A novel chemical class of DYRK1A inhibitors for the treatment of Alzheimer’s Disease

Abstract

A novel chemical class of DYRK1A inhibitors for the treatment of Alzheimer’s Disease
Hélène BENYAMINE, Séverine COUTADEUR, Laurence DELALONDE, Thierry BESSON, Bertrand LEBLOND, Laurent DESRE
The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is located within the Down Syndrome (DS) critical region on chromosome 21. Overexpression of DYRK1A has been proposed to be a significant contributor to the underlying neurodevelopmental abnormalities associated with DS. DYRK1A protein has been found to be associated with neurofibrillary tangles (NFTs) in sporadic Alzheimer’s Disease (AD) and in vitro and in vivo studies implicate the DYRK1A kinase in the generation of both amyloid and tau pathologies associated with the early onset AD that is observed in DS. Several substrates of DYRK1A play a role in neurodegeneration and AD. Among them, DYRK1A is important for phosphorylation of tau protein on multiple AD-relevant sites. In addition, DYRK1A phosphorylates APP and presenilin-1 and enhances APP cleavage by ß- and γ-secretase and the production of Aß peptides. Thus, DYRK1A may be an important therapeutic target for pharmacological interventions seeking to modify the course of tau and Aβ pathologies in AD.
Here, Tau phosphorylation and Aβ production were stimulated by overexpression of DYRK1A. To prevent these pathological events, Diaxonhit has developed a novel class of DYRK1A inhibitors and characterized new chemical entities with subnanomolar kinase inhibitory activities associated with a high degree of selectivity over 400 kinases. Among more than 100 NCEs from this series, lead compounds were identified that exhibited potent efficacy as Dyrk1A inhibitors in a Tau kinase assay. By Western blot and ELISA, these inhibitors were found to reduce Tau phosphorylation at multiple AD-relevant sites in SH-SY5Y cells and reduce Aβ production from APP-HEK293 cells without affecting cell viability.
The most active compounds identified in this program are being evaluated in various tau and Aβ cellular models in order to identify the most suitable candidates for in vivo testing and establish the usefulness of these new inhibitors to target DYRK1A as a novel, high-potential, alternate therapy for AD.

Full text

A novel chemical class of DYRK1A inhibitors for the treatment of Alzheimer’s Disease
Hélène Benyamine1*, Séverine Coutadeur1*, Laurence Delalonde1, Thierry Besson2, Anne-Sophie Casagrande1, Bertrand Leblond1, Matthew P. Pando1, Laurent Desiré1
1Diaxonhit, Paris, France; 2Université de Rouen, Rouen, France;*: equal contribution – Contact: laurent.desire@diaxonhit.com
ResultsBackground
Dyrk Family Lead compounds selectivity
Results
The dual-specificity tyrosine-regulated
kinases (DYRKs)
•Conserved family of eukaryotic kinases
within the CMGC group of the kinome
•Mammalian DYRK subfamily consists of 5
members
Selectivity within Dyrk family
EHT 5372
Kinome activity map
Expressed in adult central nervous system -
cortex, hippocampus, striatum and spinal cord
Located with APP on chromosome 21 and
overexpressed in Down syndrome (DS)
DYRK1A
IC50 (nM)
Com
p
ound D
y
rk1
A
D
y
rk1B D
y
rk2 D
y
rk3 D
y
rk4
Pharmacological inhibition of DYRK1A reduces tau
phosphorylation – cellular models
LiCl
EHT 5372 (µM)
EHT 5372 (µ M)
LiCl
Primary cortical neurons
members
Dual-specificity of DYRK kinases
•Autophosphorylation of a conserved
tyrosine residue in the activation loop of the
catalytic domain catalysed by a
translational intermediate form of DYRK
•Substrate phosphorylation by mature
