Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons in cortex, brain stem, and spinal cord die progressively, resulting in muscle wasting, paralysis, and death. Currently, effective therapies for ALS are lacking; however, identification of pathological TAR DNA-binding protein 43 (TDP-43) as the hallmark lesion in sporadic ALS suggests new therapeutic targets for pharmacological intervention. Pathological TDP-43 phosphorylation appears to drive the onset and progression of ALS and may result from upregulation of the protein kinase CK-1 in affected neurons, resulting in postranslational TDP-43 modification. Consequently, brain penetrant specific CK-1 inhibitors may provide a new therapeutic strategy for treating ALS and other TDP-43 proteinopathies. Using a chemical genetic approach, we report the discovery and further optimization of a number of potent CK-1δ inhibitors. Moreover, these small heterocyclic molecules are able to prevent TDP-43 phosphorylation in cell cultures, to increase Drosophila lifespan by reduction of TDP-43 neurotoxicity, and are predicted to cross the blood-brain barrier. Thus, N-(benzothiazolyl)-2-phenyl-acetamides are valuable drug candidates for further studies and may be a new therapeutic approach for ALS and others pathologies in which TDP-43 is involved.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons in cortex, brain stem, and spinal cord die progressively, resulting in muscle wasting, paralysis, and death. Currently, effective therapies for ALS are lacking; however, identification of pathological TAR DNA-binding protein 43 (TDP-43) as the hallmark lesion in sporadic ALS suggests new therapeutic targets for pharmacological intervention. Pathological TDP-43 phosphorylation appears to drive the onset and progression of ALS and may result from upregulation of the protein kinase CK-1 in affected neurons, resulting in postranslational TDP-43 modification. Consequently, brain penetrant specific CK-1 inhibitors may provide a new therapeutic strategy for treating ALS and other TDP-43 proteinopathies. Using a chemical genetic approach, we report the discovery and further optimization of a number of potent CK-1δ inhibitors. Moreover, these small heterocyclic molecules are able to prevent TDP-43 phosphorylation in cell cultures, to increase Drosophila lifespan by reduction of TDP-43 neurotoxicity, and are predicted to cross the blood-brain barrier. Thus, N-(benzothiazolyl)-2-phenyl-acetamides are valuable drug candidates for further studies and may be a new therapeutic approach for ALS and others pathologies in which TDP-43 is involved.
Described in 1874 by
the French neurologist Charcot, amyotrophic
lateral sclerosis (ALS) is a neurodegenerative disease where no effective
treatment exists today. Riluzole is the only palliative drug approved
by the U.S. Food and Drug Administration (FDA), which moderates disease
progression by extending survival 2–3 months without benefits
to motor function.[1]ALS affects lower
and upper neurons in the brain stem, spinal cord
and in the motor cortex, respectively.[2] This neuron loss causes atrophy and paralysis of skeletal muscles
because of the lack of communication between the nervous system and
voluntary muscles of the body. In general, patients die between 3
and 5 years after symptom onset.[3]ALS can be classified as familial (fALS) or sporadic (sALS), although
the majority of the cases are sporadic (90%). The comparative lack
of understanding of ALS etiology has hindered effective therapy development.[4]Among the fALS, different mutations in
many different genes have
been discovered including the genes encoding super oxide dismutase
(SOD1),[5] the Tar DNA binding protein 43
(TDP-43),[6] and the recently discovered
C9ORF72.[7,8]TDP-43 was identified in 2006 as the
major component of protein
aggregates of ALS and frontotemporal lobar degeneration (FTLD).[9] In 2008, the role of TDP-43 in both sporadic
and familial ALS was confirmed by the identification of mutations
in the exon 6 of the TDP-43 encoding gene.[10] The pathogenesis of TDP-43 mutation in ALS has been validated in
a variety of animal and cell models. Overexpression of mutant TDP-43
causes neuronal death in worms (Caenorhabditis elegans),[11] flies (Drosophila
melanogaster),[12] zebrafish,[13] mice,[14] rats,[15] monkeys,[16] and cultured
human motor neurons differentiated from reprogrammed stem cells.[17] Insoluble intracellular aggregates of phosphorylated
TDP-43 are observed in fALS and sALSpatients.[18] Pathological TDP-43 phosphorylation seems to be a relatively
early event in the onset and progression of ALS and FTLD-TDP. There
is some evidence that TDP-43 phosphorylation may result in differential
degradation and/or toxicity of the protein, but the precise role of
TDP-43 phosphorylation in mechanisms of disease remains unclear.[19] Regardless, TDP-43 phosphorylation at S409/410
is the most robust and consistent marker of pathological TDP-43 in
humanpatients[20] and drives neurodegeneration
in animal models of TDP-43proteinopathy.[21−23]The protein
kinase CK-1 was the first kinase reported to phosphorylate
TDP-43 directly,[24] and up-regulation of
its activity is reported on ALS spinal cord tissue.[25] CK-1 is a Ser/Thr kinase that is ubiquitously expressed
in eukaryotic organisms.[26] At least seven
isoforms (α, β, γ1–3, δ, and ε)
and their various splice variants have been characterized in different
organisms.[27] During recent years, several
studies have highlighted the importance of CK-1 in neurodegenerative
diseases[28] and CK-1δ has been determined
to phosphorylate many different sites on TDP-43 in vitro.[29] Consequently, brain penetrant specific CK-1δ
inhibitors may provide a new therapeutic strategy for treating ALS
and other TDP-43 proteinopathies. We have developed a number of potent
CK-1δ inhibitors and demonstrate that CK-1δ inhibition
prevents TDP-43 phosphorylation in vitro and neurotoxicity in vivo.
Results
and Discussion
Discovery of New CK-1δ Inhibitors
Biological
screening of chemical libraries has been shown to be an effective
methodology to discover new and chemically diverse hit compounds for
a specific target. Here, we have followed a forward chemical genetic
approach[30] exploiting the use of small
molecules as pharmacological tools to discover and validate new biological
targets for further pharmacological intervention. Moreover, this chemogenomic
approach has the advantage of revealing simultaneously some new lead
compounds for further pharmacological development. First, we performed
a blind screening of 25 chemically diverse heterocyclic small molecules
from our in-house chemical library (Figure 1). A luminescent-based method to determine CK-1 inhibition using
CK-1δ human recombinant enzyme was used.[31] For the initial screening, we tested all the compounds
at the same concentration (10 μM), and only two compounds, MR-3.55 and MR-2.16, showed significant inhibition
in the micromolar range, with IC50 calculated values of
10.12 and 7.29 μM, respectively.
Figure 1
Chemically diverse heterocyclic
compounds selected from in-house
chemical library in the first CK-1δ screening.
Chemically diverse heterocyclic
compounds selected from in-house
chemical library in the first CK-1δ screening.On the basis of the chemical structure of these
two hits (MR-3.55 and MR-2.16), we selected
a small focused
subset from another chemical library. Thus, five more compounds with
similar thiazole and benzothiazole scaffolds in their chemical structure
bearing different linkers between the heterocycle and the aromatic
ring were selected and evaluated against the CK-1δ enzyme (Table 1). In this second screening, almost all the evaluated
compounds inhibited CK-1δ at low micromolar level, with MR-3.15 being the most potent hit, with an IC50 value of 0.85 μM.
