Discovery of CDK5 Inhibitors through Structure-Guided Approach

Article abstract of DOI:10.1021/acsmedchemlett.9b00029

Specific abrogation of cyclin-dependent kinase 5 (CDK5) activity has been validated as a viable approach for the development of anticancer agents. However, no selective CDK5 inhibitor has been reported to date. Herein, a structure-based in silico screenin

Full text of DOI:10.1021/acsmedchemlett.9b00029

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Letter
Discovery of CDK5 Inhibitors through Structure-Guided Approach
Nishat Zareen Khair, Jimma Likisa Lenjisa, Solomon Tadesse, Malika Kumarasiri,
Sunita K. C. Basnet, Laychiluh Bantie Mekonnen, Manjun Li, Sarah Al Haj
Diab, Matthew James Sykes, Hugo Albrecht, Robert Milne, and Shudong Wang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00029 • Publication Date (Web): 20 Mar 2019
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Discovery of CDK5 Inhibitors through Structure-Guided Approach
Nishat Z. Khair,ϯ Jimma L. Lenjisa,ϯ Solomon Tadesse, Malika Kumarasiri, Sunita KC. Basnet,
Laychiluh B. Mekonnen, Manjun Li, Sarah Diab, Matthew J. Sykes, Hugo Albrecht, Robert Milne, and
Shudong Wang*
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Centre for Drug Discovery and Development, School of Pharmacy and Medical Sciences, Cancer Research Institute,
University of South Australia, Adelaide, SA 5001, Australia.
KEYWORDS: AML, apoptosis, CDK5, CDK5 inhibitors, cell cycle, virtual screening
ABSTRACT: Specific abrogation of cyclin dependent kinase 5 (CDK5) activity has been validated as a viable approach for the
development of anticancer agents. However, no selective CDK5
inhibitor has been reported to date. Herein, a structure-based in
silico screening was employed to identify novel scaffolds from a
library of compounds to identify potential CDK5 inhibitors which
would be relevant for drug discovery. Hits, representatives of
three chemical classes, were identified as inhibitors of CDK5.
Structural modification of hit-1 resulted in 29 and 30. Compound
29 is a dual inhibitor of CDK5 and CDK2, whereas 30
preferentially inhibits CDK5. Both leads exhibited anticancer
activity against acute myeloid leukemia (AML) cells via a
mechanism consistent with targeting cellular CDK5. This study
provides an effective strategy for discovery of CDK5 inhibitors as
potential anti-leukemic agents.
A growing body of evidence supports cyclin-dependent kinases
(CDKs) as highly attractive molecular targets for the
development of anti-cancer drugs.1-3 CDKs are members of the
serine/threonine family of protein kinases with the primary
functions of regulating the cell cycle and transcription.1, 4 The
majority of these CDKs are highly deregulated in human
malignancies, and a number of recently discovered small-
molecule CDK inhibitors, including palbociclib, abemaciclib
and ribociclib, are instrumental in target validation and drug
development.1, 5 6 This has driven significant research efforts to
exploit CDKs for the development of a new generation of
targeted cancer therapeutic agents.
most cancers studied so far, there is strong evidence supporting
the specific targeting of CDK5 as a viable strategy for the
discovery of anticancer therapeutic agents that possibly
circumvent the apparent drawbacks of the currently available
therapies; in particular, the lack of efficacy, drug resistance and
14
toxicity to healthy tissues.13,
However, the discovery of
pharmacologic inhibitors of CDK5 with sufficient potency and
selectivity remains a highly challenging task. This is linked to a
high amino-acid sequence similarity between CDK5 and other
CDKs, particularly CDK2, where the two kinases differ in their
ATP binding pockets by only two amino-acid residues.15,
16
Roscovitine (seliciclib) and dinaciclib are currently the
prototype examples of inhibitors in clinical trials that target
multiple kinases including CDK5 (Figure S1). Several phase I
and phase II clinical trials in cancer patients have been
completed for these inhibitors.10 A recent Phase III clinical trial
demonstrated the potential anti-cancer activity of dinaciclib.17
However, both inhibitors were shown to target multiple kinases
in addition to CDK5. This in turn leads to serious off-target
toxicities. There are currently no specific CDK5 inhibitors at
any stage of drug development despite very strong demand to
investigate and unravel the role of CDK5 in the growth and
proliferation of cancer cells.
