Development of highly selective casein kinase 1δ/1ε (CK1δ/ε) inhibitors with potent antiproliferative properties

Article abstract of DOI:10.1016/j.bmcl.2013.05.075

The development of a series of potent and highly selective casein kinase 1δ/ε (CK1δ/ε) inhibitors is described. Starting from a purine scaffold inhibitor (SR-653234) identified by high throughput screening, we developed a series of potent and highly kinas

Full text of DOI:10.1016/j.bmcl.2013.05.075

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4374–4380
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of highly selective casein kinase 1d/1e (CK1d/e)
inhibitors with potent antiproliferative properties
Mathieu Bibian a, , Ronald J. Rahaim a, , Jun Yong Choi a, Yoshihiko Noguchi a, Stephan Schürer d,
Weimin Chen c, Shima Nakanishi b, Konstantin Licht b, Laura H. Rosenberg c, Lin Li c, Yangbo Feng a,
Michael D. Cameron c, Derek R. Duckett c, John L. Cleveland b, William R. Roush a,
⇑
a Department of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
b Department of Cancer Biology, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
c Department of Molecular Therapeutics, Scripps Florida, 130 Scripps Way, Jupiter, FL 33458, United States
d Department of Molecular and Cellular Pharmacology and Center for Computational Science, University of Miami, 1120 NW 14th St., Miami, FL 33136, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of a series of potent and highly selective casein kinase 1d/e (CK1d/e) inhibitors is
Received 7 April 2013
Revised 20 May 2013
Accepted 21 May 2013
Available online 31 May 2013
described. Starting from a purine scaffold inhibitor (SR-653234) identified by high throughput screening,
we developed a series of potent and highly kinase selective inhibitors, including SR-2890 and SR-3029,
which have IC50 6 50 nM versus CK1d. The two lead compounds have 6100 nM EC50 values in MTT assays
against the human A375 melanoma cell line and have physical, in vitro and in vivo PK properties suitable
for use in proof of principle animal xenograft studies against human cancer cell lines.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Casein kinase 1d/
e inhibitor
Selective CK1d/ inhibitor
e
Purine scaffold kinase inhibitor
Antiproliferative agent
Potent growth inhibitor of A375 melanoma
cell line
The casein kinase 1 (CK1) family of serine/threonine-specific ki-
Casein kinases 1d and 1e are highly expressed in some cancers
nases is comprised of seven members (
a
, b1, d,
e
,
c
1,
c
2 and
c
3);
and appear to control tumor cell growth, apoptosis, metabolism
and differentiation.10,12 For example, forced expression of kinase-
impaired mutants of CK1d blocks SV40-induced cell transforma-
each isoform has a preference for pre-phosphorylated substrates.1
CK1 kinases regulate diverse processes including Wnt signaling,2,3
membrane trafficking,4 the actin cytoskeleton,5 the DNA damage
response,6 and circadian rhythms.7 Importantly, aberrant CK1d
tion and mammary carcinogenesis in vivo.13 Further, CK1
e
is re-
quired for the survival of breast cancer subtypes that rely on
aberrant b-catenin activity, and active, myristoylated CK1 is suffi-
cient to provoke transformation via stabilization of b-catenin and
activation of Wnt transcription targets.14 CK1d/
-directed stabil-
ization of b-catenin may occur via CK1d/ -directed phosphoryla-
tion of lipoprotein receptor-related protein 5/6 (Lrp5/6) and/or
and CK1
e
activity is implicated in human pathologies, including
e
neurodegenerative diseases, sleep disorders and cancer. CK1 ki-
nases are ubiquitously expressed in the central nervous system
and CK1d is thought to play roles in dopamine signaling, neuro-
transmitter release and the phosphorylation of neurotransmitter
receptors.8,9 Further, CK1d expression is elevated in Alzheimer’s
disease tissue and CK1d phosphorylates tau, which initiates micro-
tubule destabilization and neuronal cell death.5,10 These kinases
may also play roles in cleavage of the amyloid precursor protein
(APP),9 as CK1 inhibitors disrupt APP cleavage and a constitutively
e
e
dishevelled (dvl/dsh).15–18 CK1d and CK1
e also play roles in ovarian
cancer19 and pancreatic adenocarcinoma.20
These important biological roles have stimulated considerable
effort to develop CK1d/
e
inhibitors.10,21–24 Included among the
many small molecule inhibitors of CK1d that have been reported
are CKI-7,25 D4476,26,27 IC261,28 (R)-DRF053,22 Bischof-524 (com-
pound 5 in Ref. 24) and PF-670462 (see Fig. 1).29,30 CKI-7 is a
active form of CK1e
augments APP peptide production.9,11 Finally,
the up-regulation of CK1 isoforms in Alzheimer’s patients makes
CK1 an attractive target for the treatment of Alzheimer’s disease.9
6 l
M CK1 inhibitor, but does not readily pass cell membranes.25,26
IC261, D4476 and (R)-DRF053 are cell-permeable yet have limita-
tions. Specifically, D4476 is a 0.3
M CK1 inhibitor in vitro,26 has
low activity (20–50
M) in cell-based assays,27,29 and also inhibits
p38
, raising concerns regarding off-target effects.10,26,27 Further,
l
⇑
Corresponding author. Tel.: +1 561 228 2450; fax: +1 561 228 3052.