Table 1
Focalized Structures
Subset Chosen
from Our In-House Chemical Library and Biological Evaluation on CK-1δ
Hit-to-Lead Optimization
Given its potency against
CK-1δ, N-(benzothiazol-2-yl)-2-(3-chlorophenyl)
acetamide (MR-3.15) was selected for further biological
activity optimization. Representative chemical features of MR-3.15 are the 2-aminobenzothiazole moiety linked to a substituted phenyl
ring through a carbonyl and methylene group spacer. On the basis of
that scaffold, we designed different structures depicted in Figure 2 to determine the relationship between chemical
structure and biological activity toward CK-1δ enzyme. Several
substituents such halides, alkyloxy, or trifluoromethyl were introduced
in the heterocycle core or the phenyl ring. Moreover, the influence
of the nature of the heterocyclic moiety in CK-1δ inhibition
was studied. Thus, the benzothiazole scaffold was changed by benzimidazole,
1,3,4-thiadiazole, pyrimidine, 1,3-thiazole, and benzoxazole. Finally,
the length and nature of the linker between the heterocycle and the
phenyl ring was also analyzed.
Figure 2
Structural modifications proposed from
the hit compounds MR-3.15.
Structural modifications proposed from
the hit compounds MR-3.15.The preparation of all the proposed derivatives was performed
by
a convergent synthesis (Scheme 1), using as
starting material the corresponding amino heterocycle. The coupling
reaction with different organic acid derivatives was accomplished
under microwave irradiation. In that case, reactions were performed
at 110–150 °C for 5–20 min with excellent yields
(see Experimental Section). In other cases,
reaction of the amino heterocycle with carboxylic acids or N-aryl-isocyanates yielded the desired compound. All the
synthesized derivatives were characterized using NMR, HPLC, and elemental
analysis techniques.
Scheme 1
General Synthetic Procedure for the New
Designed CK-1 Inhibitors
Reactions conditions: (i) THF
or solvent free, MW, 110–150 °C, 5–20 min; (ii)
DMF or CH2Cl2, EDC, DMAP, N(Et)3,
rt or reflux, 5–30 h; (iii) THF, MW, 110 °C, 0.5–4
h.
General Synthetic Procedure for the New
Designed CK-1 Inhibitors
Reactions conditions: (i) THF
or solvent free, MW, 110–150 °C, 5–20 min; (ii)
DMF or CH2Cl2, EDC, DMAP, N(Et)3,
rt or reflux, 5–30 h; (iii) THF, MW, 110 °C, 0.5–4
h.All the synthesized compounds were evaluated
on CK-1δ human
recombinant enzyme at a fixed concentration of 10 μM. In the
cases where the inhibitory effect toward the enzyme was higher than
60%, the IC50 was calculated. All the data are collected
in Tables 2, 3, and 4.
Table 2
N-Heteroaryl-phenyl-acetamides
Derivatives Synthesized and Evaluated as CK-1δ Inhibitors
Table 3
Benzothiazoles Derivatives Synthesized
and Evaluated as CK-1δ Inhibitors
compd
X
Z
Y
R1
R2
R3
R4
%inh @10 μM (%)
CK-1δ IC50 (μM)
MR-3.15
NH
CO
CH2
H
H
Cl
H
>60
0.85 ± 0.10
9
O
CO
CH2
H
H
H
Cl
20
10
NH
CO
CF3
OMe
H
H
25
11
NH
CH2
CH2
CF3
OMe
H
H
25
12
NH
CO
CHPh
H
H
H
H
>60
1.96 ± 0.83
13
NH
CO
CHPh
OEt
H
H
H
>60
2.82 ± 0.43
14
NH
CO
CH2CH2
H
H
Cl
H
>60
3.58 ± 0.21
15
NH
CO
CH2CHPh
H
H
H
H
>60
2.50 ± 0.33
16
NH
CO
NH
CF3
H
H
OMe
>60
5.50 ± 0.11
Table 4
N-Benzothiazolyl-2-phenyl-acetamides
Derivatives Synthesized and Evaluated as CK-1δ Inhibitors
compd
R
R1
R2
R3
R4
R5
%inh @10 μM (%)
CK-1δ IC50 (μM)
MR-3.15
H
H
H
Cl
H
H
>60
0.85 ± 0.10
17
H
Me
H
Cl
H
H
>60
0.083 ± 0.003
18
Me
H
H
Cl
H
H
20
19
Cl
H
H
Cl
H
H
20
20
H
CF3
H
Cl
H
H
>60
0.023 ± 0.002
21
H
OMe
H
Cl
H
H
>60
0.53 ± 0.07
22
H
OCF3
H
Cl
H
H
>60
0.54 ± 0.02
23
H
OEt
H
Cl
H
H
>60
1.21 ± 0.09
24
H
CF3
Cl
H
H
H
>60
0.068 ± 0.007
25
H
OMe
Cl
H
H
H
>60
9.71 ± 0.99
26
H
OEt
Cl
H
H
H
>60
17.43 ± 1.21
27
H
CF3
H
H
Cl
H
>60
0.065 ± 0.003
28
H
OMe
H
H
Cl
H
>60
0.75 ± 0.09
29
H
OEt
H
H
Cl
H
>60
1.11 ± 0.29
30
H
Br
OMe
H
H
H
>60
0.26 ± 0.02
31
H
Cl
OMe
H
H
H
>60
0.32 ± 0.03
32
H
F
OMe
H
H
H
>60
1.17 ± 0.51
33
H
Me
OMe
H
H
H
>60
0.29 ± 0.03
34
H
CF3
OMe
H
H
H
>60
0.010 ± 0.001
35
H
OMe
OMe
H
H
H
>60
2.22 ± 0.30
36
H
OCF3
OMe
H
H
H
>60
0.62 ± 0.06
37
H
OEt
OMe
H
H
H
>60
5.76 ± 0.61
38
H
CF3
H
OMe
H
H
>60
0.042 ± 0.050
39
H
OMe
H
OMe
H
H
>60
0.42 ± 0.06
40
H
OEt
H
OMe
H
H
>60
0.99 ± 0.07
41
H
CF3
H
CF3
H
H
>60
0.087 ± 0.033
42
H
CF3
H
H
OMe
H
>60
0.033 ± 0.002
43
H
OMe
H
H
OMe
H
>60
0.57 ± 0.08
44
H
OEt
H
H
OMe
H
>60
1.09 ± 0.12
45
H
H
H
H
H
H
>60
0.33 ± 0.03
46
H
CF3
H
H
H
H
>60
0.047 ± 0.005
47
H
CF3
H
Cl
Cl
H
>60
0.056 ± 0.002
48
H
OMe
H
Cl
Cl
H
>60
1.24 ± 0.17
49
H
OEt
H
Cl
Cl
H
>60
3.43 ± 0.69
50
H
OCF3
H
Cl
Cl
H
>60
0.59 ± 0.04
51
H
CF3
OMe
H
H
OMe
>60
0.19 ± 0.05
52
H
OCF3
H
OMe
OMe
OMe
>60
0.079 ± 0.007
53
H
OMe
H
OMe
OMe
OMe
>60
1.12 ± 0.41
54
H
OEt
H
OMe
OMe
OMe
>60
1.43 ± 0.35
55
H
CF3
H
OMe
OMe
OMe
>60
0.015 ± 0.007
Modification of the nature of the heterocyclic moiety
present in
the hit compound MR-3.15 by introduction of N-methyl-benzimidazole, benzoxazole, pyrimidine, 1,3,4-thiadiazole,
or thiazole led to inactive or less potent derivatives (compounds 1–8) (Table 2).