In this study, we report the identification of CDK5 inhibitory
molecular scaffolds using structure-based virtual screening and
optimization. Evaluation of 28 virtual screening hits (Table S2)
with our in-house CDK5 biochemical assay (Table S1) revealed
three hits with moderate inhibition, and their anti-proliferative
CDK5 is an atypical member of the mammalian CDKs which
has long been known for its role in the central nervous system.7,
10 It has been attracting considerable attention as its selective
inhibition could offer an exciting therapeutic benefit in clinical
10
oncology.7,
CDK5 is unique within its family members
because it is activated by the non-cyclin proteins, p35, p39 and
their respective truncated products p25 and p29.8 These
activators are abundantly expressed in the brain, making CDK5
an important regulator of the development of the central
nervous system (CNS) and its various functions, including
neuronal migration, and survival.9 On the other hand, CDK5 is
a well-established kinase that mediates the pathophysiology of
common neurodegenerative disorders, such as Alzheimer’s and
Parkinson’s disease.9 Recently, aberrant expression of CDK5
and its activators has been observed in multiple solid and
hematological malignancies,10 but not in healthy tissues.11, 12 In
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effects were confirmed in MV4-11 and MOLM-13 AML cell assay (Table S1). The compounds were found to inhibit CDK5
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lines. Medicinal chemistry optimization of a selected hit
generated a series of compounds which led to the rapid
identification of two lead molecules, 29 and 30, which were
confirmed to target CDK5 in MV4-11 AML cells by western
blot analysis. The results demonstrated a successful application
of structure-based approach in identifying CDK5-targeting
scaffolds that can form the basis for medicinal chemistry
optimization.
with varying strength encompassing moderate (> 60%), to weak
(< 60%) inhibitory activities (Table S2). Ki values were further
determined for the compounds which showed ≥ 60% inhibition.
The compounds hit-1, hit-2, and hit-3, the representative of
three
tetrahydroquinazolin-2-amine, N-(3-(pyrrolidin-1-ylmethyl)
phenyl)-1H-indazole-3-carboxamide and N-phenyl-6,7-
chemical
classes
i.e.
N-phenyl-5,6,7,8-
dihydro-5H-cyclopenta[d]pyrimidin-2-amine (Figure 1B),
inhibited CDK5 with Ki values of 0.88, 2.03, and 1.72 µM,
respectively. These compounds were profiled against other
members of the CDK family including CDKs 1, 2, 4, 6, 7 and 9
in which hit-1 showed higher potency and selectivity for CDK5
compared to the other two (Figure 1C). Finally, their anti-
proliferative activities against MV4-11 and MOLM-13 AML
cells were confirmed (Figure 1D). To provide additional insight
into our findings, the binding modes of the three hits,
roscovitine and an inactive compound hit-16 as a negative
control were studied using Glide XP docking. Hit-1 forms two
hydrogen bonds with the amino and carbonyl of Cys83 residue
in the hinge region of CDK5 (Figure 2A). A similar type of
interaction was observed with hit-3 and hit-2 (Figure S2). Hit-
16, however, does not reach the hinge region and rather it forms
a hydrogen bond with Asn144 (Figure 2A). The hits have been
found to exhibit a similar binding mode as roscovitine, with the
exception that the latter formed one extra interaction with
Gln130 which is a likely explanation for its enhanced inhibitory
activity against CDK5 (Figure S1, IC50 of 0.27 µM for
roscovitine vs 1.76 µM for hit-1).