E-mail address: roush@scripps.edu (W.R. Roush).
These authors contributed equally to this work.
l
a
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.075

M. Bibian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4374–4380
4375
interface.11 These efforts led to SR-1277 being designated as Probe
ML177 in the MLPCN system.32 However, SR-1277 has poor solu-
bility, sub-optimal PK properties and metabolic liabilities due to
the thiophene unit and especially the aryl nitro substituent.33,34
Therefore, we have performed and report herein additional SAR
studies that led to the identification of several analogs (including
SR-2890 and SR-3029) that are appropriate for progression into
murine xenograft studies against human cancers.
We adopted the general procedure published by Schultz for
synthesis of analogs of SR-653234 and SR-1277.35–37 As depicted
in Figure 3 for the synthesis of SR-653234, the N-thienyl interme-
diate 2 was accessed via a Chan–Lam coupling reaction of commer-
cially available dichloropurine 1 and 3-thienylboronic acid.36,38,39
A one-pot double nucleophilic substitution sequence then con-
verted intermediate 2 into the targeted CK1d/e inhibitor. The regi-
oselectivity of the latter sequence is excellent, with the first amine
nucleophile adding to C(6) of the purine scaffold as has been dem-
onstrated previously.35–37
The substituted 2-(aminomethyl)benzimidazoles (6) used in
this study that are not commercially available were synthesized
as summarized in Figure 4. Thus, a substituted phenylenediamine
3 (prepared by reduction of the corresponding ortho-nitroaniline,40
if not commercially available) was coupled to N-Boc-glycine using
EDC and HOBt as the coupling reagents to give a mixture of 4a and
4b. The mixture of these two amides was heated at 80 °C in acetic
acid to effect cyclization to the N-Boc protected benzimidazole 5.
Finally, the Boc group was removed by treatment of 5 with a mix-
ture of HCl (12 N in water) and dioxane at room temperature over-
night. The product 6 was obtained as the HCl salt by precipitation
from diethyl ether. This three-step procedure usually did not re-
quire any chromatographic purification steps, and provided the
substituted benzimidazoles 6 (with a range of substituents corre-
sponding to those in the inhibitors presented in Tables 1–3) having
acceptable purity for use directly in the synthesis of the targeted
Figure 1. Representative CK1d/e inhibitors.
the IC50 of IC261 is only 1 lM for CK1 inhibition in vitro and 25 lM
in cells,10 and there are off target effects as IC261 binds to tubulin
and inhibits microtubule polymerization.28 Moreover, (R)-DRF053
is a potent, dual CK1/CDK inhibitor (14 nM vs CK1), yet only exhib-
CK1d/e inhibitors according to the procedure summarized in
its weak (EC50 17.2 lM) antiproliferative activity against human
Figure 3.
neuroblastoma SH-SY5Y cells. Bischof-5 is yet another potent
(48 nM) CK1d inhibitor, but is also weakly active in cells, likely
due to poor cell penetration.29 Finally, PF-670462 is a 14 nM inhib-
itor of CK1d in vitro and was initially reported to be highly selec-
tive, at least among the 45 kinases tested.29 Subsequent studies
showed that PF-670462 also potently inhibits p38 and EGFR.30
Using this chemistry, we synthesized a series of analogs of SR-
653234 with a range of substituents in the benzimidazole ring to
probe the effect of this substitution on inhibitor activity. Substitu-
tion of the benzimidazole ring in either position 4 (R1) or position 5
(R2) led to an increase of CK1d inhibition compared to the unsub-
stituted parent compound SR-653234 (Table 1). A trifluoromethyl
group at R1 modestly enhanced CK1d inhibition (compare entries
1 and 2) while nitro and methanesulfonyl substituents at this posi-
tion led to significantly more active analogs SR-1277 and SR-2805
(entries 7 and 11). Improvements of CK1d inhibitor activity were
also achieved by incorporating a range of substituents at R2. Substi-
tution with a trifluoromethyl group (SR-1273, entry 3), a nitro
group (SR-1278, entry 8), a cyano group (SR-1276, entry 6), a
methoxy group (SR-1279, entry 9) or a methanesulfonyl group
(SR-2797, entry 10) led to significant improvement of CK1d inhib-
itor activity.
