Camptothecin-psammaplin A hybrids as topoisomerase I and HDAC dual-action inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.11.021

Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of camptothecin derivatives and HDAC inhibitors. To exploit this synergy in a single active compound, we designed new dual-acting multivalent molecules simultaneously targeting topoisomerase I and HDAC. In particular, a selected compound containing a camptothecin and the psammaplin A scaffold showed a broad spectrum of antiproliferative activity, with IC₅₀ values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Full text of DOI:10.1016/j.ejmech.2017.11.021

Accepted Manuscript
Camptothecin-psammaplin a hybrids as topoisomerase I and HDAC dual-action
inhibitors
Raffaella Cincinelli, Loana Musso, Roberto Artali, Mario Guglielmi, Erminia Bianchino,
Francesco Cardile, Fabiana Colelli, Claudio Pisano, Sabrina Dallavalle
PII:
S0223-5234(17)30916-9
10.1016/j.ejmech.2017.11.021
EJMECH 9896
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 July 2017
Revised Date: 6 November 2017
Accepted Date: 7 November 2017
Please cite this article as: R. Cincinelli, L. Musso, R. Artali, M. Guglielmi, E. Bianchino, F. Cardile,
F. Colelli, C. Pisano, S. Dallavalle, Camptothecin-psammaplin a hybrids as topoisomerase I and
HDAC dual-action inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.021.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Camptothecin-psammaplin A hybrids as topoisomerase I and HDAC dual-action inhibitors
Raffaella Cincinelli±a, Loana Musso±a, Roberto Artalib, Mario Guglielmic, Erminia Bianchinoc,
Francesco Cardilec, Fabiana Colellic, Claudio Pisanoc, Sabrina Dallavallea*
a Department of Food, Environmental and Nutritional Sciences, Division of Chemistry and
Molecular Biology Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy.
,
b Scientia Advice, di Roberto Artali, 20832 Desio (MB), Italy
c Biogem, Research Institute, Ariano Irpino (AV), Italy
± Equal contribution
* Corresponding author. E-mail address: sabrina.dallavalle@unimi.it (S. Dallavalle).
Abstract
Recent studies have demonstrated enhanced anticancer effects of combination therapy consisting of
camptothecin derivatives and HDAC inhibitors. To exploit this synergy in a single active compound,
we designed new dual-acting multivalent molecules simultaneously targeting topoisomerase I and
HDAC. In particular, a selected compound containing a camptothecin and the psammaplin A
scaffold showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar
range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma
primary cell line MM487 orthotopically xenografted in CD-1 nude mice and very high tolerability.
1.
Introduction
The epigenetic control of chromatin organization by the covalent modification of DNA and histone
proteins plays a major role in the regulation of cell differentiation, proliferation and survival.
Several human diseases such as cancers, neurodegenerative diseases or metabolic disorders result
from abnormal gene expression due to the disequilibrium between epigenetic enzymes activities.
Histone N-3-lysine acetylation was identified as a key player, a reaction controlled by histone acetyl
transferases (HAT) in balance with histone deacetylases (HDAC).
Keywords: Psammaplin; HDAC inhibitors; camptothecin; antiproliferative activity; antitumor
activity; dual-action inhibitors.
1

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In recent years, HDAC inhibitors have emerged as highly attractive targets for anticancer therapies
for their overexpression in several cancer cell lines, resulting in tumor suppressor gene silencing [1,
2].
Among the myriad of HDAC inhibitors, the cytotoxic marine natural product psammaplin A (1)
from the sponge Pseudoceratina sp. [3-5] has gained interest due to its structure, and potent HDAC
and DNA methyltransferase inhibition [6].
Psammaplin A displays an intriguing structure, being a symmetrical disulfide with a cystamine
linker functionalized on both sides by α-(hydroxyimino)acyl moieties (Chart 1). It functions as a
natural prodrug, requiring reduction of its disulfide functionality to the corresponding thiol
monomer in order to potently inhibit HDACs [7].
Since the initial report by Crews and co-workers on psammaplin A as a potent HDAC inhibitor,
crystal structures of analogues have been obtained in order to perform SAR studies on various
epigenetic targets [8]. These studies showed that both the disulfide bridge and the α-
hydroxyiminoamide moieties are necessary for HDAC inhibition, whereas structural variations on
the aromatic ring are allowed [9].
During the last years we have been involved in the development of hybrid bifunctional agents.