In this study, initially, the ChemBridge library containing 1.1
million drug-like molecules was screened using the Glide
docking module of the Schrödinger suite. The crystal structure
of active CDK5 protein bound with its ligand roscovitine (PDB
ID: 1UNL) was used as a template for the screening. Figure 1A
is the schematic representation of the screening process. The
ligand library was first screened using Glide high throughput
virtual screening (HTVS) which was subsequently followed by
Glide standard precision (SP) and Glide extra precision (XP)
(SI). The top 2700 molecules from the XP screening were
filtered for exclusion of pan assay interference compounds
(PAINS) and chosen for inspection. These molecules were
clustered based on their calculated tanimoto coefficient using
the 2D fingerprint in Schrödinger Canvas 2.8. Subsequently,
based on the analysis of physicochemical properties, docking
scores and consideration of Lipinski’s rule of five, 250
molecules were chosen for further analysis. From these 250
molecules, 28 were selected based on visual inspection for
biochemical kinase screening at 10 µM against CDK5 using the
ADP-Glo kinase
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Figure 1. Hit identification. A) Schematic representation of the high throughput virtual screening steps. B) CDK5 inhibition potency of the
three hits and chemical structures of inactive hit-16. C) Selectivity for CDK5 over other members of CDK family at a concentration of 10
µM. D) Anti-proliferative effects in MOLM-13 and MV4-11 leukemia cell lines. GI50 was determined by a 72 h resazurin assay and
represents mean ± SD of three independent experiments. 5/p25: CDK5/p25; 1/B1: CDK1/Cyclin B1; 2/A: CDK2/Cyclin A: 4/D1:
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CDK4/Cyclin D1; 6/D3: CDK6/Cyclin D3; 7/HMAT: CDK7/Cyclin H/MAT1; 9/T1: CDK9/Cyclin T; GI50: the concentration for 50%
inhibition of cell proliferation, and the Ki values were obtained from three independently repeated experiments
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Next,
the
chemical
scaffold
N-phenyl-5,6,7,8-
acompound; b not determined; c: not active.
tetrahydroquinazolin-2-amine of hit-1 was selected for further
optimization due to its better selectivity profile and potency
towards CDK5. Hit-1 is a topological structure of quinazoline,
which resulted in EGFR inhibitors including gefitinib and
erlotinib. It also consists of an aminopyrimidine moiety which
has been an important scaffold to generate kinase inhibitors e.g.
abemaciclib and imatinib etc.2 However, the kinase activity of
our scaffold has not been reported so far. Therefore, exploration
of the current scaffold may lead to the identification of potent
and selective inhibitors. The docking of hit-1 to CDK5 revealed
the presence of unoccupied space around its aniline and
tetrahydroquinazoline rings. This enabled substitution with
various functional groups that can potentially target amino acid
residues in the immediate vicinity (Figure 2A). For example,
Asp84 and Asp86 in CDK5 can be targeted by introducing
substitutions around the aniline ring and Ile10 and Phe80, can
be targeted by modifying the cyclohexane moiety. Moreover,
our previous studies of a class of N-phenyl-4-(thiazol-5-
yl)pyrimidin-2-amine derivatives revealed that meta-
substitution e.g. m-SO2NH2, m-NO2 on the aniline ring was
important for CDK2 inhibition.18 Since CDK5 has high
sequence similarity with CDK2, these substituents might
facilitate CDK5 inhibitory activity. Therefore, in the current
study, we explored the SAR through the synthesis of
compounds 29-45 (Table 1 and SI), with different groups at the
-ortho, -meta and -para positions of the aniline ring. The
compounds were firstly screened for their % inhibition against
CDK5/p25 and CDK2/A at 10 µM. Compounds achieving ≥
70% inhibition of CDK5/p25 were then chosen for Ki
activity study. Interestingly, compounds with meta-substituent
aniline at the 2C-position of 5,6,7,8-tetrahydroquinazolin; e.g.
