New Dual CK2/HDAC1 Inhibitors with Nanomolar Inhibitory Activity against Both Enzymes

Article abstract of DOI:10.1021/acsmedchemlett.9b00561

Four potent CK2 inhibitors derived from CX-4945 are described. They also provided nanomolar activity against HDAC1, therefore having promising utility as dual-target agents for cancer. The linker length between the hydroxamic acid and the CX-4945 scaffold plays an important role in dictating balanced activity against the targeted enzymes. The seven-carbon linker (compound 15c) was optimal for inhibition of both CK2 and HDAC1. Remarkably, 15c showed 3.0 and 3.5 times higher inhibitory activity than the reference compounds CX-4945 (against CK2) and SAHA (against HDAC1), respectively. Compound 15c exhibited micromolar activity in cell-based cytotoxic assays against multiple cell lines.

Full text of DOI:10.1021/acsmedchemlett.9b00561

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Letter
New dual CK2/HDAC1 inhibitors with nanomolar
inhibitory activity against both enzymes
Loganathan Rangasamy, Irene Ortín, José María Zapico,
Claire Coderch, Ana Ramos, and Beatriz de Pascual-Teresa
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00561 • Publication Date (Web): 07 Apr 2020
Downloaded from pubs.acs.org on April 9, 2020
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New dual CK2/HDAC1 inhibitors with nanomolar inhibitory activity
against both enzymes
Loganathan Rangasamy,‡ Irene Ortín,‡ José María Zapico, Claire Coderch, Ana Ramos,* Beatriz de
Pascual-Teresa.*
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Departamento de Química y Bioquímica, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities,
Urbanización Montepríncipe, 28925, Alcorcón, Madrid, Spain.
KEYWORDS. Multi-target drugs, CK2, HDAC1, Dual inhibitors, docking, molecular dynamics
ABSTRACT: Four potent CK2 inhibitors derived from CX-4945 are described. They are provided also of nanomolar activity against
HDAC1, therefore having promising utility as dual-target agents for cancer. The linker length between the hydroxamic acid and the
CX-4945 scaffold plays an important role in dictating balanced activity against the targeted enzymes. The seven-carbon linker
(compound 15c) was optimal for inhibition of both CK2 and HDAC1. Remarkably, 15c showed 3.0 and 3.5 times higher inhibitory
activity than the reference compounds CX-4945 (against CK2) and SAHA (against HDAC1), respectively. Compound 15c exhibited
micromolar activity in cell-based cytotoxic assays against multiple cell lines.
Cancer is a multifactorial complex disease that is caused by
multiple dysfunctions in genes or pathways.1 Combination
therapies directed to two or more molecular targets have been
widely used in the treatment of this disease.2 Nevertheless,
combination therapy has serious disadvantages such as patient
incompliance, difficulty to predict side effects and to optimize
the dose ratio, as well as unwanted drug-drug interactions, and
unpredictable pharmacokinetics.2 Multi-target single agents are
expected to improve the efficacy of the treatments, by
exploiting synergistic interactions, avoiding problems of drug-
drug interactions, decreasing drug resistance, and making the
pharmacokinetic studies easier to perform.3
the treatment of solid cancers have been disappointing.4-6
Interestingly, a SAR study confirms that the cap group in
HDACi is flexible and tolerates modification. Thus, the zinc-
binding group present in most of them can be easily linked to
other anticancer scaffolds providing numerous multi-targeting
agents with higher potency than the parent compounds.4-6, 14, 15
Some examples are CUDC-101,16 CUDC-90717 and 4SC-20218
(Figure 1) which have already entered clinical trials.14 These
results strongly support that
a single compound that
simultaneously inhibits HDAC1 and other oncological target
could be beneficial in cancer resistance over single-acting
agents, through the interference with multiple pathways.