Hybrid agents are compounds designed to inhibit simultaneously multiple cellular targets relevant
to tumour growth/survival. The interest toward multivalent ligand design is rapidly increasing, due
to the potential advantages of such bifunctional molecules [10]. The benefits of drugs with multiple
targets include the improvement of the efficacy by exploiting synergistic interactions, the
enhancement of the selectivity resulting in a better tolerability and modulation of drug resistance
[11]. Additionally, multivalent molecules are expected to provide pharmacokinetic and
pharmacodynamic advantages over the separate administration of the two drug components.
Specifically, we were interested in developing bifunctional compounds containing the camptothecin
scaffold [12]. Camptothecins are clinically validated topoisomerase I (Top1) inhibitors [13]. The
parent compound camptothecin (CPT, chart 1) was isolated for the first time by Wall et al. [14]
from the tree Camptotheca acuminata, a plant of the Nyssaceae family originating from China. Two
semisynthetic CPT derivatives have been approved by US FDA for cancer chemotherapy,
irinotecan (CPT-11) and topotecan. Belotecan has been approved in South Korea for treatment of
NSCLC. (Figure 1). Other derivatives are in different steps of clinical development, such as NSC-
603071 (9-amino-camptothecin), 9-CN or 9-nitrocamptothecin, GG-211 (GI 147211), and DX-
8591f. Although the conventional CPT derivatives have made a significant contribution to cancer
treatment, the dose-limiting toxicities and drug resistance remain significant hurdles in the use of
these drugs.
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O
Br
S
O
N
S
H
N
NOH
HO
N
HN
HON
O
O
CPT
Psammaplin A
OH
O
Br
OH
N
N
HO
N
O
O
O
N
N
O
N
N
O
O
Topotecan
Irinotecan
OH
O
OH
O
Chart 1. Structures of psammaplin A and camptothecin topoisomerase I inhibitors
.
Recently, it has been reported that co-treatment with HDAC inhibitors and camptothecin derivatives
(such as topotecan or irinotecan) synergistically block cell proliferation [15-20]. To exploit this
synergy in a single active compound, we designed new dual-acting multivalent molecules
containing a camptothecin and the psammaplin A scaffold. There are examples in the literature of
dual-acting molecules containing camptothecin and SAHA-like templates [21]. However, to the
best of our knowledge, the conjugation of a psammaplin to a CPT has not been attempted before.
The following criteria have guided the design of the conjugates: a) a CPT moiety substituted in a
suitable position to maintain cytotoxic activity; b) a proper linker between the psammaplin A
pharmacophore and the CPT skeleton. In previous works we have reported that CPT
functionalization at C-7 is highly tolerable [22-24]. Particularly, we have synthesized 7-
oxyiminomethyl derivatives showing potent in vitro and in vivo activity, even when bulky and
long-chain substituents were introduced [22, 24]. For this reason, suitably substituted (E)-7-
oxyminomethyl CPTs were selected for conjugation to the psammaplin A active fragment through
an amide bond (Figure 1). The ability of the compounds to act as both HDAC and Topoisomerase-I
ligands was studied by molecular modeling. The choice of the spacer length (n = 5, Figure 1) was
based on a preliminary virtual screening study in which all compounds with n ranged from 1 to 10
were taken into account. The results obtained prompted us to select compounds 1a-c for
topoisomerase I - HDAC dual inhibitory activity investigation.
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O
H
N
S
R
O
N
S
N
5
H
O
N
OH
HO
R1
O
N
1a
R = H, R1 = H
1b R = Br, R1 = H
1c
N
O
R = Br, R1 = OH
HO
O
Fig. 1. Structure of hybrids CPT–psammaplin A 1a-c.
2. Results and discussion
2.1 Chemistry
The reaction of 7-(dimethoxymethyl)-camptothecin 2a [25] with 6-aminooxyhexanoic acid
hydrobromide [26] in acetic acid afforded compound 3a in 73% yield (Scheme 1). Following a
similar procedure, compound 3b was obtained starting from 10-hydroxy-7-formylcamptothecin 2b
[27]. The choice of synthesizing a 10-hydroxyderivative starting from 2b is based on the presence
of this structural feature in both the CPT derivatives in clinical use, topotecan and irinotecan.