29 (R2=SO2NH2), 30 (R2=CH3), 31 (R2=OCH3), 32 (R2=NO2)
and 33 (R2=CH2CH3) inhibited CDK5 in a range of 78-98%
(Table 1), with an exception of 34 (R2=CF3) which show little
activity. All other derivatives with ortho- or para-substituted
aniline e.g. 35-39 and 41-44 showed low activity against both
enzymes. However, 40 (R1=CH3) and 45 (R3=CH3) achieved
82% and 77% inhibition against CDK5, respectively. Overall,
there is a similar trend of CDK5 and CDK2/A inhibition, and
the meta-substituted aniline was favourable for both CDK5 and
CDK2 inhibition. Further evaluation of the selected compounds
(% inhibition ≥ 70%) showed that 29, 30 and 40 inhibited
CDK5 activity potently with Ki value ≤ 1 μM, whereas the rest
(31-33 and 45) has a Ki value ≥ 1 μM (Table 2). 29 was
identified as the most potent inhibitor with a Ki = 0.16 µM.
However, 29 was not selective for CDK5/p25 over CDK2/A.
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determination
and
cellular
antiproliferative
Table 1. Structures activity relationship of N-phenyl-5,6,7,8-
tetrahydroquinazoline-2-amine derivatives.
% Inhibition
(10 µM)
R1
R2
R3
Cpda
5/p25
2/A
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
H
H
SO NH
H
H
98
87
85
83
78
5
95
Figure 2. Binding modes of compounds with CDK5/p25 (PDB:
1UNL) (A-C) and CDK2/A (PDB: 4NJ3) (D). A) Hit-1 (green)
and hit-16 (yellow, Table S2) B) Roscovitine (gray), C) 29 (blue)
and 30 (pink); D) 29 and 30 in CDK2. All representations are
predicted by Glide XP docking in Schrödinger suite.
2
2
CH
72
93
87
39
7
3
H
OCH
H
3
H
NO
H
2
H
CH CH
H
2
3
H
CF
H
3
Table 2. The CDK selectivity profiles
CH
NHSO CH
H
63
22
4
NDb
3
2
3
Cytotoxicityb,
(GI50) μM
CDK inhibition K ,a μM
Cpd
OCH
H
H
H
H
H
H
F
H
2
i
3
OCH CH
H
2
NAc
5/p25 1/B 2/A
0.16 2.61 0.27
0.80 2.36 3.32
1.01 ˃ 5 1.11
1.26 ˃ 5 1.10
2/E
1.0
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
4/D1 6/D3 7/HMAT1 9/T1
MV4-11
2
3
OCF
H
9
29
30
31
32
33
40
45
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
3.01
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
1
2.55 ± 0.14
3.29 ± 0.17
2.28 ± 0.00
3.50 ± 0.70
2.60 ± 0.11
> 10
3
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
˃ 5
CF
H
10
82
45
21
1
8
3
CH
H
54
NAc
7
3
OCH
Cl
OCH
OCH
1.23 ˃ 5
˃ 5
3
0.99 ˃ 5 4.12
1.44 ˃ 5 4.54
OCH
3
3
3
6.00 ± 0.40
H
H
H
H
H
H
3
aThe Ki values show the average of at least two experiment. %
residual activity for Ki values >5 is presented in Table S5.
CF
6
7
3
CH
77
58
3
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bCytotoxicity (GI50) was determined by a 72 h resazurin assay and
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represents mean ± SD of three independent experiments.
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Compound 30 inhibited CDK5/p25 with a Ki value of 0.8 μM,
and exhibited more than four-fold increase in potency for
CDK5/p25 over CDK2/A (p < 0.05, Table S3). Interestingly, all
these compounds demonstrated considerable selectivity for
CDK5/p25 over CDK2/E as well as other CDKs such as 1/B,
4/D1, 6/D3, 7/H and 9/T1. The compounds were tested for their
anti-proliferative activity in MV4-11 cells, showing GI50 in the
range of 2.28-6.0 µM, with the exception of 40 (GI50 > 10 M).