O
Histone deacetylases (HDACs) are a family of epigenetic
enzymes that control the transcription and regulation of genes
as well as cell proliferation, differentiation, migration, death,
and angiogenesis.4-6 Also, overexpression of HDACs has been
found in many human cancers.7 Therefore, inhibiting HDACs
have been recognized as a promising approach for treating
cancer. To date, four HDAC inhibitors (HDACi) were
approved. by the FDA:6 vorinostat (SAHA), romidepsin (FK-
228), belinostat, (PXD101) and panobinostat (LBH589), and
another one was approved by the Chinese FDA:8 chidamide
(tucidinostat, HBI-8000). On the other hand, almost 18 HDACi
are in clinical trials.6
O
OH
HN
N
N
N
HN OH
O
O
O
N
S
NH
N
CUDC-101
H3CO
CUDC-907
EGFR: IC50 = 2.4 nM
HDAC1: IC50 = 4.4 nM
HER2: IC50 = 15.7 nM
PI3Ka: IC50 = 19 nM
HDAC1: IC50 = 1.7 nM
O
S
O
N
H
N
N
N
O
H2N
Protein kinase 2 (CK2) is an ubiquitously expressed and
4SC-202, Domatinostat
constitutively
active
serine/threonine
kinase
that
KDM1A/LSD1: IC50 = 0.6 -1.2 µM
HDAC1: IC50 = 1.2 µM
phosphorylates an impressive array of substrates including
10
HDACs.9,
Overexpression of CK2 is involved in several
Figure 1. HDACi based dual inhibitors in clinical trials
human cancers and has also been linked to poor prognosis and
disease progression.11 Several CK2 inhibitors have been
discovered in the past, but among them only two inhibitors, CX-
4945 (NCT03904862) and CIGB-300 (NCT01639625) have
recently entered into Phase II clinical trials as potential
anticancer drugs.12
Protein Kinase 2 (CK2) regulates the dimerization of HDAC1
and HDAC2 during mitosis19. Under hypoxic conditions, CK2
phosphorylates HDAC leading to HDAC activation. Activated
HDAC contributes to tumor growth and vasculogenesis by
Hippel-Lindau protein (pVHL) downregulation and, hence, to
While many HDACi are in preclinical and clinical studies, they
have been found to induce drug resistance13 and their results in
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ACS Medicinal Chemistry Letters
maintained, whereas the original hydrogen bonds established by
Page 2 of 8
the increased expression and stabilization of hypoxia-inducible
factor HIF-1 protein.20 Since both CK2 and HDAC are
involved in the related cancer-relevant biological pathways,21-24
we anticipated that simultaneously inhibiting these two targets
by a multi-target single molecule should improve efficacy
compared to single-target agents.
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the hydroxamic acid varied, but always maintained an
interaction with the entrance of the binding site (Figure S3,
Supporting information). These results demonstrate that the
incorporation of an alkyl chain with a hydroxamic acid to the
CX-4945 core does not impair the proper orientation of the
molecules in the ATP binding site, as well as the establishment
of the essential interactions that guarantee the high potency
found for the reference compound CX-4945.
In previous publications,25,26 we developed a series of dual
CK2/HDAC1 inhibitors using TBB and DMAT as a scaffolds
to promote CK2 inhibition and a hydroxamate zinc binding
group (ZBG) to interact with the zinc present in the active site
of HDAC1 and simulating the structure of SAHA, a potent
inhibitor of HDAC1. The synthesized dual-acting agents
exhibited promising inhibitory activities with the best
compound showing IC50 of 5 M for both enzymes and
micromolar activity in cell-based assays (Figure 2).
9
The binding of compounds 15a-d to HDAC1 was similar to that
of SAHA (Vorinostat®) to different HDACs in the reference
crystal structures.29,30,31 (Figure S4, Supporting information).