OH
O
N
O
R1
N
R2
R1
O
O
a
N
N
N
O
O
HO
HO
O
O
3a
2a R1 = -CH(OCH3)2, R2 = H
2b
R1 = H
3b R1 = OH
R1 = -CHO, R2 = OH
Scheme 1. Synthesis of camptothecin derivatives 3a-b. Reagents and conditions: a) for 3a: 6-aminooxyhexanoic acid
hydrobromide, acetic acid, 3h, 80 °C, 73%; for 3b: ethanol, pyridine, 4h, reflux, 75%.
The synthesis of camptothecin – psammaplin A hybrid compounds (1a-c) is described in Scheme 2.
Compound 4a was synthesized in two steps from the commercially available 4-
hydroxyphenylpyruvic acid [28]. Following a procedure reported in the literature [29], compound
4a was brominated with KBr and KBrO3 to give hydroxyiminopropionic acid 4b. Condensation of
acids 4a-b with Boc-protected cystamine 5 afforded the corresponding amides, which in turn were
treated with trifluoroacetic acid in dichloromethane to yield the desired intermediates 6a-b.
Condensation of the camptothecin derivative 3a with the psammaplin fragments 6a-b by WSC and
HOBt, afforded the hybrids 1a and 1b in 74% and 89% yield, respectively. The 10-OH derivative
3b was condensed with the amine 6a to give the hybrid 1c in 57% yield.
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HO
R
HO
N
H2N
S
OH
+
S
NHBoc
O
4a R = H
5
4b
R =Br
a,b
S
HO
R
HO
N
H
N
S
NH2 .TFA
O
6a R =Br
6b
+
R = H
c
OH
1a-c
O
N
O
R1
O
N
N
O
R1 = OH, H
HO
O
3a
R1 = H
3b R1 = OH
Scheme 2. Synthesis of CPT- psammaplin hybrids 1a-c. Reagents and conditions: a) NHS, DCC, DMF, 24h; b) TFA,
CH2Cl2, 1h; c) i: WSC, HOBt, DMF, 3-24h, rt; ii: 6a or 6b, 2-3 h, rt, 1a: 74%, 1b: 89%, 1c: 57%.
2.2 Molecular Modeling Studies
It has been previously reported that in terms of its HDAC activity, psammaplin A functions as a
natural prodrug, the active free thiol being revealed following disulfide reduction upon cell uptake
[30, 31]. Thus, the proposed binding models take into account the reduction of the disulfide bridge
in cell cultures, resulting in the release of reduced psammaplin and CPT fragments.
DNA-Topoisomerase-I complexes. Molecular modeling studies were performed to explore the
binding mode for the CPT fragment released from compounds 1a-b and the 10-OH CPR fragment
released from compound 1c to the DNA-topoisomerase-I complex. The two fragments are properly
positioned inside the intercalation binding site, created by conformational changes of the
phosphodiester bond between the +1 (upstream) and -1 (downstream) base pairs of the uncleaved
strand, which effectively ‘‘open’’ the DNA duplex, with multiple strong π-π stacking interactions
between the aromatic A-D rings of the fragments and both the -1 and +1 base pairs. Compounds
show a hydrogen bond between the nitrogen atom of ring B and R364 residue, which in turn forms
an additional hydrogen bond with the OH group of the lactone ring E. The lactone ring is also
involved in another hydrogen bond with the OH group of the lactone ring E and D533.
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Table 1. Binding free energies (kcal/mol) and distances (in Å) between the two CPT-fragments and the binding site
residues of the DNA-topoisomerase-I system.
∆Gbinding D533Oδ2
R364N2
E356Oε2
K436Nζ
2.97
DA113N6
-
CPT-
fragment
10 OH
-12.51
2.61
3.03-2.74
-
CPT-
-13.29
2.55
3.30-2.88
2.56
3.05
3.30
fragment
The interaction pattern of the 10-OH CPT fragment within the binding site is completed by a
hydrogen bond between the phenolic OH and E356, an interaction clearly impossible for the other
CPT fragment. The side chain of both CPT derivatives is located in the rear region of the binding
site, resting on the DNA duplex and forming a series of interactions that contribute to the stability
of the complexes. In both complexes, the lateral chain is arranged to form a hydrogen bond between
the amidic C=O group and K436N. Moreover, in the phenolic derivative, the lateral chain shows a
slight twist that allows for the formation of a hydrogen bond between the oxime nitrogen and
DA113N6. The 3D structures of the complexes with the DNA-Topoisomerase-I system are given in
Figure 2.