29 (most potent) and 30 (most selective) were selected for
further studies of their binding modes and anti-cancer mode of
action. To understand the difference in potency and selectivity
observed with 29 and 30, we analysed the binding modes of 29
and 30 in CDK5/p25 (PDB: 1UNL) and CDK2/A (PDB: 4NJ3).
It was observed that both compounds bind with Cys83 at the
hinge region of CDK5 via two hydrogen bonds as shown earlier
by hit-1. However, 29 formed one extra hydrogen bond with
Asp84 of CDK5 which might account for the improved potency
when compared to hit-1 and 30 (Figure 2C). It also showed
similar binding mode in CDK5 and CDK2/A, which correlated
with comparable potencies against both enzymes (Table 2). In
contrast, 30 in CDK2/A2 formed a hydrogen bond with Val64
(Figure 2D). This finding is in agreement with the in vitro
results where 30 was found to be more potent for CDK5/p25
over CDK2/A. In addition to these, the subtle differences in the
conformation and plasticity of the active site of the two
enzymes might also play a role.19
in MV4-11 AML cells using western blot analysis. Incubation
of the cells with 29 or 30 for 24 h suppressed the level of FAK
phosphorylation (Figure 3A) in a concentration-dependent
manner. Our data confirmed the ability of these compounds to
inhibit cellular CDK5 kinase activity. Next, the anti-
proliferative effects of 29 and 30 were measured with cell
viability assays after 72 h of treatment by employing resazurin
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and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) in leukemia and adherent (solid tumor) cell
lines, respectively. Of note, both compounds showed potent
anti-proliferative activity against MOLM-13 and MV4-11
AML cells with GI50 values ranging from 2.16 to 3.29 µM
(Table S4). As both cell lines are carrying FLT3-ITD mutation,
29 and 30 were subsequently tested in FLT3-ITD kinase assays,
showing IC50 = 1.48 µM for 29 and > 10 µM for 30. In addition,
we have shown that both compounds didn’t target cellular FLT3
using western blot analysis (Figure S3). However, in this type
of AML cells, the high proliferation rate induced by oncogenes
e.g. MYC or FLT3-ITD can lead to the phenomenon called
replicative stress.22 This in turn causes high degree of genomic
instability and upregulation of DNA damage repair machineries
including ataxia–telangiectasia mutated (ATM) and p53, both
of which are known targets of CDK5. This might be the cause
of preferential sensitivity of MV4-11 and MOLM-13 cells
towards the inhibition of CDK5 compared to solid cancer cell
and FLT3 wild-type AML cells lines (Table S4). Nevertheless,
their activity in solid tumor cell lines was comparable to that of
roscovitine.23 The advantage of our compounds over
roscovitine is their improved selectivity for CDK5 over other
members of CDK family.
Following this, target confirmation study in cellular context
was undertaken for 29 and 30 by monitoring the level of
phosphorylation status of FAK at Serine 732.20, 21 This was
assessed
Figure 3. Primary target confirmation and anticancer mechanisms of 29 and 30. A) 29 and 30 inhibits CDK5 in MV4-11 cells. B) and
D) The effects of 29 and 30 on cell cycle progression in MV4-11 and MOLM-13 cell lines, respectively. C) Molecular mechanisms of cell
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cycle arrest by 29 and 30 in MV4-11 cells. E) Comparison of CDK5 and related proteins expression in MOLM-13 and MV4-11 cells. F)
Induction of apoptosis in MOLM-13 cells. G) Caspase 3/7 activity in MOLM-13 cells. H) Western blot analysis showing mechanism of cell
death in MOLM-13 cells. All the data were generated from at least two independently repeated experiments, and numerical data in B, D and
G represent the mean value.