Thus, the benzothiazole framework was selected as fixed scaffold for
the following compound set synthesis in which the nature and length
of the central linker was explored. Data collected in Table 3 show that the inhibitory
potency on CK-1δ is completely lost when the nitrogen directly
attached to the benzothiazole ring is substituted by an oxygen atom
(compound 9), when the carbonyl group of the linker is
directly attached to the aromatic moiety (compound 10), or when the carbonyl group is replaced by a methylene group (compound 11). Although other possibilities such as the increase in
the linker length and/or steric volume are tolerated by the enzyme
(compounds 12–16), a reduction in
the inhibitory potency of these compounds, in comparison with the
hit MR-3.15, is observed. Thus we maintained the atom
sequence NHCOCH2 as a spacer in the third group of synthesized
compounds to explore different substitutions both in the benzothiazole
and phenyl moiety (Table 4).Several N-(benzothiazolyl)-2-phenylacetamides
were then prepared which incorporate diverse substituents in different
positions of both aromatic rings. One striking observation is the
influence that the introduction of a substituent at position 6 of
benzothiazole has in the CK-1δ inhibition. Thus, while a methyl
or trifluoromethyl group increases 1 order of magnitude, the inhibitory
potency of the acetamides (compounds 17 and 20), the methoxy or trifluoromethoxy moieties maintain the biological
activity (compounds 21 and 22) and the sterically
greater ethoxy group (compound 23) slightly decrease
the enzymatic inhibition in comparison with the hit compound MR-3.15. This part of the inhibitor chemical structure may
fit in a hydrophobic cavity of the enzyme, and when a methyl group
or chlorine atom is attached to position 4 of benzothiazole, the enzymatic
inhibition is lost (compounds 18 and 19)
due to steric hindrance. On the other hand, substituents in the phenyl
ring have little influence on biological activity, suggesting that
hydrophobic interactions may be more relevant in the interaction with
the enzyme than electrostatic interactions (compounds 24–55). Moreover, derivatives 56 and 57 were synthesized (Figure 3) to test
this hypothesis. Both compounds are CK-1δ inhibitors in the
submicromolar range, suggesting hydrophobic interactions produced
by the phenyl ring are crucial for enzymatic inhibition.
Figure 3
N-Benzothiadizolyl-2-alkyl-acetamides 56 and 57 as CK-1δ inhibitors.
N-Benzothiadizolyl-2-alkyl-acetamides 56 and 57 as CK-1δ inhibitors.In the light of these results, we conclude that N-(benzothiazolyl)-phenyl acetamides derivatives are potent CK-1δ
inhibitors, with IC50 values in the low micromolar to nanomolar
range. The preferred benzothiazole ring substituent is the trifluoromethyl
group attached to the 6 position, and almost all the compounds bearing
this moiety have IC50 values for CK-1δ inhibition
in the nanomolar range. These compounds have emerged as promising
leads for further studies.
Binding Mode Studies of CK-1δ Inhibitors
Once
we established that N-(benzothiazolyl)-2-phenylacetamides
are potent CK-1δ inhibitors, we began to explore structural
reasons for their inhibitory activity. A two-step docking study was
performed to gain insight into the nature of small molecule/enzyme
interactions. First, a blind/ensemble docking was done following by
a docking refinement (see Experimental Section for details).There are three humanCK-1δ crystallographic
structures collected in the Protein Data Bank (PDBs entries: 3UYS, 3UYT, 3UZP). The docking study
was performed over the whole CK-1δ (blind docking), with the
potent CK-1δ inhibitor 20 (IC50 = 23
nM) and the inactive compound 11 (25% inhibition at 10
μM) employing the crystal structure of CK-1δ (PDB code: 3UYS). Docking analysis
of the best solutions for compound 20 showed the most
populated cluster and lowest energy poses in the ATP binding site,
giving us a clue of the preferred location for N-(benzothiazolyl)-phenylacetamides
inhibitors (Figure 4A). The same docking study
applied to the CK-1δ inhibitors 24 and 34 confirms that the catalytic enzyme site is the preferred binding
site for this new family of compounds (Figure
S1 Supporting Information). A detailed inspection of the ATP
site shows that Ile 23 and 148, Asp 91, 132, and 149, Leu 135, Lys
130, and Asn 133 are the most important amino acids for the drug interaction
with the enzyme. The amide group present in this class of inhibitors
is essential for the interaction with the aspartyl residues 132 and
91 (inhibitor 20) and Asp 149, Asn 133, and Lys 130 (inhibitors 24 and 34). This fact is corroborated by the
analysis of the best docking found for the inactive compound 11. It is also located in the ATP region (Figure 4B), but in that case, the absence of the carbonyl
group in the linker does not allow stable interaction between the
compound and enzyme, leading to a very weak drug–enzyme complex.
Moreover, the benzothiazole moiety with trifluoromethyl group substituent
facilitates the hydrophobic interactions between 20 and
the hydrophobic area created by Leu 135, Ile 23, and Ile 148. Inhibitors 24 and 34 also presented affinity to hydrophobic
area but differently to compound 20 showed hydrogen bonding
interaction with Asn 133 and Lys 130.
Figure 4
Docking studies with CK-1δ (PDB
code: 3UYS)
of compound 20 (A) and 11(B).
Docking studies with CK-1δ (PDB
code: 3UYS)
of compound 20 (A) and 11(B).To confirm the results obtained with the “blind
docking”
study, experimental enzymatic kinetic studies were performed. Three
different CK-1δ inhibitors were chosen, 20, 24, and 34, to analyze the competition with ATP.
As standard reference, the commercially available compound IC261, a known ATP competitive CK1δ inhibitor (IC50 =
1.0 μM), was used. Kinetic experiments were performed by varying
concentrations of both ATP (from 1 to 50 μM) and compounds.
Double reciprocal plotting of the data is depicted in Figure 5. The intercept of the plot in the vertical axis
(1/V) does not change when the IC261 concentration increases (from 0.25 to 0.5 μM), which means
that this compound acts as an ATP competitive inhibitor as it was
described in the literature.[32]
Figure 5
Kinetic data
determined for IC261 and the N-benzothiazolyl-phenyl-amides
derivatives 20, 24 and 34.
ATP concentrations in the
reaction mixture varied from 1 to 50 μM. Compound concentrations
used are depicted in the plot, and the concentration of casein, the
substrate used in the phosphorylation reaction, was kept constant
at 12.5 μM. Each point is the mean of two different experiments,
each one analyzed in duplicate.
Kinetic data
determined for IC261 and the N-benzothiazolyl-phenyl-amides
derivatives 20, 24 and 34.