Both PF-670462 and PF-4800567 (Pfizer’s CK1
e
inhibitor)30 lack
anti-cancer activity.28
A high-throughput screening (HTS) campaign under the aus-
pices of the MLPCN program at Scripps Florida, targeting inhibitors
of Wee1 degradation,31 identified SR-653234 as a promising hit.
Extensive mechanistic and biochemical profiling studies demon-
strated that SR-653234 and especially its analog SR-1277 (Fig. 2)
are highly selective CK1d/e inhibitors and that CK1d plays a crucial
role in regulating the activity of Wee1 at the G2/M cell cycle
A thiophene substituent, especially when not substituted at
positions 2 and/or 4, is generally considered to be a liability in view
of the potential for production of highly reactive metabolites.41,42
To avoid this potential problem, we sought other groups that could
be used at the purine 9-position (R3) without significant loss of
CK1d inhibitory activity (Table 2).43 Replacement of the thiophene
ring of SR-653234 by a cyclopentyl group led to a more potent
inhibitor, SR-2149 (entry 1). Although the furan-containing ana-
logs SR-2850 and SR-2007 had excellent potency, the furan ring
is also a known metabolic liability, especially when not substituted
at positions 2 and 4.43 On the other hand, several inhibitors bearing
fluoro-substituted phenyl rings at position R3 had very interesting
properties. As depicted by the results in entries 4–7 of Table 2, the
position of the fluorine substituent dramatically influenced the
Figure 2. CK1d/e inhibition data for SR-1277 and SR-653234.

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M. Bibian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4374–4380
Figure 3. Strategy for synthesis of purine-scaffold CK1d/e inhibitors, illustrated by the synthesis of SR-653234. Conditions: (a) ArB(OH)2, CuOAc2, MS 4 Å, NEt3, CH2Cl2, 24 h,
23 °C, 27 %; (b) 2-(aminomethyl)benzimidazole, (i-Pr)2NEt, i-PrOH, 30 min, 90 °C, microwave; (c) morpholine, 130 °C, 30 min, microwave, 70%.
Table 2
Activity of CK1d inhibitors with other substituents at N-9 (R3)
Entry Compound number R1
R2 R3
IC50 CK1da (nM)
1
2
3
4
5
6
7
SR-2149
SR-2850
SR-2007
SR-2362
SR-2364
SR-2366
SR-2368
H
H
H
Cl
H
H
H
H
H
Cyclopentyl
3-Furyl
3-Furyl
57
23
19
>1000
57b
114b
NO2
NO2
NO2
NO2
NO2
4-F-Phenyl
3-F-Phenyl
2-F-Phenyl
Figure 4. General method for synthesis of substituted 2-(aminomethyl)benzimi-
dazoles. Conditions: (a) EDCÁHCl, HOBtÁH2O, Boc-GlyOH, CH2Cl2, 23 °C, 2 h; (b)
AcOH (neat), 80 °C, 2 h; (c) HCl (12 N), dioxane, (1/1), overnight, 23 °C.
3,5-DiF-phenyl 75b
a
CK1d inhibition data obtained by Reaction Biology Corp., unless indicated
otherwise.
Table 1
Structure–activity relationship data for CK1d inhibitors with substituted benzimid-
azole units
b
CK1d inhibition data determined from an in-house kinase inhibition assay.
Table 3
Activity of CK1d inhibitors with other substituents at purine C-2 (R4)
Entry
Compound number
R1
R2
IC50 CK1da (nM)
1
2
3
4
5
6
7
8
9
SR-653234
SR-1272
SR-1273
SR-1274
SR-1275
SR-1276
SR-1277
SR-1278
SR-1279
SR-2797
SR-2805
H
CF3
H
H
H
H
NO2
H
H
H
H
H
CF3
Cl
F
CN
H
NO2
OMe
SO2Me
H
160
128
13
105
50
11
49
21
17
Entry
Compound number
R4
R2
IC50 CK1d (nM)
1
2
3
4
5
SR-1292
SR-1294
SR-2876
SR-2875
SR-2915
N-Piperazine
4-(N-Me)-piperazine
N-Piperazine
4-Amino-piperidine
3-Methyl-piperazine
H
H
Cl
Cl
H
3a
11a
51b
119b
199b
a
CK1d inhibition data obtained by Reaction Biology Corp.