All compounds established with HDAC1 a bidentate chelation
to the catalytic Zn2+ ion stabilized by hydrogen bonds with the
side chains of His140 and Tyr303, and oriented the CX-4945
moiety to establish different interactions with amino acids in the
surface of the protein that also varied depending on the length
of the linker (Figure S5, Supporting information). The stability
of the binding mode of compound 15c to HDAC1 (Figure 4)
was also assessed by means of MD simulations. During the
simulation the CX-4945 moiety explored the surface of the
protein establishing different interactions through the
carboxylic acid moiety but mainly establishing π-stacking
interactions with the side chain of Phe205 (Figure S6,
Supporting information). The movement brought about by the
linker and the CX-4945 moiety destabilized some of the initial
hydrogen bonding interactions between the hydroxamate
moiety and the side chains of His140 and Tyr303; however, a
new and stable hydrogen bond is established with the side chain
of His141. These results suggested that the interaction between
the CX-4945 cap and residue Phe205, and an optimal linker
length to establish bidentate Zn2+ coordination is important for
the HDAC1 inhibitory potency.
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Br
H
Br
Br
HN
O
N
OH
N
N
N
N
HO
NH
O
Br
Br
N
Br
Br
N
N
Br
N
TBB
HDAC1 IC50 = 5.0 M
CK2 IC50 = 5.4 M
CK2 IC50 = 0.5 M
Pharmacophore
hybridization
Cl
First HDAC/CK2 dual inhibitor
O
O
HN
n
HN
HN
HN
OH
n = 1- 4
N
N
N
N
OH
OH
O
O
Second generation of
HDAC/CK2 inhibitors
Silmitasertib
CX-4945
SAHA
IC50 = 33 nM
HDAC1
(
)
CK2 IC50 = 1 nM
These results predict an overall good affinity of 15a-d for CK2
and HDAC1, so they were forwarded for synthesis and
enzymatic studies.
Figure 2. Design strategy for dual CK2/HDAC1 inhibitors.
Encouraged by these results, we have designed a new series of
CK2/HDAC1 dual inhibitors where the TBB scaffold has been
substituted by CX-4945, a 500- fold more potent CK2 inhibitor
than TBB.12, 27 Herein we describe the computational design and
synthesis of these compounds, together with some promising
preliminary assays of their biological activity.
RESULTS AND DISCUSSION
Computational docking and molecular dynamics
The binding to the target proteins was analyzed by means of
docking and molecular dynamics (MD). The binding mode to
CK2 of 15a-d was similar to that of CX-4945 in the reference
crystal structure28 (PDB code 3PE1) (Figure S1, Supporting
information). The carboxylic acid moiety interacts with the side
chain of Lys68 and the backbone NH of Asp175; whereas, the
pyridine nitrogen interacts with the backbone NH of Val116 in
the hinge region. The linkers oriented the hydroxamic moieties
towards the entrance of the nucleotide-binding site establishing
different hydrogen bonds with the amino acids side chains
depending on the length of the linker (Figure S2, Supporting
information). The stability of the binding mode of compound
15c (Figure 3) was assessed by a MD simulation that proved
that, despite the high mobility of the linker, the initial binding
mode and the interactions of the CX-4945 moiety were
Figure 3. PyMOL stick and cartoon representation of the best
docking pose of compound 15c to CK2. For the sake of clarity,
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only polar hydrogens are shown, and hydrogen bonds have been
highlighted with dashed lines.
3 and 6 were synthesized from commercially available
carboxylic acids 1 and 2 as described in Scheme 2. Then, lactam
1
2
3
4
5
6
7
8
9
7
was
obtained
through
a
one-pot
Suzuki
coupling/intramolecular amide cyclization between the boronic
ester 6 and the ethyl ester 3. Treatment of lactam 7 with
phosphorous oxychloride afforded chloroquinoline 8 which was
ready for the substitution with the different amines 12a-d.
O
O
O
NO2
NO2
Br
NO2
Br
O
O
O
HO
a
b
O
B
5
88%
c
4
88%
O
O
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2
O
O
NH2
OH
O
d
N
+
N
O
B
Br
Br
O
6
88%
3
88%
1
e
Cl
O
N
NH
f
N
N
O
O
8
88%
O
7
O
88%
Scheme 2. Synthesis of compound 8.