ATP concentrations in the
reaction mixture varied from 1 to 50 μM. Compound concentrations
used are depicted in the plot, and the concentration of casein, the
substrate used in the phosphorylation reaction, was kept constant
at 12.5 μM. Each point is the mean of two different experiments,
each one analyzed in duplicate.The same experiments were done with the three new compounds 20, 24, and 34 here reported (Figure 5). In all the cases, these compounds have a similar
behavior than IC261, showing an ATP competitive inhibition
of the enzyme and validating the in silico docking studies of these
compounds.
Kinases Selectivity Study
The human
‘‘Kinome’’
describes the total protein kinase component encoded by the human
genome and includes more than 500 genes.[33] The close homology within the ATP binding pocket is a defining characteristic
for most protein kinases and constitutes the promise and challenge
for kinase drug discovery. Considering that compounds here reported
are ATP competitive, it is important to determine their selectivity
against an extensive panel of kinases to avoid unexpected off-target
effects and undesirable side effects in future drug development.Compounds 20 and 24 were screened at 10
μM using the KinomeScan approach on a panel of 456 protein kinases
(Tables S2 and S3 Supporting Information). Results are depicted in Figure 6. Both
compounds were highly selective for CK-1δ inhibition, with a
final selectivity score or “S” score (a quantitative
measure of compound selectivity which is calculated by dividing the
number of kinases that compounds bind to by the total number of distinct
kinases tested, excluding mutant variants) of 0.04. Other commercialized
kinases inhibitors such as Sunitinib and Lapatinib, present an S score
of 0.57 and 0.010, respectively, in a 290 kinases panel.[34]
Figure 6
Kinome tree of compounds 20 and 24 (A
and B, respectively). Figures were generated with DiscoverRx TREEspot version 4. The original results were shown as percent
control to DMSO, and targets exhibiting less than 1% remaining activity
were selected in the figures. The sizes of the red circles are proportional to the strength of the binding; the larger
circles imply higher affinity.
Kinome tree of compounds 20 and 24 (A
and B, respectively). Figures were generated with DiscoverRx TREEspot version 4. The original results were shown as percent
control to DMSO, and targets exhibiting less than 1% remaining activity
were selected in the figures. The sizes of the red circles are proportional to the strength of the binding; the larger
circles imply higher affinity.Both compounds also inhibited CDC like kinase 1 and 4 (CLK1,
CLK4),
the protein kinase CK-1 family (CK-1α1, CK-1δ, CK-1ε,
CK-1γ2), the dual-specificity tyrosine-(Y)-phosphorylation regulated
kinase (DYRK1A, DYRK1B), fms-related tyrosine kinase 1 (FLT1), myosin
light chain kinase 3 (MLCK), and platelet-derived growth factor receptor
(PDGFRB). These results delineated an excellent selectivity kinase
profile for the N-(benzothiazolyl)-phenylamides, 20 and 24, in spite of their competition for
the ATP.
Cellular Assays of CK-1δ Inhibitors
To determine
whether small molecule inhibition of CK-1 kinase activity by novel
CK-1δ inhibitors will prevent TDP-43 phosphorylation in vitro,
we utilized a mammalian cultured cell model of induced TDP-43 phosphorylation
driven by glutathione depletion. HEK293 cells were pretreated with
CK-1δ inhibitors for 1.5 h. Phosphorylation of endogenous cellular
TDP-43 was then induced with exposure to ethacrynic acid.[22,35] Cells were harvested, lysed, and tested for changes in TDP-43 phosphorylation
state by immunoblotting. Candidate inhibitors were tested at an extended
concentration range between 1.25 and 160 μM, with levels of
TDP-43 phosphorylation determined by immunoblotting (Figure 7). Compound 46 exhibited the strongest
inhibition of TDP-43 phosphorylation, with nearly complete absence
of phosphorylation at 2.5 μM and higher. Amide 38 exhibited significant reduction of TDP-43 phosphorylation by 5 μM
inhibitor, while 41 had greater than 2-fold reduction
in TDP-43 phosphorylation by 10 μM. Compound 24 had moderate inhibition at 1.25 μM and above, while compounds 20 and 34 demonstrated inhibition at concentrations
above 80 μM. Relatively low doses of the selected CK-1δ
inhibitors are required to dramatically reduce TDP-43 phosphorylation
in mammalian cells, indicating that inhibition of CK-1δ may
be viable strategy to reduce neurotoxicTDP-43 phosphorylation in
more complex in vivo systems as well.
Figure 7
CK-1δ inhibitors prevent TDP-43
phosphorylation in HEK293
cells. HEK293 cells are incubated with increasing concentrations of
CK-1δ inhibitors for 1.5 h, then treated with 150 μM ethacrynic
acid to induce TDP-43 phosphorylation. Increasing concentrations of
compounds (A) 20, (B) 24, (C) 34, (D) 38, (E) 41, or (F) 46 prevent ethacrynic acid induced TDP-43 phosphorylation.
CK-1δ inhibitors prevent TDP-43
phosphorylation in HEK293
cells. HEK293 cells are incubated with increasing concentrations of
CK-1δ inhibitors for 1.5 h, then treated with 150 μM ethacrynic
acid to induce TDP-43 phosphorylation. Increasing concentrations of
compounds (A) 20, (B) 24, (C) 34, (D) 38, (E) 41, or (F) 46 prevent ethacrynic acid induced TDP-43 phosphorylation.
Blood–Brain Barrier Penetration
Blood–brain
barrier (BBB) penetration is an essential property for any compound
developed to target neurodegenerative diseases. Thus the next step
in development for CK-1δ inhibitors is to determine CNS activity
and BBB penetration, so selection of lead compounds for in vivo studies
can proceed. The BBB is a unique barrier, controlling the selective
and specific transport of both exogenous and endogenous materials
to the brain. Because of its biological structure, lipophilic compounds
with low molecular weight (MW< 550 Da) are more likely to cross
the BBB and may pass directly through via passive diffusion between
the capillary walls. Determination of BBB penetration and other druglike
properties at early stages during drug discovery is of utmost importance
to select good candidates for in vivo studies and further pharmacological
development. Parallel artificial membrane permeability assay (PAMPA)
is a high-throughput technique developed to predict passive permeability
through biological membranes.[36] To explore
the ability of N-(benzothiazolyl)-phenylacetamides
to penetrate into the brain, we used the PAMPA-BBB method employing
a porcine brain lipid membrane. First, an assay validation was made
comparing the reported permeabilities (Pe) values
of commercial drugs with the experimental data obtained by using this
methodology (Figure S2 Supporting Information). A good correlation between experimental-described values was obtained Pe (exp) = 1.1202 (bibl) – 0.7413 (R2 = 0.9689). From this equation and following the pattern
established in the literature for BBB permeation prediction,[37] we could classify compounds as CNS+ when they
present a permeability >3.74 × 10–6 cm·s–1. The in vitro permeabilities (Pe)
of commercial drugs through lipid membrane extract together with those
belonging to several CK-1δ inhibitors with IC50 values
below 90 nM were determined and described in Table 5. Prediction of BBB permeability
for compound 24 was not possible due to the low solubility
of this compound in the assay vehicle. With exception of derivative 47, all the N-(benzothiazolyl)-phenylacetamides
tested were predicted to cross the BBB by PAMPA assay. These compounds
are thus the preferred candidates for further pharmaceutical development
including in vivo studies.
