centre for Genomic Regulation (CRG) - The Barcelona Institute of Science and Technology -
Dr. Aiguader 88 - Barcelona 08003
SPAIN
<julia.roewenstrunk@crg.es>
Ulli ROTHWEILER
The Norwegian Structural Biology Centre - Department of Chemistry - Faculty of Science and
Technology - University of Tromsø - 9037 Tromsø
NORWAY
<ulli.rothweiler@uit.no>
“DYRK1A, related kinases & human disease” 67
Michel ROUX
IGBMC 1 rue Laurent Fries 67404 Illkrich
FRANCE
<mjroux@igbmc.fr>
Nikolai SEPETOV
Nanosyn, 3100 central Expressway Santa Clara CA 95051
USA
<nsepetov@nanosyn.com>
Rajeev SINGH
Hans Meerwein Str. 3 - Marburg 35043
GERMANY
<singhr@staff.uni-marburg.de>
Despina SMIRLIS
Hellenic Pasteur Institute - Vasilissis Sofias 127 - Athens 11521
GREECE
<penny.smirlis@gmail.com>
Samantha STORA
Fondation Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<Samantha.stora@institutlejeune.org>
Benoit SOUCHET
Inserm U1169, Bât. C. Bernard 84 rue du Gal Leclerc - 94276 Le Kremlin Bicêtre
FRANCE
<benoit.souchet@inserm.fr> <benoit.souchet@cea.fr>
John S. SVENDSEN
Department of Chemistry UiT - The Arctic University of Norway
NORWAY
<john-sigurd.svendsen@uit.no>
Nicholas TONKS
Cold Spring Harbor Laboratories -1 Bungtown Road Cold Spring Harbor NY 11724
USA
<tonks@cshl.edu>
Piyush TRIVEDI
73 Indralok colony R.T.O. Road Indore
INDIA
<prof_piyushtrivedi@yahoo.com>
Bregje VAN BON
Clinical geneticist, Radboud University Medical Center, Nijmegen, the Netherlands
THE NETHERLANDS
<bregje.vanbon@radboudumc.nl>
“DYRK1A, related kinases & human disease” 68
Marie VINCENT
CHU Nantes Service de Génétique médicale 1 place Alexix Ricordeau
44093 Nantes Cedex
FRANCE
<marie.vincent@chu.nantes.fr>
Bridget WAGNER
Broad Institute, Chemical Biology and Therapeutics Science Program, 415 Main Street,
Cambridge, MA 02142
USA
<bwagner@broadinstitute.org>
Hervé WALTI
Fondation Jérôme Lejeune - 37 rue des Volontaires 75015 Paris
FRANCE
<hervé.walti@institutlejeune.org>
Marjolaine WILLEMS
35 rue Jean Moulin 34000 Montpellier
FRANCE
<m-willems@chu-montpellier.fr>
Kiyotsugu YOSHIDA
3-25-8, Nishi-shinbashi - Minato-ku - Tokyo 105-8461
JAPAN
<kyoshida@jikei.ac.jp>
“DYRK1A, related kinases & human disease” 69
PARTICIPANTS BY COUNTRY
AUSTRALIA
Athena PHOA
Jonathan MORRIS
BRASIL
Jon ELKINS
CHINA
Fei LIU
FRANCE
Jean-Pierre BAZUREAU
Thierry BESSON
Baptiste BILLOIR
Henri BLEHAUT
Marc BLONDEL
Jérôme BRAUDEAU
Véronique BRAULT
Frédéric BURON
François CARREAUX
Morgane CAM
Cécile CIEUTA-WALTI
Thierry DE LA VILLEJEGU
Pauline DE LAFFOREST
Jean-Maurice DELABAR
Nadia DI FRANCO
Aline DUBOS
Gaëlle FRIOCOURT
Jean-Louis GUEANT
Yann HERAULT
Marie-Louise JUNG
Jean-Marie LE MENÉ
Catherine LEMONIER