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caspases from the complex resulting in cell death. Previously,
down regulation of p21CIP1 has been observed in a mouse model
In the study of potential anticancer mechanisms of 29 and 30,
we observed that the compounds, at concentrations of 2.5 and
10 µM, arrested MV4-11 cells in the G1-phase of the cell cycle
(Figure 3B) after 24h of treatment. Interestingly, both
compounds caused a substantial accumulation of MOLM-13
cells in sub-G1 phase at the higher concentration i.e. 10 µM
(Figure 3D) and this is consistent with the effects of CDK5
inhibition in other cancers.10 In western blot analysis we found
that 29 and 30 reduced the protein levels of pAkt (S473),
pRB(S780), and Cyclin D1, and increased that of p27KIP1 in
MV4-11 cells (Figure 3C) and MOLM-13 cells (Figure S4)
which is consistent with the observed G1 cell cycle effects. Our
findings are supported by literature reports that CDK5
of thyroid carcinoma in which CDK5 kinase activity has been
suppressed.10 This might support that inactivation of PI3K-Akt
pathway as
a result of CDK5 inhibition caused a
downregulation of p21CIP1, which in turn activates caspases to
trigger apoptosis. Our findings in the CDK5-targeted cellular
mode of action are consistent with what others had reported
using CDK5 knockout/down cells in colorectal, prostate,
ovarian and breast cancers.7, 10, 20, 25, 27
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In summary, we have employed the approaches of structure-
based in silico screening and medicinal chemistry optimization
to identify a new class of CDK5 inhibitors. The initial hit-1, hit-
2 and hit-3 inhibited CDK5/p25 with Ki values of 0.88 – 2.03
M. Further structure–guided design and optimization of the
hit-1 resulted in a series of 5, 6, 7, 8-tetrahydroquinazolin
derivatives that enabled us to establish the structure-activity
relationship. Lead compounds 29 and 30 exhibited potent
activity against CDK5/p25 and the latter also demonstrated an
appreciable selectivity for CDK5/p25 over other members of
CDK family. Cellular mechanistic investigation confirmed that
29 and 30 targeted cellular CDK5 and caused cell cycle arrest,
and substantial apoptotic cell death in AML cells. Taken
together, this study describes an effective strategy to identify
CDK5 inhibitors that have potential to be developed as anti-
leukemic agents.
knockout/down
by
siRNA/shRNA
abrogated
the
phosphorylation of Akt at S473, leading to G1 cell cycle arrest
in the cancer of prostate, breast and ovary.10, 24 Intriguingly, the
PI3K-Akt pathway has been established as the primary
mediator of an early adaptation and resistance of cancer cells
7
during the clinical use of CDK4/6 inhibitors,1, suggesting
CDK5 inhibitor as potential new therapy to overcome this
problem. Furthermore, AnnexinV and PI double staining shows
that both compounds increased the proportion of apoptotic
MOLM-13 cells (Figure 3F), but had little effect on the MV4-
11 cells (Figure S5). Similarly, other groups reported that
knockout/down of CDK5 through SiRNA/shRNA caused
apoptosis in cells derived from solid tumors including breast,
prostate, and ovarian cancers.7, 10, 25 However, given the very
high genetic and mutational similarity between the two
leukemic cell lines, it was surprising to see such a discrepancy
in response to 29 and 30. A very recent study demonstrated
ASSOCIATED CONTENT
Supporting Information
Experimental procedure for the synthesis and characterization of
compounds; molecular modelling methods; procedures for
biological evaluation, additional tables and figures. (PDF)
AML cells with low level FAK expression are more susceptible
24
to killing by suppressed FAK activity.