Table 5
Permeability (Pe
10–6 cm·s–1) in the PAMPA-BBB
Assay for 10 Commercial Drugs, Used in the Experiment Validation,
and Different N-(Benzothiazolyl)-phenylamides with
Their Predictive Penetration in the CNS
compd
bibl
Pea (10–6 cm·s–1)
BBB
prediction
atenolol
0.8
0.2 ± 0.1
caffeine
1.3
0.9 ± 0.1
desipramine
12
14.6 ± 0.6
enoxacine
0.9
0.2 ± 0.1
hydrocortisone
1.9
1.1 ± 0.8
ofloxacine
0.8
0.4 ± 0.3
piroxicam
2.5
0.5 ± 0.1
promazine
8.8
12.7 ± 1.3
testosterone
17
21.8 ± 4.0
verapamil
16
24.6 ± 1.5
17
5.6 ± 0.8
CNS+
20
11.2 ± 2.0
CNS+
27
9.6 ± 0.1
CNS+
34
11.2 ± 2.0
CNS+
38
11.2 ± 2.0
CNS+
41
6.4 ± 3.0
CNS+
42
14.6 ± 0.1
CNS+
46
4.4 ± 2.9
CNS+
47
1.4 ± 1.5
CNS–
52
2.6 ± 0.8
CNS+/CNS–
55
7.2 ± 2.5
CNS+
PBS:EtOH (70:30) was used as solvent.
Data are the mean ± SD of three independent experiments.
PBS:EtOH (70:30) was used as solvent.
Data are the mean ± SD of three independent experiments.
Efficacy on Drosophila Transgenic
TDP-43 flies
As the N-(benzothiazolyl)-phenylacetamides
here described target CK-1δ and are able to cross the BBB, we
decided to evaluate their therapeutic potential in vivo, using a new
transgenicDrosophila model of TDP-43proteinopathies.[38] Several Drosophila models of TDP-43 proteinopathies, based
on the expression of humanTDP-43 (hTDP-43) protein by the Gal4/UAS
binary expression system, were recently characterized.[39] Collectively, these models showed that in flies,
hTDP-43 expression recapitulates several key features of the humanTDP-43 proteinopathies, including axon and neuron degeneration, impaired
motor behavior, cognitive deficits, and reduced lifespan. Additionally,
biochemical data showed that hTDP-43 proteins undergo processing and
abnormal phosphorylation at disease-specific sites in flies. In this
study, we used the Drosophila lifespan
as a phenotypic test to evaluate the neuroprotective role of N-(benzothiazolyl)-phenylacetamides against hTDP-43 potentially
produced by decreasing its phosphorylation by inhibition of CK-1δ.
Reduction of the lifespan of flies is a phenotype closely related
to the neurodegenerative process that reflects an alteration of either
neuronal functioning or cell viability. Previously, we showed that
specific expression of hTDP-43 in adult differentiated neurons, using
the panneuronal inducible GeneSwitch-GAL4 system (elav-Gal4GS),[40] drastically reduced Drosophila lifespan.[38] To check our hypothesis, we selected four compounds as chemical
probes, (20, 24, 35, and 9), with different CK-1δ inhibition potency (IC50 values of 23 nM, 68 nM, and 2.22 μM for compounds 20, 24, and 35, respectively, and
the inactive N-benzothiazolyl-phenylacetamide 9). Induction of hTDP-43 expression on adult flies was started
nine days after hatching by feeding with RU486 (Mifepristone). From
this moment, the gene-switch was “ON” and the UAS-transgene
was expressed. As shown in Figure 8, feeding
flies with food supplemented with 100 nM of compounds 20, 24, or 35 significantly extended fly
lifespan (20, mean lifespan = 37.84 days, p = 0.0 × 10+00, N = 178; 24, mean lifespan = 38.63 days, p = 0.0 × 10+00, N = 163; 35, mean lifespan
= 36.17 days, p = 4.2 × 10–6, N = 173), compared with the control group (DMSO,
mean lifespan = 33.17 days, N = 151). Interestingly,
in direct correlation with their inhibitory potency on CK-1δ
in vitro (Table 3), the benzothiazoles 20 and 24 were more efficient in reducing hTDP-43toxicity than 35. This compound is 100-fold less potent
than 20 and 24 as CK-1δ inhibitor.
Furthermore, the chemically similar inactive compound 9 did not significantly modify fly longevity (N = 102). From these experiments, we can conclude that CK-1δ inhibitors
here reported have a protective effect on in vivo hTDP-43 neurotoxicity,
showing their potential for the pharmacological treatment of humanTDP-43 proteinopathies such ALS.
Figure 8
CK-1δ inhibitors decrease TDP-43
toxicity in flies. Lifespan
of elav-Gal4GS > UAS-hTDP-43 transgenic
flies expressing hTDP-43 proteins specifically in adult differentiated
neurons and treated with candidate drugs or vehicle (DMSO, control
flies). The survival curves show the proportion of living flies as
a function of age (days). The longevity assay was performed on a large
cohort (N > 150/experimental condition, see text).
Statistical data relative to longevity experiments are described in
the text. The lifespan of the flies was significantly increased when
they were treated with 100 nM of compounds 20, 24, or 35, as judged by the log rank test.
CK-1δ inhibitors decrease TDP-43toxicity in flies. Lifespan
of elav-Gal4GS > UAS-hTDP-43transgenic
flies expressing hTDP-43 proteins specifically in adult differentiated
neurons and treated with candidate drugs or vehicle (DMSO, control
flies). The survival curves show the proportion of living flies as
a function of age (days). The longevity assay was performed on a large
cohort (N > 150/experimental condition, see text).
Statistical data relative to longevity experiments are described in
the text. The lifespan of the flies was significantly increased when
they were treated with 100 nM of compounds 20, 24, or 35, as judged by the log rank test.
Conclusions
The
search of new therapies for ALS is an urgent need. The identification
of pathological TDP-43 as the hallmark lesion in sporadic ALS open
new avenues for pharmacological intervention. Our library screening
methodology has led to the discovery and further optimization of a
new family of potent CK-1δ inhibitors able to reduce TDP-43
phosphorylation in a cellular-based assay. These compounds are heterocyclic
small molecules with IC50 on the selected kinase in the
nanomolar range and selective on a 456 kinases panel. They are predicted
to cross the blood–brain barrier, making them excellent tools
for further pharmacological studies, and they have a protective effect
on in vivo hTDP-43neurotoxicity Drosophila model. Collectively, all these data show that N-benzothiazolyl amides represent a promising family of new drugs
with potential for the pharmacological treatment of humanTDP-43 proteinopathies
such as ALS.