CK1d inhibition data determined from an in-house assay.
10
11
10
16
b
SO2Me
a
CK1d inhibition data obtained by Reaction Biology Corp.
gave potent CK1d inhibitors, with the 3-fluorophenyl compound
SR-2364 having an IC50 value of 57 nM versus CK1d. The 3,5-difluo-
rophenyl analog SR-2368 was also a reasonably potent CK1d inhib-
itor (entry 7).
CK1d inhibitor activity. The 4-fluorophenyl analog SR-2362 was
essentially inactive whereas inhibitors with 2-fluorophenyl (SR-
2366, entry 6) and 3-fluorophenyl (SR-2364, entry 5) substitutions

M. Bibian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4374–4380
4377
Table 4
IC50 data, cell-based activity and in vitro PK data for selected CK1d inhibitors
Compound
Biochemicala
Cell activity
In vitro PK properties
Microsome stability (h/r/m) T1/2b (min) Solublityc
CK1dInh IC50 (nM)
CK1e
Inh IC50 (nM)
MTT assay A375 EC50 (nM)
(
lM)
Cyp inhibitiond
SR-653234
SR-1277
SR-2848
SR-2849
SR-2889
SR-2890
SR-3029
PF-670462
D4476
160
49
30
11
5
540
260
111
22
89
3
2
38
86
>10,000
10,000
2300
1570
11/6/1
7/5/2
28
1
e
>10
>10
>10
>10
>10
>10
l
l
l
l
l
l
M
M
M
M
M
M
31/26/15
6/5/1
25/NA/8
44/NA/11
18/NA/5
25
18
71
60
13
4
44
13
167
29
—
260
90
350
199
—
Bischof-5
AC220
Sunitinib
54/21/21
a
b
c
Data obtained by Reaction Biology Corporation (RBC).
Microsome stability using human, rat, and mouse liver microsomes, with sunitinib as the reference. ‘NA’ = data not obtained.
Solubility in DMEM/10% FBS.
Cyp assay versus 1A2, 2C9, 2D6, and 3A4.
d
e
86% Inhibition of 1A2 at 10 lM.
Another liability of lead compound SR-1277 is its low solubility
lM in PBS buffer). In an attempt to address this problem, addi-
(1
tional analogs with piperazine and piperidine substituents R4 were
synthesized (see Table 3). Inhibitor SR-1292 with an unsubstituted
piperazine ring at this position is a 3 nM inhibitor of CK1d (entry
1), approximately sixteen-fold more potent than SR-1277. The N-
methylpiperazine derivative (SR-1294) was also highly potent
(11 nM inhibitor of CK1d, entry 2). However, use of several other
amines at this position led to loss of inhibitor activity (SR-2875
and SR-2915, entries 4 and 5). The data for these compounds sug-
gests that the positioning or the steric environment of the pipera-
zine ammonium group (protonated at physiological pH) is critical
for high CK1d inhibitor activity. Not surprisingly, solubility of SR-
Figure 5. Poses of SR-1277 (left) and SR-1292 (right) docked into the CK1d active
site.
1292 and SR-2876 (75 lM and 54 lM in PBS, respectively) was
substantially greater than that for analogous compounds with
morpholine substituents at R4.
target activity of our CK1d/e inhibitors (vida infra). The significant
A number of additional, potent analogs were synthesized by
combining two or more of the structure elements highlighted in
Tables 1–3. Data for several such analogs are summarized in
Table 4.
increase in inhibitor activity associated with use of piperazine or
N-methyl piperazine as the R4 substituent (e.g., SR-1292 and SR-
1294, Table 3) is consistent with the positively charged ammonium
unit of these agents interacting with the amide carbonyl of Asp-
132. Finally, the R3 thiophene, furan, and 3-fluorophenyl groups
that are associated with high inhibitor potency (Table 2) bind in
a relatively tight recognition pocket deep in the ATP binding site.