Reagents and conditions: a) MeOH, H2SO4, reflux, 12 h; b)
KOAc, Pd(dppf)Cl2, Bis(pinacolate)diboron, DCM, Dioxane,
80 ºC, overnight; c) H2, Pd/C 10%, EtOAc, RT, 12 h; d) MeOH,
H2SO4, reflux, 12 h; e) NaOAc, Pd(dppf)Cl2, DMC, Dioxane,
125 ºC, 12 h; f) neat POCl3, reflux, 2h.
Figure 4. PyMOL stick and cartoon representation of the best
docking pose of compound 15c to HDAC1. For the sake of
clarity, only polar hydrogens are shown, and hydrogen bonds
have been highlighted with dashed lines.
Chemistry
Amines 12a-b were prepared as depicted in scheme 3.
Nucleophilic substitution of the commercially available
bromo carboxylic acids 9a-d gave the corresponding azido
carboxylic acids 10a-d. Amide coupling of 10a-d with the O-
benzyl-hydroxylamine followed by selective catalytic
hydrogenation of the formed azides 11a-d, gave the desired
amines 12a-d. The use of ethyl acetate as solvent is the key
condition to achieve selective reduction of the azide, without
deprotecting the benzyl group present in the molecule. When
more polar solvents such as methanol were used both reductions
occurred.
A convergent strategy was developed for the synthesis of this
new generation of dual inhibitors. First, chloroquinoline 8
which possesses a chlorine atom that allows the substitution
with different nucleophiles was synthesized. Then, different
amine benzyloxy-protected compounds 12a-d were selected
and synthesized in order to assess the linker that best fits in the
catalytic site of HDAC1 (Scheme 1).
Cl
H
n
N
N
H2N
OBn
N
O
n
n
O
OH
n = 1, 2, 3, 4
OH
N3
Br
a
O
O
O
12
(HDAC scaffold)
8
(CK2 scaffold)
10a
, n = 1, 84%
, n = 2, 87%
, n = 3, 84%
, n = 4, 92%
9a
9b
9c
9d
, n = 1
, n = 2
, n = 3
, n = 4
10b
10c
10d
H
n
N
HN
OH
O
N
b
N
OH
H
H
n
n
N
N
O
H2N
OBn
N3
OBn
c
CK2/HDAC dual inhibibitor
O
, n = 1, 89%
, n = 2, 52%
, n = 3, 53%
, n = 4, 94%
O
12a
12b
12c
12d
11a
11b
11c
11d
, n = 1, 60%
, n = 2, 73%
, n = 3, 83%
, n = 4, 97%
Scheme 1. Convergent strategy for dual inhibitor synthesis.
Compound 8 was efficiently synthesized adapting the
procedure described in the literature32 (Scheme 2). Compounds
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Scheme 3. Synthesis of amine (benzyloxy)amino
SAHA
33.534
33.134
1
2
3
4
5
6
7
8
compounds 12a-d as HDAC1 scaffold.
CX-4945
1.8 ± 1.2
Reagents and conditions: a) NaN3, DMF, 77 ºC, 48 h; b)
HCl.H2N-OBn, HBTU, DIPEA, DMF, RT, 12 h; c) H2, Pd/C,
EtOAc, RT, 2 h.
The four compounds presented an inhibitory activity on CK2 in
the low nanomolar range. This result demonstrates that the
introduction of a carbon chain containing the hydroxamic group
in the CX-4945 scaffold does not interfere in the interaction of
these compounds with the active site of CK2 and supports our
hypothesis that CX-4945 is an appropriate scaffold for the
design of dual CK2/HDAC1 inhibitors.
The best result was found for 15c, with a value of IC50 = 1.7
nM, similar to that obtained for the control compound (CX-
4945). What is more interesting, 15c is also a potent inhibitor
of HDAC1, with a value of IC50 = 3.3 nM, which is 3.4-fold
lower than the value obtained for SAHA in our hands. In the
design of dual inhibitors, it is especially important to achieve a
good balance in the activity against the two target enzymes.
This is the case of 15c, a promising compound with a
remarkable potency for both enzymes.