Experimental Section
Chemistry
Substrates were purchased from commercial
sources and used without further purification. Melting points were
determined with a Mettler Toledo MP70 apparatus. Crude residues were
purified with the indicated solvent as eluent by flash column chromatography
carried out at medium pressure using silica gel (E. Merck, grade 60,
particle size 0.040–0.063 mm, 230–240 mesh ASTM) or
IsoleraOne flash purification system from Biotage. Compounds were
detected with UV light (254 nm). 1HNMR spectra were obtained
on the Bruker AVANCE-300 spectrometer working at 300 MHz or on a Varian
INOVA 400 spectrometer working at 400 MHz. Typical spectral parameters:
spectral width 16 ppm, pulse width 9 μs (57°), data size
32 K. 13CNMR experiments were carried out on the Bruker
AVANCE-300 spectrometer operating at 75 MHz or on a Varian INOVA 400
spectrometer working at 100 MHz. The acquisition parameters: spectral
width 16 kHz, acquisition time 0.99 s, pulse width 9 μs (57°),
data size 32 K. Chemical shifts are reported in values (ppm) relative
to internal Me4Si, and J values are reported
in Hz. HPLC analyses were performed on Alliance Waters 2690 equipment,
with a UV detector photodiode array Waters 2996 with MS detector MicromassZQ
(Waters), using an Sunfire column C18, 3.5 μm (50 mm ×
4.6 mm) and acetonitrile and Milli-Q water (with 0.1% formic acid)
as mobile phase. The standard gradient consisted of a 5 min run from
15% to 95% of acetonitrile at a flow rate of 1 mL/min. Elemental analysis
results of all the new compounds were recorded on Heraeus CHN-O-rapid
analyzer performed by the analytical department at CENQUIOR (CSIC),
and values were within ±0.4% of the theoretical values for all
compounds; therefore, these compounds meet the criteria of ≥95%.
Additionally, purity of all final compounds was found to be ≥95%
by LC/MS analysis. The microwave assisted syntheses were carried out
using a Biotage Initiator 8 single-mode cavity instrument from Biotage.
Experiments were performed with temperature control mode in sealed
microwave process vials. The temperature was measured with an IR sensor
on the outside of the reaction vessel. Stirring was provided by an
in situ magnetic stirrer.
General Procedure for 1–8, 10, 13, 17–44, and 46–57
A
mixture of
the corresponding acid (1 equiv) and SOCl2 (1.5 equiv)
was heated at 80 °C for 6 h. Afterward, the excess of SOCl2 was removed under reduced pressure and the acyl chloride
obtained was used in the next step of the synthesis without further
purification.A mixture of the amine derivative (1 equiv), the
corresponding acyl chloride previously synthesized (1 equiv), and
THF if necessary, was heated under microwave irradiation (5–20
min, 110–150 °C). After cooling to room temperature, 50
mL of CH2Cl2 were added and the mixture was
extracted with HCl 0.1 M (50 mL × 3). Afterward, the organic
layer was washed with a saturated solution of NaHCO3 (50
mL × 3) and with a saturated solution of NaCl (50 mL × 3).
Finally, the organic phase was dried over sodium sulfate and the solvent
evaporated under reduced pressure. The residue was chromatographed
as indicated in each case.
Reagents: 6-(trifluoromethyl)benzothiazol-2-amine
(1.1 mmol) and 3,3-dimethylbutanoyl chloride (1.1 mmol). Reaction
conditions: 5 min at 150 °C. The crude product was purified by
IsoleraOne (AcOEt/hexane 1:1) to afford a white solid (146.4 mg, 40%),
mp 189–191 °C. 1HNMR (300 MHz, DMSO-d6): δ 12.50 (s, 1H), 8.47 (s, 1H), 7.88
(d, J = 8.5 Hz, 1H), 7.72 (dd, J = 8.6, 1.6 Hz, 1H), 2.40 (s, 2H), 1.02 (s, 9H). 13CNMR
(75 MHz, DMSO-d6): δ 171.4, 160.9,
151.2, 131.9, 124.6 (q, J = 271.7 Hz), 123.6 (q, J = 31.9 Hz), 122.8 (q, J = 3.0 Hz), 120.8,
119.8 (q, J = 4.1 Hz), 48.0, 31.0, 29.4. HPLC: purity
>99%. m/z (ES) 317 [M + H]. Anal.
(C14H15F3N2OS) C, H, N,
S.
General Procedure for 12, 14–15, and 45
A
mixture of the corresponding
acid derivative (1 equiv), the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) (1.3 equiv), 4-dimethylaminopyridine (DMAP) (0.2 equiv), and
triethylamine (TEA) (1 equiv) if necessary, in CH2Cl2 or DMF (20–40 mL) was stirred at room temperature
for 1 h. Afterward, the amine derivative (1 equiv) was added and the
reaction mixture was heated at the temperature and during the time
indicated in each case. After cooling, the solvent was evaporated
under reduced pressure. The residue was dissolved in AcOEt (50 mL)
and extracted with HCl 0.1 M (50 mL × 3). Then the organic layer
was washed with a saturated solution of NaHCO3 (50 mL ×
3) and with a saturated solution of NaCl (50 mL × 3). Finally,
the organic phase was dried over sodium sulfate and the solvent evaporated
under reduced pressure. The residue was chromatographed as indicated
in each case.
N-(Benzothiazol-2-yl)-2,2-diphenylacetamide
(12)
Reagents: 2,2-diphenyl-acetic acid (1.7
mmol), EDC (2.2 mmol), DMAP (0.3 mmol), CH2Cl2 (40 mL), and benzothiazol-2-amine (1.7 mmol). Reaction conditions:
48 h at room temperature. The desired product was obtained directly
after the extractions affording a white solid (417.9 mg, 73%), mp
212–213 °C. 1HNMR (300 MHz, DMSO-d6): δ 12.83 (s, 1H), 7.98 (d, J = 7.4 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.57–7.08
(m, 12H), 5.42 (s, 1H). 13CNMR (75 MHz, DMSO-d6): δ 171.4, 158.2, 148.8, 139.2, 131.8, 128.9,
127.6, 126.6, 124.1, 122.1, 121.0, 56.6. HPLC: purity 97%. m/z (ES) 345 [M + H]. Anal. (C21H16N2OS) C, H, N, S.
N-(Benzothiazol-2-yl)-3-phenylpropanamide (14)
Reagents: 3-(3-chloro-phenyl)propanoic acid (1.7
mmol), EDC (2.2 mmol), DMAP (0.3 mmol), CH2Cl2 (40 mL), and benzothiazol-2-amine (1.7 mmol). Reaction conditions:
17 h at reflux temperature. The crude product was purified by IsoleraOne
(AcOEt/hexane 1:1) to afford a white solid (166.2 mg, 32%), mp 158–156
°C. 1HNMR (300 MHz, DMSO-d6): δ 12.37 (s, 1H), 7.97 (d, J = 7.9 Hz, 1H),
7.73 (d, J = 8.1 Hz, 1H), 7.48–7.39 (m, 1H),
7.36 (s, 1H), 7.34–7.19 (m, 4H), 3.02–2.90 (m, 2H),
2.88–2.77 (m, 2H). 13CNMR (75 MHz, DMSO-d6): δ 171.6, 158.1, 148.9, 143.7, 133.3,
131.8, 130.6, 128.6, 127.4, 126.5, 126.4, 123.8, 122.0, 120.8, 36.8,
30.1. HPLC: purity >99%. m/z (ES)
317 [M + H]. Anal. (C16H13ClN2OS)
C, H, N, S.