Fig. 2. (A) - Schematic representation of the proposed top-score binding mode for the CPT fragments in the DNA-
Topoisomarase-I complex. Topoisomerase-I is represented as Solvent Accessible Surface (SAS) coded by interpolated
charges, while the ligand is rendered in CPK. (B,C) - Top-score conformations of the two CPT fragment complexes,
with the aminoacid/nucleotide side chains relevant to the discussion rendered in line and the ligands in stick. Hydrogen
bonds are shown as green dotted lines.
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HDAC-II complexes.
The complexes are obtained by molecular docking of psammaplin fragments from compounds 1a-c
using the Class I Rpd3-like protein HDAC2 as a model.
The analysis of the complexes obtained by molecular docking shows the key interactions of these
moieties with the enzyme. The free thiol group forms a H-bond (2.45
the Zn2+ ion, while the oxime group forms a hydrogen bond (2.30 ) with D104Oδ2 (see Figure 3),
Å
) with H146Hε2 and chelates
Å
and the 4-hydroxyphenyl group gives few hydrophobic contacts with Y209, F155 and F210 belonging
to the entrance of the binding site.
Fig. 3. Top-score conformations of the of reduced psammaplin A fragment (in blue) and bromine-free analogue (in red)
complexes to HDAC-II model (5IWG). The aminoacids side chains relevant to the discussion are rendered in line and
the ligands in stick. Hydrogen bonds are shown as green dotted lines. The zinc ion is shown in CPK.
2.3. Biological activity
The antiproliferative activity was evaluated on a series of human solid tumor cell lines (NCI-H460:
non small cell lung cancer; CAPAN 1: human pancreatic ductal adenocarcinoma; A431: human
epidermoid carcinoma; HeLa: human epithelioid cervix carcinoma; HT29: human colon
adenocarcinoma; DU145: human prostate cancer; HepG2: liver hepatocellular carcinoma; A2780:
human ovarian carcinoma; A2780-Dox: doxorubicin-resistant human ovarian carcinoma. The
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activity was assessed after 72 h with the SRB assay. Irinotecan, SAHA and trichostatin a were used
as reference standards.
Compounds 1a-b showed significant antiproliferative activity in the nanomolar range (0.05 µM <
IC50 < 0.8 µM), while SAHA (IC50 > 0.9 µM) and irinotecan (IC50 > 1.5 µM) were less effective
(Table 3). The introduction of the hydroxy group on the CPT scaffold (compound 1c) had a
deleterious effect, causing a reduction of the activity on H460 and CAPAN1 cell lines. The removal
of the bromine atom in the psammaplin A pharmacophore did not cause any significant change in
activity (1a vs 1b). Interestingly, compound 1b seemed to be poorly affected by the multidrug-
resistant protein PGP, as showed in Table 3 by comparing the IC50 against A2780 with respect to
resistant A2780-Dox (RI: 3.8).
Thus, 1b was selected for further investigation. The compound was very effective against a number
of hematologic cancer cell lines (DG-75 Burkitt lymphoma; MAVER, MINO, JECO-1 mantle cell
lymphoma; KM-H2 and L-428 Hodgkin lymphoma, OCI-LY3 and U-2932 diffuse large B-cell
lymphoma, NB4 acute promyelocytic leukemia). Indeed, 1b showed a cytotoxic potency mostly in
the nanomolar range (0.03 µM < IC50 < 0.6 µM) and greater than SAHA and irinotecan except for
DG-75 cell line (Table 4).
Table 3. Human solid tumor cell lines exposed to Top1, HDAC and dual Top1 / HDAC inhibitors.
Irinotecan SAHA
Compound
Cell lines
1a
1b
1c
IC50 ± SD (µM)a
NCI-H460 2.4 ± 0.1 1.0 ± 0.1
0.22 ± 0.005 0.17 ± 0.006 0.88 ± 0.003
0.075 ± 0.005 0.05 ± 0.002 0.2 ± 0.01
CAPAN1
A431
1.5 ± 0.1 1.8 ± 0.2
4.1 ± 0.1 3.5 ± 0.1
5.8 ± 0.6 2.7 ± 0.2
4.8 ± 0.4 2.2 ± 0.3
0.31 ± 0.04
0.58 ± 0.2
0.46 ± 0.1
0.18 ±0.02
0.47 ± 0.1
0.66 ± 0.2
0.11 ± 0.01
0.76 ± 0.2
0.22 ± 0.02
0.83 ± 0.2
N.e.
N.e.
N.e.
N.e.
N.e.
N.e.
N.e.