Finally, as depicted in scheme 4, chloroquinoline 8 and amines
12a-d were reacted to yield methyl benzo[c][2,6]naphthyridine-
8-carboxylates 13a-d. After the O-debenzylation by catalytic
hydrogenation at high pressure in methanol, these compounds
were further converted to the corresponding carboxylic acids
15a-d that were purified by HPLC and isolated as
trifluoroacetic salts with the necessary purity (>95%) for
biological assays.
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H
N
Cl
HN
OBn
n
H
N
O
N
H2N
OBn
N
n
a
O
N
N
O
O
O
8
O
Interestingly, this compound showed potent activity in HDAC6
(see table 1). SAHA is also highly potent against both HDAC1
(IC50: 34 nM) and HDAC6 (IC50: 33 nM) while is remarkably
less potent in other isoforms such as HDAC4 (IC50: >1000 nM)
and HDAC8 (IC50: 776 nM).34
13a
, n = 1, 43%
13b
13c
13d
, n = 2, 61%
, n = 3, 68%
, n = 4, 46%
b
When the chain connecting the two selected pharmacophores
was shortened (compound 15b) or elongated (compound 15d)
the inhibitory potency in CK2 was maintained, while some
activity in HDAC1 was lost. These results demonstrated that the
best length for a dual interaction is provided by a seven-carbon
chain. Our efforts in the design of the next series of inhibitors
will concentrate in the modification of the nature of the
connecting chain, keeping the same length, and the
modification of the ZBG.
H
N
H
N
HN
n
OH
HN
OH
n
TFA
O
O
c
N
N
N
N
O
OH
14a
, n = 1, 52%
, n = 2, 38%
, n = 3, 43%
, n = 4, 94%
O
O
14b
14c
14d
15a
15b
15c
15d
, n = 1, 37%
, n = 2, 59%
, n = 3, 22%
, n = 4, 40%
Cytotoxic activity in cells
The cytotoxicity of the four compounds 15a-d in comparison
with reference compounds SAHA and CX-4945 under identical
conditions was investigated by using the resazurin assay.35 In
vitro cytotoxicity for CX-4945 was reported already against a
panel of cancer cell lines, including LNCaP, PC3, MCF-7 and
A549 and in vivo efficacy was tested in PC3 xenografts.32
Similarly, the HDAC inhibitor SAHA showed low micromolar
cytotoxicity against LNCaP, PC3, MCF-7 and A549 cell
lines.36-38 To directly compare the potency of our compounds
with CX-4945 and SAHA, we selected these four human cancer
cell lines, namely lung (A549), breast (MCF-7), and prostate
(PC3 and LNCaP) cell lines (Table 2).
Scheme 4. Synthesis of dual inhibitors 15a-d isolated as TFA
salts.
Reagents and conditions: a) DMF, K2CO3; 135 ºC, 55 min,
MW irradiation; b) for 14a-c: H2, Pd/C 10%, MeOH, 4 bar, RT,
12 h, for 14d: H2 atmosphere and MeOH:THF 1:1 as solvent,
12 h; c) LiOH, RT, 96 h.
Enzymatic inhibitory evaluation
The inhibitory activities of the synthesized compounds were
determined against CK2 and HDAC1 and the IC50 values are
collected in Table 1. The activity towards human recombinant
HDAC1 was tested using a fluorometric method.25 CK2
inhibition was measured by a luminometric assay using ADP-
Glo Kinase Assay (Promega).33
Table 2. IC50 values of compounds 15a-d, SAHA and CX-4945
against four human tumor cell lines.
IC50 values (M)
Table 1. Inhibitory activity (IC50, nM) on HDAC1, HDAC6
and CK2
Cpd
15a
15b
15c
LNCaP
>100
PC3
>100
>100
MCF-7
>100
A549
>100
>100
Cpd
15a
15b
15c
HDAC1
190 ± 100
140 ± 20
3.3 ± 1.6
130 ± 30
5.6 ± 2.0
HDAC6
CK2
>100
>100
3.5 ± 3.3
14 ± 19
1.7 ± 1.0
19 ± 24
16.31 
0.7
40.42 
4.4
52.48 
104.73 
10.0
7.9
13 ± 1
15d
>100
>100
>100
66.70 
5.0
15d
TSA
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SAHA
0.69  0.1 2.03  0.1 1.26  0.1 3.14  0.2
1
2
3
The Supporting Information is available free of charge on the ACS
Publications website.