Reagents: 3,3-diphenyl-propanoic acid (1.7
mmol), EDC (2.2 mmol), DMAP (0.3 mmol), CH2Cl2 (40 mL), and benzothiazol-2-amine (1.7 mmol). Reaction conditions:
17 h at reflux temperature. The crude product was purified by IsoleraOne
(AcOEt/hexane 1:1) to afford a white solid (195.7 mg, 33%), mp 197–198
°C. 1HNMR (300 MHz, DMSO-d6): δ 12.43 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H),
7.72 (d, J = 8.2 Hz, 1H), 7.42 (dd, J = 10.7, 4.6 Hz, 2H), 7.37–7.22 (m, 8H), 7.22–7.11
(m, 2H), 4.65 (t, J = 8.0 Hz, 1H), 3.31 (d, J = 8.1 Hz, 2H). 13CNMR (75 MHz, DMSO-d6): δ 170.7, 158.0, 148.8, 144.1, 131.7,
127.9, 126.7, 126.4, 123.9, 122.0, 120.8, 46.7, 41.3. HPLC: purity
97%. m/z (ES) 359 [M + H]. Anal.
(C22H18N2OS) C, H, N, S.
N-(Benzothiazol-2-yl)-2-phenylacetamide (45)
Reagents: 2-phenylacetic acid (1.7 mmol), EDC
(2.2 mmol), DMAP (0.3 mmol), DMF (20 mL), and benzothiazol-2-amine
(1.7 mmol). Reaction conditions: 17 h at reflux temperature. The crude
product was purified by IsoleraOne (AcOEt/hexane 1:1) to afford a
yellow solid (159.1 mg, 34%), mp 162–164 °C (lit.[41] 160–161 °C). 1HNMR (300
MHz, DMSO-d6): δ 12.58 (s, 1H),
7.95 (d, J = 7.9 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.50–7.18
(m, 6H), 3.82 (s, 2H). 13CNMR (75 MHz, DMSO-d6): δ 170.2, 157.9, 148.5, 134.6, 131.4, 129.3,
128.4, 126.9, 126.1, 123.5, 121.7, 120.5, 41.8. HPLC: purity >99%. m/z (ES) 269 [M + H]. Anal. (C15H12N2OS) C, H, N, S.
(Benzothiazol-2-yl)-2-(4-chlorophenyl)acetate
(9)
To a solution of benzothiazol-2-ol (1.2
mmol) and pyridine
(4.6 mmol) in anhydrous CH2Cl2 (17 mL), 2-(4-chlorophenyl)acetyl
chloride (1.2 mmol) was added drop by drop at room temperature. The
reaction was heated vigorously under reflux for 6 h. To get reaction
to completion, a catalytic amount of DMAP was added and stirred overnight
at room temperature. Afterward, the mixture was extracted with a 0.1
M solution of HCl (50 mL × 3). The organic phase was dried over
sodium sulfate and the solvent evaporated under reduced pressure.
The residue was purified by column chromatography using AcOEt/hexane
(1:2) as eluent to afford a white solid (74.1 mg, 21%), mp 137–139
°C. 1HNMR (300 MHz, DMSO-d6): δ 8.15 (dd, J = 7.8, 1.8 Hz, 1H), 7.72
(dd, J = 7.1, 2.1 Hz, 1H), 7.49–7.19 (m, 6H),
4.42 (s, 2H). 13CNMR (75 MHz, DMSO-d6): δ 172.3, 169.9, 136.3, 134.1, 131.3, 128.1, 126.4,
123.2, 122.7, 122.6, 111.4, 39.8. m/z (ES) 304 [M + H]. Anal. (C15H10ClNO2S) C, H, N, S.
A solution of 6-(trifluoromethyl)benzothiazol-2-amine
(1.1 mmol) and 1-isocyanate-4-methoxybenzene (1.1 mmol) in THF (0.4
mL) was heated under microwave irradiation (1 h, 110 °C). After
cooling to room temperature, 50 mL of CH2Cl2 were added and the mixture was extracted with a saturated solution
of NaCl (50 mL × 3). The organic phase was dried over sodium
sulfate and the solvent evaporated under reduced pressure. The residue
was purified by column chromatography using AcOEt/hexane (1:3) as
eluent to afford a white solid (30.0 mg, 7%), mp 194–196 °C. 1HNMR (300 MHz, DMSO-d6): δ
10.97 (s, 1H), 8.99 (s, 1H), 8.39 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.72–7.62 (m, 1H), 7.41 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 3.72 (s,
3H). 13CNMR (75 MHz, DMSO-d6): δ 162.6, 160.8, 155.4, 151.8, 132.3, 131.1, 125.3 (q, J = 39.6 Hz), 124.8 (q, J = 242.6 Hz),
122.8 (q, J = 2.7 Hz), 120.9, 119.5 (q, J = 4.2 Hz), 119.5, 114.1, 55.2. HPLC: purity >99%. m/z (ES) 368 [M + H]. Anal. (C16H12F3N3O2S) C, H, N, S.
Molecular Modeling
Ligand Preparation
Molecular modeling
of the compounds
(12, 20, 24, and 34) was performed using Spartan 10 V.1.1.0 (Wave function Inc. Irvine,
CA, 2000) software in order to obtain the most favorable conformations
for each ligand. Structures were minimized in vacuum with mechanical
molecular force field MMFF.[42] Then the
lower energy compounds were submitted an equilibrium geometry calculation
using a semiempirical PM6 method.[43] Optimized
structures models were used to the docking studies.
Docking Studies
Initial protein structures were set
up using Sybyl 8.0 software[44] adding hydrogens,
removing ligands, cofactors, waters, and capping N and C-terminal
residues and finally removing clashes and amide bumps. To carry out
blind docking experiments, the resulting proteins structures were
used with Autodock Vina software.[45,46] To this end,
grids of points covering the whole protein were generated with Box
size: 126 × 126 × 126 points with a standard space of 0.375
Å. The center of the box is the protein center. The resulting
docking solutions were clustered, and only the lower energy binding
pose in each protein structure was considered for further analysis.
Visual inspection of solutions for all ligands in all structures led
us to select only the solutions obtained for structure 3UYS, which provided
a common binding mode for all ligands compatible with the substrate
binding site. Finally, the most interesting complexes were minimized
with MMFF94 force field implemented in Sybyl 8.0 until a 0.01 kcal/mol
gradient was reached in order to carry out a detailed analysis.
Biology
CK-1δ Assay Protocol
The “Kinase-Glo”
Kit from Promega was used to screen compounds for activity against
CK-1δ. Kinase-Glo assays were performed in assay buffer using
black 96-well plates. In a typical assay, 10 μL of test compound
(dissolved in dimethyl sulfoxide [DMSO] at 1 mM concentration and
diluted in advance in assay buffer to the desired concentration) and
10 μL (16 ng) of enzyme were added to each well followed by
20 μL of assay buffer containing 0.1% casein as substrate and
4 μM ATP. The final DMSO concentration in the reaction mixture
did not exceed 1%. After 60 min incubation at 30 °C, the enzymatic
reaction was stopped with 40 μL of Kinase-Glo reagent. Glow-type
luminescence was recorded after 10 min using a FLUOstar Optima (BMG
Labtechnologies GmbH, Offenburg, Germany) multimode reader. The activity
is proportional to the difference of the total and consumed ATP. The
inhibitory activities were calculated on the basis of maximal activities
measured in the absence of inhibitor. The IC50 was defined
as the concentration of each compound that reduces a 50% the enzymatic
activity with respect to that without inhibitors.