Similarly, we and
others26 found that MOLM-13 cells express low level of FAK
protein (Figure 3E) compared to MV4-11 cells and this could
be one of the reasons for the increased susceptibility of
MOLM-13 cells to killing by 29 and 30. Finally, the molecular
mechanisms of 29 and 30 mediated killing effects were
analyzed in MOLM-13 cells. Past evidence revealed caspase
activation as one of the major mechanisms of apoptosis
induction by CDK5 inhibition,23, 25, 27 and thus, we assessed
caspase activity by Apo-ONE® Homogeneous Caspase-3/7 Glo
Assay. Treatment of MOLM-13 cells with 29 or 30 at the
concentrations shown for 24 h resulted in a robust increase in
caspase 3/7 activity (Figure 3G). Moreover, caspases 3 and 7
were activated as demonstrated by the massive increase in the
cleaved (Cl) caspase 3 and 7 bands in western blot analysis
(Figure 3H). This might be linked to down regulation of p21CIP1
protein (Figure 3H) following CDK5 inhibition by 29 and 30. It
has recently been reported that p21CIP1appears to play an
important role in protecting cells from apoptosis beyond its
conventional tumor suppressive role. In the cytoplasm, p21CIP1
forms complexes with anti-apoptotic elements such as
AUTHOR INFORMATION
Corresponding Author
* Professor Shudong Wang; e-mail: shudong.wang@unisa.edu.au
Author Contributions
ϯ These authors contributed equally to this work. The manuscript
was written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
NZK and JLL acknowledge the support from the Australian
Government Research Training Program (RTP) Scholarship.
ABBREVIATIONS
AML; acute myeloid leukemia, ATM; ataxia–telangiectasia
mutated, EGFR; epidermal growth factor receptor, FAK;focal
adhesion kinase, GSK-3β; glycogen synthase kinase-3 β, HER2;
procaspase 3 and renders them inactive to ensure the survival of
28, 29
cancer cells.
Thus, inhibition of p21CIP1 releases pro-
5
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(20) Shao, H.; Shi, S.; Foley, D. W.; Lam, F.; Abbas, A. Y.; Liu, X.;
human epidermal growth factor receptor 2, HTVS; high
throughput virtual screening, mTOR; mammalian target of
Huang, S.; Jiang, X.; Baharin, N.; Fischer, P. M.; Wang, S. Synthesis,
structure–activity relationship and biological evaluation of 2,4,5-
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dynamics simulations on the inhibition of Cyclin-dependent kinases 2
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2
3
4
5
6
rapamycin,
MTT;
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, PAINS; pan assay interference
compounds, RB; retinoblastoma protein, SP; standard precision,
XP; extra precision
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Figure 1. Hit identification. A) Schematic representation of the high throughput virtual screening
steps. B) CDK5 inhibition potency of the three hits and chemical structures of inactive hit-16. C)
Selectivity for CDK5 over other members of CDK family at a concentration of 10 µM. D) Anti-
proliferative effects in MOLM-13 and MV4-11 leukemia cell lines. GI50 was determined by a 72
h resazurin assay and represents mean ± SD of three independent experiments. 5/p25: CDK5/p25;
1/B1: CDK1/Cyclin B1; 2/A: CDK2/Cyclin A: 4/D1: CDK4/Cyclin D1; 6/D3: CDK6/Cyclin D3;
7/HMAT: CDK7/Cyclin H/MAT1; 9/T1: CDK9/Cyclin T; GI50: the concentration for 50%
inhibition of cell proliferation, and the Ki values were obtained from three independently repeated
experiments.
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Figure 2. Binding modes of compounds with CDK5/p25 (PDB: 1UNL) (A-C) and CDK2/A (PDB: 4NJ3)
(D). A) Hit-1 (green) and hit-16 (yellow) B) Roscovitine (gray), C) 29 (blue) and 30 (pink); D) 29 and 30 in
CDK2. All representations are predicted by Glide XP docking in Schrödinger suite.
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Figure 3. Primary target confirmation and anticancer mechanisms of 29 and 30. A) 29 and 30 inhibits CDK5
in MV4-11 cells. B) and D) The effect on cell cycle progression in MV4-11 and MOLM-13 cell lines,
respectively. C) Molecular mechanism for the cell cycle effects in MV4-11 cells. E) Comparison of CDK5 and
related proteins expression in MOLM-13 and MV4-11 cells. F) Induction of apoptosis in MOLM-13 cells. G)
Caspase 3/7 activity in MOLM-13 cells. H) Confirmation of caspase activation by western blot analysis. All the
data were generated from at least two independently repeated experiments, and numerical data in B, D and G
represent the mean values.
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