Alice LEON
Astrid LUNN
Laurent MEIJER
Clotilde MIRCHER
Thu Lan NGUYEN
Nassima OUMATA
Amélie PITON
Michel ROUX
Benoit SOUCHET
Samantha STORA
Marie VINCENT
Hervé WALTI
Marjolaine WILLEMS
GERMANY
Walter BECKER
Franz BRACHER
Stefan KNAPP
Reinhardt KÖSTER
Conrad KUNICK
Christian LECHNER
Rosana MEINE
Rajeev SINGH
GREECE
Maria GAITANOU
Despina SMIRLIS
INDIA
Piyush TRIVEDI
JAPAN
Masatoshi HAGIWARA
Akiko KOBAYASHI
Nobuhiro KURABAYASHI
Yoshihiko MIYATA
Kyotsugu YOSHIDA
THE NETHERLANDS
Bregje VAN BON
NORWAY
Ulli ROTHWEILER
John-Sigurd SVENDSEN
POLAND
Szymon DZIOMBA
SOUTH KOREA
Sungchan CHO
Miri CHOI
SPAIN
Mariona ARBONES
Marco CALZADO CANALE
Susana DE LA LUNA
Chiara DI VONA
Maribel LARA CHICA
Rosario MORRUGARES
Mireia ORTEGA
Julia ROEWENSTRUNK
SWEDEN
Mattias LINDBERG
SWITZERLAND
Martin MEHNERT
UNITED KINGDOM
Simon COOK
Laureano DE LA VEGA
Scott HENDERSON
Rachael HUNTLY
Robin KETTELER
Glynn MARTIN
Tyler NICHOLS
USA
Rahul BHANSALI
Robert DE VITA
Edward GELMANN
Olga ISSAKOVA
Bernard KHOR
Larissa LITOVCHICK
Benoit MELCHIOR
Gopi Kumar MITTAPALLI
Kassandra ORI-McKENNEY
Nikolai SEPETOV
Nicholas TONKS
Bridget WAGNER
“DYRK1A, related kinases & human disease” 70
SPONSORS
We are grateful to the following companies, foundations and organizations for
generous support
Adipogen www.adipogen.com
AgenT www.agent-biotech.com
BPI France www.bpifrance.fr/
Conseil Régional de Bretagne www.bretagne.bzh
Covalab www.covalab.com/
C.RIS PHARMA www.c-rispharma.com
DYRK1A syndrome community http://dyrk1a.org/
Enamine www.enamine.net
Evotec International Gmbh www.evotec.com
Fondation Jérôme Lejeune www.fondationlejeune.org
Fonds Unique Interministériel http://competitivite.gouv.fr/les-financements-des-projets-
des-poles/les-appels-a-projets-de-r-d-fui-375.html
HCS Pharma http://hcs-pharma.com/
IJMS www.mdpi.com/journal/ijms
Ipsen www.ipsen.com/
www.j-alz.com/.
Kinexus www.kinexus.ca
Ligue contre le Cancer 35 www.ligue-cancer.net/cd35
ManRos Therapeutics www.manros-therapeutics.com
Marine Drugs www.mdpi.com/journal/marinedrugs
Médecine/Sciences www.medecinesciences.org/fr/
Nony www.nony.fr/
Pôle Mer Bretagne Atlantique www.pole-mer-bretagne-atlantique.com/fr/
Prestwick Chemical Inc. www.prestwickchemical.com/
Proqinase www.proqinase.com
Servier www.servier.fr/
Starlab www.starlab-france.com
Synovo www.synovo.com
Very special thanks for great organizational help to:
Pauline DE LAFFOREST, Astrid LUNN and Noémie DE PAUW
“DYRK1A, related kinases & human disease” 71
Cover : Whip Coral (Ellisella sp.) - showing polyp detail. Found throughout the Indo-Pacific.
Photo taken off Anilao, Philippines. Within the Coral Triangle.
http://www.oceanwideimages.com