Kinetic Studies
on CK-1δ
To investigate the inhibitory
mechanism of ATP competitivity of benzothiazoles on CK-1δ, several
kinetic experiments were performed. Lineweaver–Burk plots of
enzyme kinetics are shown in Figure 5. Kinetic
experiments varying both ATP (from 1 to 50 μM), compound used
as control IC261 (from 0.25 to 0.5 μM), and benzothiazoles 20, 24, and 34 (from 0.025 to 0.05
μM) concentrations were performed using the ADP-Glo Kinase Assay.[47]
Cell Cultures Assays
HEK293 cells
(ATCC, Manassas,
VA) were cultured in 6-well dishes under standard culture conditions
in Dulbecco Modified Eagle Medium (DMEM), 10% defined fetal bovine
serum (FBS), and penicillin (50 IU/mL)–streptomycin (50 mg/mL).
CK-1δ inhibitors were diluted in dimethyl sulfoxide (DMSO),
and 8 μL of inhibitor + DMSO or DMSO alone (control) were added
to cells at the indicated concentrations. After 1.5 h of exposure
to the CK-1δ inhibitor alone, ethacrynic acid was added at a
final concentration of 150 μM and cells were incubated for 4
h to induce phosphorylation of endogenous TDP-43. Cells were harvested,
washed in phosphate buffered saline, and snap frozen prior to preparation
for immunoblot. Cell lysates were loaded and resolved on precast 4–15%
gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis
gels and transferred to polyvinylidene difluoride membrane as recommended
by the manufacturer (Bio-Rad, Hercules, CA). On immunoblots, phosphorylated
humanTDP-43 (pS409/410) was detected with a monoclonal antibody (TIP-PTD-M01,
Cosmo Bio, Carlsbad, CA). Actin (load control) was detected with a
monoclonal antibody (A4700, Sigma-Aldrich, St. Louis, MO).
In Vitro
Parallel Artificial Membrane Permeability Assay (PAMPA)
Ten
commercial drugs, phosphate buffer saline solution at pH 7.4
(PBS), ethanol, and dodecane were purchased from Sigma, Acros Organics,
Merck, Aldrich, and Fluka. The porcine polar brain lipid (PBL) (catalogue
no. 141101) was from Avanti Polar Lipids. The donor plate was a 96-well
filtrate plate (Multiscreen IP Sterile Plate PDVF membrane, pore size
0.45 μM, catalogue no. MAIPS4510) and the acceptor plate was
an indented 96-well plate (Multiscreen, catalogue no. MAMCS9610),
both from Millipore. Filter PDVF membrane units (diameter 30 mm, pore
size 0.45 μm) from Symta were used to filtered the samples.
A 96-well plate UV reader (Thermoscientific, Multiskan spectrum) was
used for the UV measurements. Test compounds [(3–5 mg of caffeine,
enoxacine, hydrocortisone, desipramine, ofloxacine, piroxicam, and
testosterone), (12 mg of promazine), and 25 mg of verapamil and atenolol]
were dissolved in EtOH (1000 μL). Then 100 μL of this
compound stock solution was taken and 1400 μL of EtOH and 3500
μL of PBS pH 7.4 buffer were added to reach 30% of EtOH concentration
in the experiment. These solutions were filtered. The acceptor 96-well
microplate was filled with 180 μL of PBS/EtOH (70/30). The donor
96-well plate was coated with 4 μL of porcine brain lipid in
dodecane (20 mg mL–1), and after 5 min, 180 μL
of each compound solution was added. Then 1–2 mg of every compound
to be determined their ability to pass the blood–brain barrier
were dissolved in 1500 μL of EtOH and 3500 μL of PBS pH
= 7.4 buffer, filtered, and then added to the donor 96-well plate.
Then the donor plate was carefully put on the acceptor plate to form
a “sandwich”, which was left undisturbed for 2 h and
30 min at 25 °C. During this time the compounds diffused from
the donor plate through the brain lipid membrane into the acceptor
plate. After incubation, the donor plate was removed. UV plate reader
determined the concentration of compounds and commercial drugs in
the acceptor and the donor wells. Every sample was analyzed at three
to five wavelengths, in three wells and in two independent runs. Results
are given as the mean [standard deviation (SD)], and the average of
the two runs is reported. Ten quality control compounds (previously
mentioned) of known BBB permeability were included in each experiment
to validate the analysis set.
Drosophila Strains and
Evaluation of Lifespan
To generate
the UAS-hTDP-43transgenic line, PCR-amplification of humanTDP-43
cDNA was achieved with the primers 5′-CGCAGGGCCGGACGGGCCCAAAATGTCTGAATATATTCGGGTAACCG-3′
and 5′-CGCAGGGCCCCAGTGGCC CTACATTCCCCAGCCAGAAGACTTAGAATCC-3′.
The PCR product was sub cloned into the pUASTattBSfiI vector as a
Sfi fragment and then sequenced. The pUASTattBSfiI, kindly provided
by Dr. Hervé Tricoire, had been generated by subcloning the
SfiI cassette (described in Lasbleiz et al., 2005)[48] into the pUASTattB vector digested with EcoRI/BglII. Transgenic strain was generated by BestGene
Inc. (Chino Hills, CA, USA), according to standard methods, using
the y1M ZH-2A w*; M ZH-51C (cytological region 51C on the second chromosome,
strain identifier at BestGene: 24482) as recipient strain. Drosophila were maintained on a 12:12 light/dark
cycle on standard cornmeal–yeastagar medium at 25 °C.
UAS-hTDP43transgenic males were crossed with elav-GAL4-GeneSwitch (ElavGal4GS) females.[40] Then 50:50 males and females (0–2 days old) of the progeny
were collected (clusters of 25–30) into food tubes containing
instant Drosophila medium (Carolina
Biological Supply Company, Burlington, NC). Vials were changed on
a 2–3 day cycle. After 7 days, flies were treated with 100
μg/mL RU486 (Mifepristone, Betapharma-Shanghai Co., Ltd., China)
in the presence of 100 nM candidate drugs or vehicle (DMSO). When
flies were fed with RU486, Gene-Switch was “On” and
the UAS-transgene was expressed. Every 2–3 days, the flies
were transferred to new tubes with fresh media and dead flies were
counted. Survival curves were established using the Kaplan–Meier
method, and differences between curves were assessed using a Log Rank
Test (OASIS web application).[49]
Authors: Luc Régal; Ludo Vanopdenbosch; Petra Tilkin; Ludo Van den Bosch; Vincent Thijs; Rafael Sciot; Wim Robberecht Journal: Arch Neurol Date: 2006-02
Authors: Mootaz M Salman; Zaid Al-Obaidi; Philip Kitchen; Andrea Loreto; Roslyn M Bill; Richard Wade-Martins Journal: Int J Mol Sci Date: 2021-04-28 Impact factor: 5.923
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