Throughout the progression of this work, new inhibitors were
subjected to an MTT assay against the human melanoma A375 cell
line.45 Those compounds that exhibited significant anti-prolifera-
tive activity in this assay (EC50 <200 nM) were taken forward to a
core set of in vitro DMPK assays (microsome stability, inhibition
of cytochrome P450 1A2, 2C9, 2D6, and 3A4) to assess the drug-
like characteristics of the increasingly optimized candidates.46
The data summarized in Table 4 demonstrates that we have
accomplished the synthesis of a number of very potent CK1d inhib-
itors (e.g., SR-2848, SR-2849, SR-2889, SR-2890, and SR-3029),
Kinetic analysis demonstrated that the purine scaffold CK1d/
e
inhibitors that are the subject of this paper are ATP competitive;
Ki’s measured for SR-1277, SR-2890, and SR-3029 are 69 nM,
14 nM, and 97 nM, respectively. This insight enabled us to perform
modeling studies of inhibitors bound to CK1d using the published
CK1d-PF670462 co-crystal structure as the template for struc-
ture-based design.44 Docking poses of SR-1277 and SR-1292 in
the CK1d-PF670462 active site are shown in Figure 5. Our working
hypothesis is that the ability of the substituents on the benzimid-
azole unit (either R1 or R2, as defined in Table 1) to interact with
Arg-13 is responsible for the significant improvement in CK1d inhi-
bition activity associated with these substitutions, leading to en-
hanced selectivity versus FLT3, which is the most significant off-

4378
M. Bibian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4374–4380
Figure 6. Dendrogram presentation of results of DiscoverRXÒ KINOMEscanÒ kinase binding selectivity analysis of PF-670462, SR-1277, SR-2890 and SR-3029. Data are
presented for all kinases that have <10% control activity at 10
of the inhibitor).
lM (% control is the percentage of kinase remaining bound to the bead-bound active-site ligand in the presence
Table 5
Kinases showing less than 10% activity in the presence of 10
SR-3029
l
M SR-1277, SR-2890 or
SR-3029
some of which have low nM EC50’s as inhibitors of melanoma A375
cell growth. The data in Table 4 indicates that inhibitors with high
microsome stability—approaching that of the reference compound
sunitinib—are those that have piperazine derivatives at R4. In addi-
Target
SR-1277
SR-2890
68
0.2
10
1.5
BMP2K
6.2
56
56
39
4.4
19
38
100
22
22
1.5
0.4
3.7
42
41
19
12
44
26
7.3
23
22
2.1
98
37
91
20
55
88
54
100
24
35
34
CDK4/cyclin D1
CDK4/cyclin D3
CDK7
tion, the new CK1d/
demonstrate less than 50% inhibition of cytochrome P450 1A2,
2C9, 2D6, and 3A4 at a 10 M test concentration.
The exceptional activity of the purine scaffold CK1d/
e inhibitors presented in Table 4 consistently
72
l
CDK13
CSNK1A1
1
63
88
10
e
inhibitors
in MTT assays versus human melanoma A375 cells is striking (Ta-
ble 4), especially since other potent CK1d inhibitors are much less
active in this cell-based assay. For example, we have determined
that the EC50’s of PF-670462,29,30 D4476,26,27 and Bischof-524 in
CSNK1A1L
CSNK1D [CK1d]
9.6
4.2
0.5
3.4
7.3
11
3.7
5.2
11
100
100
100
47
2.6
38
2.5
1.6
0.1
CSNK1E [CK1
FLT3
e]
13
FLT3 (D835H)
FLT3 (D835Y)
FLT3 (ITD)
FLT3 (K663Q)
FLT3 (N841L)
LATS2
MARK2
MAST1
MELK
MYLK4
NLK
PCTK3
PDGFRB
PFTAIRE2
PFTK1
PRKCQ
ROCK2
37
10
15
13
13
3.1
96
6.2
7.1
85
5.6
6.6
9.4
9.3
4.6
5
the A375 melanoma MTT assay is >10 lM, 10 lM, and 2.3 lM,
respectively (see Table 4). The significantly reduced activity of
D4476 and Bischof-5 may be rationalized by poor cell penetration,
as has been noted elsewhere.24,27 However, the lack of anti-mela-
Table 6
IC50 values (nM) for inhibition of off-target kinases by SR-1277, SR-2890 and SR-
3029a,b
Off-Target Kinase
SR-1277 (nM)
SR-2890 (nM)
SR-3029 (nM)
62
8.2
47
FLT3
305
1340
—
—
311
109
809
283
391
1240
4420
—
3000
576
368
428
427
—
CDK4/cyclin D1
CDK4/cyclin D3
CDK6/cyclin D1
CDK6/cyclin D3
CDK9/cyclin K
40
100
100
9.6
57
8.8
99
1
RIOK3
SGK
a
Data obtained by Reaction Biology Corporation (RBC).