DYRK on serine or threonine residues
IC
50
<1
nM
Lead compound EHT 5372 targets DYRK1A and DYRK1B and
shows
g
ood selectivit
y
p
rofile on a 339 kinases
p
anel
Overexpression in mouse exhibit impairments
in spatial learning and memory tasks; synaptic
plasticity in the hippocampus is also altered
Transgenic models indicate that DYRK1A may
play a significant role in developmental brain
defects and in early onset AD disease that is
uniformly observed in DS
Dyrk1A is very dose-dependent: 1.5 fold
increases produce major Down Syndrome
-
like
p
y
y
y
y
y
EHT 5372 0.22 0.28 10.80 93.20 No inhibition
AL11-28 0.36 0.59 3.16 21.10 1570
AF10-314 2.76 3.66 31.63 54.55 >10000
AF10-249 3.60 6.55 26.75 275.5 No inhibition
0
20
40
60
80
100
120
10nM 100nM 1µM 10µM 25µM
Cell survival
(% of control)
0.1
pS356
pT212
pS231
Cont
AT8
(pS202 and pT205)
1510
LiCl
20 mM
EHT
5372
(µM)
55 kDa -
0.1Cont 1 5 10
EHT
5372
(µM)
55 kDa -
55 kDa -
55 kDa -
LiCl
20 mM
Actin
Tau -4 6
55 kDa -
42 kDa -
DYRK1A in neurodegenerative disorders and AD DYRK1A controls tau phosphorylation
IC
50
<
1
nM
Minoractivity
gyp
p
increases
produce
major
Down
Syndrome
like
pathologies in the mouse
•APP, tau, Presenilin, Septin-4 at
various AD-relevant sites
•α-s
y
nuclein
(
Parkinson’s disease
)
Shown to promote pathological hallmarks of these diseases by promoting phosphorylation of
their most pathologivally key components
0
20
40
60
80
100
120
pS396Tau(%control)
pS396Tau
Cellviability
SH-SY5Y cells
transient transfection
of DYRK1A and tau
Cont Veh Sc 10 25 50 nM
siRNA DYRK1A
Impact of DYRK1A knockdown
on pS396 tau levels Analysis of tau phosphorylation in different cell types. Cells were treated for 24h with various
concentrationsof EHT 5372 or with LiCl (GSK3 inhibitor).
In rat cortical neurons,phosphorylated Tau was detected using various AD-relevant anti-phospho tau
antibodies. Global tau (phosphorylated and non phosphorylated) was detected by Tau-46.
In SH-SY5Y cells, an ELISAspeci fic for pS396 tau was used.
0.1Cont 1 5 10
EHT 5372 (µM)
pS396-
LiCl
20 mM
SH-SY5Y – tau441 cells
y( )
•Hip-1 (Huntington’s disease)
Priming kinase, allowing the
substrate to be further
phosphorylated by GSK3
Phosphorylates alternative
splicing factors ASF/SF2 and
SC35, causing increased non-
physiological 3R/4R tau ratio and
tau aggregation
Ph h l t RCAN1 th b
0
100
200
300
400
500
600
NT(400ng) Dyrk1A(80ng) Tau(320ng) D80+T320
pS396Taulevels(pg/ml)
0
Cont LiCl20mM 0.1 1 5 µMEHT5372
DYRKA is also involved in amyloid-beta Aβ
production
siRNA knockdown Impact of knockdown on Aβlevels
0
20
40
60
80
100
120
pS396 tau (% of control)
pS396 tau ELISA
pS396 tau ELISA
-Dyrk1A
Cell viability was determined wi th MTT in both model s
HEK293 cells were transfected with the indicated plasmid and SH-
SY
5
Y
tau
441
cells
were
transfected
with
the
indicated
siRNA
After
48
h
Ph
osp
h
ory
l
a
t
es
RCAN1
,
th
ere
b
y
enhancing its ability to inhibit the
phosphatase activity of
calcineurin, leading to enhanced
tau phosphorylation
Contributes to Aβ production by
controlling APP and presenilin
(PS1) phosphorylation
A positive feedback between
DYRK1A expression and Aβmay
(from B. Smith. et al. ACS Chem. Neuroscienc e, 2012)
DYRK1A inhibition
could lead to novel drugs
Pharmacological inhibition of DYRK1A reduces tau
phosphorylation-tau kinase assay
0
25
50
75
100
125
150
Cont Veh Scrambled
siRNA
siRNA DYR1B siRNA DYR1A
Aβ levels (% control)
siRNA DYRK1A
Cont Veh Sc 5 10 25 50 nM
ELISAAβ1‐40
NT 0,01µM 0,1µM 1µM 10µM
-Dyrk1A
EHTcompond(µM)
S396/t 5
SY
5
Y
-
tau
441
cells
were
transfected
with
the
indicated
siRNA
.