CX-4945
6.52  0.6
10.87 
0.6
9.41  0.3
11.60 
1.8
4
5
6
7
8
9
AUTHOR INFORMATION
Corresponding Author
Interestingly, only 15c showed activity in the cell experiments,
which is in accordance with the docking and enzymatic assay
results. The best activity was observed in LNCaP, an androgen-
sensitive human prostate adenocarcinoma cell line, with an IC50
of 16.31 M. The activity in this cell line was lower than the
activities obtained for the parent compounds. Considering the
results of inhibitory activity of 15c in isolated enzymes, we
postulate that the lower activity exerted by 15c in cells could be
due to a low permeability, which we will try to overcome in the
next series of inhibitors.
* E-mail: For Ana Ramos aramgon@ceu.es and for Beatriz de
Pascual-Teresa bpaster@ceu.es
ORCID
Beatriz de Pascual-Teresa: 0000-0002-1101-0373
Ana Ramos: 0000-0003-2984-4672
Loganathan Rangasamy: 0000-0002-1339-0330
Irene Ortin: 0000-0001-8507-2641
José Mª Zapico: 0000-0003-1980-4749
Claire Coderch: 0000-0001-7229-8289
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In order to evaluate the effect on the cytotoxicity of the TFA
salts,39, 40 we carried out a cell assay on LNCaP cell line using
CX-4945 as a TFA salt. At the concentrations used in the
cytotoxicity study we observed slight differences compared to
the free compound. (CX-4945 6.52 M; CX-4945 as a TFA salt
3.70 M).
In conclusion, we found four remarkably potent CK2 inhibitors,
showing that the introduction of chains of different lengths
provided with hydroxamate groups, does not affect the
interaction of the CX-4945 ring with the active site of CK2.
Author Contributions
‡L.R and I. O 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.
ACKNOWLEDGMENT
This work was supported by MICIU/FEDER, UE grant number
RTI2018-093539-B-I00. This project received funding from the
European Union´s Horizon 2020 research and innovation
programme under the Marie-Sklodowska-Curie grant agreement
number DUALITY 746225.
The presence of the hydroxamate allows the interaction with
HDAC1, providing only one promising dual CK2/HDAC1
inhibitory agent 15c, with nanomolar activity in both enzymes.
The modelling work has provided plausible binding modes that
can account for these results.
ABBREVIATIONS
HDAC, histone deacetylase; CK2, Protein kinase type 2; SAHA,
suberoylanilide hydroxamic acid (Vorinostat); TBB, 4,5,6,7-
tetrabromo-2H-benzotriazole; DMAT, 2-dimethylamino-4,5,6,7-
Preliminary assays in human cell lines confirm the interest of
this compound as a cytotoxic agent. Future studies are
necessary to improve the pharmacokinetic profile of this new
hit compound and examine its activity in other human cell lines.
tetrabromo-1H-benzimidazole;
dppf,
1,1'-
Bis(diphenylphosphino)ferrocene; DMF, dimethylformamide;
MW, microwave; RT, room temperature; THF, tetrahydrofurane;
ZBG, Zinc Binding Group; TFA, trifluoroacetic acid; TSA,
ASSOCIATED CONTENT
Supporting Information
Trichostatin A;
HBTU, O-(Benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate; DIPEA,
N
N-
Synthetic procedures, spectroscopic and analytical data for all
compounds; in vitro enzymatic assay, resazurin assay and
computational methods are described.
Diisopropylethylamine; DCM, dichloromethane; THF,
tetrahydrofurane; PDB, protein data bank; ATP, adenosine 5'-
triphosphate.
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