Comparative data, also from RBC, for inhibition of CK1d and CK1e are provided
TAOK2
TAOK3
84
85
7.4
4.8
b
in Table 4.

M. Bibian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4374–4380
4379
Table 7
Mouse PK data for selected CK1d/e inhibitors (IV dosing, 1 mg/kg, 10/10/80 DMSO/Tween/water)
Compound
Cmax
(lM)
Cl (ml/min/kg)
AUC (
lM h)
T1/2 (h)
%F
Brain penetration (%)
SR-653234
SR-1277
SR-2890
SR-3029
3.2
1.2
4.6
7.3
2.2
2.8
8.4
5.5
1.75
1.26
4.16
6.35
0.73
1.42
1.50
0.90
0
0
10
13
13
24
<1
12
noma activity for PF-670462 is a concern, as this compound is
widely believed to be a highly selective kinase inhibitor.
of SR-3029 suggests that this compound could be useful in animal
studies of brain cancers.
To address the question of kinase selectivity, and especially to
investigate if our compounds are hitting other kinase target(s) that
might be responsible for the potent antiproliferative effects, SR-
1277, SR-2890, and SR-3029 were subjected to the DiscoverRXÒ
KINOMEscanÒ analysis of 442 kinases. The Pfizer inhibitor PF-
In summary, we have developed a series of potent and highly
selective CK1d/e inhibitors with potent antiproliferative activity
by optimization of HTS-derived SR-653234. These efforts led to
the identification of SR-2890 and SR-3029 that have in vitro and
in vivo PK properties suitable for use in xenograft studies of human
cancers, including brain cancers in the case of SR-3029. Further
studies on the development of these compounds as novel antican-
cer agents, as well as studies targeting their use in appropriate
models of Alzheimer’s disease, will be reported in due course.
670462 was also tested. This assay, run at 10 lM, assesses the de-
gree to which the inhibitor competitively displaces a bead-bound
active-site ligand from a DNA-tagged kinase using quantitative
PCR. It is clear from the dendrogram depiction of these kinome
selectivity analyses (Fig. 6, which includes all kinases inhibited
P90% at 10
inhibitor. Among the 44 kinases that are inhibited P90% by
10 M PF-670462 are the pro-apototic kinases JNK, p38, and EGFR
isoforms (and strongly so). In contrast, these studies established
the high selectivity of SR-1277, SR-2890, and SR-3029 as CK1d/
inhibitors, with only 6 off-target kinases inhibited P90% at
10 M by SR-3029, and that SR-2890 is less kinase selective than
l
M) that PF-670462 is a very non-selective kinase
Acknowledgments
l
This research was supported by the NIH Molecular Library
Screening Center Network grant U54MH074404 (Dr. Hugh Rosen,
Principal Investigator), the Rendina Family Foundation, and funds
from the State of Florida to Scripps Florida. S. Nakanishi was sup-
ported by a Marie Mayer Fellowship.
e
l
either SR-1277 or SR-3029. A higher resolution snapshot of those
kinases with less than 10% activity in the presence of SR-1277,
SR-2890, or SR-3029 is provided in Table 5, with data for the ki-
nases inhibited P90% by specific inhibitors (i.e., less than 10% of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
05.075.
active kinase remaining at 10 lM) highlighted in blue. These data
show that the selectivity profile of each inhibitor is different, and
that there are only three kinases that are strongly inhibited by all
three inhibitors: CK1d, CK1e and FLT3, plus several FLT3 mutants.
Because the DiscoverRXÒ KINOMEscanÒ kinase binding assay is
a competitive active site probe displacement assay, with different
binding affinities of the active site probe for the range of kinases
studied, these data do not, and are not intended to correlate with
IC50 values. Therefore, to assess the inhibition potency of SR-
1277, SR-2890, and SR-3029 against the most important off-target
kinases identified, IC50 values were obtained by Reaction Biology
Corporation (Table 6). These data suggest that the most important
off-target kinase, FLT3, is only weakly inhibited by SR-1277, SR-
2890, and SR-3029. These data, together with the results summa-
rized in Table 4 for the MTT assay of the known FLT3 inhibitor
AC22047 against the A375 melanoma cell line, indicate that inhibi-
tion of FLT3 or FLT3 mutants is not responsible for the potent anti-
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