After
48
h
or 96h, respectively, pS396 tau levels weredetermined with an ELISA
specific for pS396.
A novel class of DYRK1A inhibitors
Patented Thiazoloquinazoline Series
Results
Reference compounds on Dyrk1A
Compound
Dyrk1A
Compound
Dyrk1A
DYRK1A
expression
and
Aβ
may
accelerate disease progression
DYRK1A
inhibition
could
lead
to
novel
drugs
to rescue DS & AD and other tauopathies
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Harmine EGCG TG003 L‐41 EHT5372 AL11‐28 AL11‐55 AF11‐411 AL11‐32 AF11‐369 AF10‐341 AF11‐408 AL11‐64 AF11‐393 DAPT
NormalizedAβlevels
Potencyinkinaseassay
100
150
n
trol)
100
120
ActivityofEHT5372 Cellviability
InactiveInactive
‐+++
00.0 10.11310
00.010.030.10.31
TAU
DYRK1A
ATP
Compound(µM)
64kDa‐
64kDa‐
‐+++
++‐ +
+‐ ++
EHT5372
AL11‐28
TAU
64kDa‐
AL11‐55
Referencecompound(µM)
p
S396/t
au
5
EHT5372
Conclusion
•Very potent and selective DYRK1A inhibitors were discovered in a novel chemical series
•
Lead
compounds
are
active
to
inhibit
tau
phosphorylation
at
multiple
AD
-
relevant
sites
in
Compound
IC50 (nM)
EHT 5372 0.22
AL11-28 0.36
AL11-55 0.94
AF11 -4 11 0.9 8
AL11-32 0.99
AF11-369 1.13
AF10-341 1.65
AF11-408 1.81
AL11
-
64
22
Compound
IC50 (nM)
L41 7.60
TG003 30.40
Harmine 34.80
EGCG 11130.00
-120 NCEs synthesized
in this first series
-WO 2013/026806
Bk i b d
-6 -4 -2 0 2
-50
0
50
EC50 1.068
EHT 5372 Log[µM]
A levels (%co
n
0
20
40
60
80
0 10nM 100nM 1µM 10µM
%viability
Aβ40 levels were determined by ELISA in
supernatents of HEK-APP cells treated for
24h with various concentrations of the
indicated compounds.
Cell viability was determined with MTT
++‐ +
+‐ ++
Harmine
EGCG
TG003
AF11‐411
AF10‐341
64kDa‐
DYRK1A
ATP
AL11‐32
AF11‐369
•
Lead
compounds
are
active
to
inhibit
tau
phosphorylation
at
multiple
AD
-
relevant
sites
in
various biochemical and cellular models
•Lead compounds also prevent Aβproduction, possibly by reducing APP phosphorylation at
T668
•These compounds are being further characterized in various cellular models relevant to
AD, and some of them are good candiates for follow-up in vivo testing in AD/DS models
Kinase activities were assayed in a radiometric based filtration binding assay, the Reaction Biology Corporation’s HotSpotSM technology. The kinase reaction is
performedi nthe presence of 33P- γ-ATP,followed by binding of the final radioisotope labeled products to filters after which unreacted phosphate can be washed
away without interfering with the detection of real phosphorylated products.
AL11
64
2
.
2
AF10-314 2.76
AF10-249 3.60
AL11-31 3.89
AF11-363 4.25
AF11-381 4.44
AF11-386 4.91
DYRK1A inhibitors show potent activty on tau phosphorylation, when compared to currently
avaialble reference compounds
-
B
ac
k
up ser
i
es
b
ase
d
on different scaffold
Western-blot analysis of tau phosphorylation induced by incubation of Tau, DYRK1A and ATP in the presence of various concentrations of
the indicated compound. Phosphorylated Tau was detected using the phospho-S396 epitope-dependent anti-tau antibody and global tau
(phosphorylated and non phosphorylated) was detected by Tau-5.
L‐41
AF11‐408
L41 TG003
Harmine EGCG