HeLa
HT29
DU145
HepG2
A2780
2.8 ± 0.07 0.87 ± 0.03 0.19 ± 0.01
4.0 ± 0.8 1.54 ± 0.1 0.61 ± 0.2
3.8 ± 0.5 2.1 ± 0.2
0.29 ± 0.03
1.6 ± 0.5
A2780-Dox 20.1 ± 2.9 3.3 ± 0.6
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RI
5.3
1.6
5.5
3.8
aAntiproliferative activity assessed upon 72 h with the SBR assay. N.e. = not evaluated.
Table 4. Human lymphoma and leukemic cell lines exposed to Top1, HDAC, and dual
Topo1/HDAC inhibitor 1b.
MINO
MAVER-2
JECO-1
U-2932
OCI-LY3
L-428
KM-H2
DG-75
NB4
IC50 ± SD (µM)a
>10
Cpd
>10
>10
>10
>10
1.0 ± 0.3
Irinotecan >10 (36%)
0.74 ± 0.1
1.8 ± 0.2
4.4 ± 0.8
(48 %)
(0 %)
(0 %)
(12 %)
(0 %)
0.87 ±
0.03
SAHA
1.1 ± 0.3
3.5 ± 0.1 2.7 ± 0.2 2.2 ± 0.3
1.54 ± 0.1 2.1 ± 0.2 3.3 ± 0.6
0.035 ±
0.01
0.3 ±
0.09
0.06 ±
0.02
0.11 ±
0.03
0.1 ±
0.01
0.6 ±
0.09
0.28 ±
0.07
8.4 ±
0.4
0.047 ±
0.01
1b
aHuman lymphoma cells were treated with Topo1, HDAC, and dual Topo1/HDAC inhibitors and the anti-proliferative
activity assessed upon 72 h with the MTT assay.
Given the promising results, compound 1b was also tested against a series of human ex-vivo
luciferase-transfected mesothelioma cell lines (MM432 sarcomatoid; MM473 hepitelioid; MM487
biphasic), showing a significant antiproliferative activity, higher than the reference compounds
SAHA and irinotecan (Table 5).
Table 5. Human ex-vivo luciferase-transfected mesothelioma cell lines exposed to 1b dual Top1 /
HDAC inhibitor.
MM432
MM473
MM487
IC50 ± SD (µM)a
Compound
1b
4.0 ± 0.6 1.9 ± 0.5 0.13 ± 0.02
6.5 ± 0.8 8.2 ± 0.6 4.1 ± 0.6
SAHA
Irinotecan 10.0 ± 2.0
-
9.4 ± 0.7
aHuman mesothelioma cells were exposed to 1b , SAHA and irinotecan. The antiproliferative activity assessed upon 72
h with the SRB assay.
In terms of HDACs inhibition, the compound showed a significant activity against four purified
HDAC isoforms, representative of class I (HDAC 1, 2) and Class IIb (HDAC 6, 10) HDACs,
displaying optimal activity against HDAC10. Conversely, 1b was inactive against HDAC 4
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(representative of Class IIa HDACs), similarly to what reported for other psammaplin A analogues
against Class IIa HDACs [29].
A similar profile of HDAC isoforms inhibition was found for compound 1c (Table 6).
Table 6. Inhibition of HDAC isoforms by compounds 1b and 1c. Trichostatin A and TMP269 were
used as reference compounds.
HDAC1
HDAC2
HDAC4
IC50 (M)
HDAC6
HDAC10
Compound
1.50E-07
3.27E-07 n.a.
7.09E-07
1.49E-06
7.55E-09
9.12E-09
1b
2.53E-07 4.10E-07 n.a.
1c
1.244E-08 2.36E-08
-
-
2.09E-09
-
3.20E-08
-
Trichostatin A
TMP269
-
3.53E-07
IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation (see SI).
n.a. = not active
1b
Ctr
SN38 SAHA
Ac-H4
H4
Ac-Tub
Tub
Fig. 4. MM473 cells were treated for 24 h with SN38 (0.5 µM), SAHA (1µM) and with 1b (0.2 and 0.5 µM ). Cells
were lysed in 0.5% NP-40 and 40µg of total proteins were loaded on NuPAGE 4-12% (Invitrogen), transferred to a
nitrocellulose paper and probed with specific antibodies
To explore the HDAC inhibitory activity of compound 1b in the cells, accumulation of both
acetylated histone H4 and acetylated tubulin was investigated in the treated cells using western blot
analysis. The in vitro exposure of MM473 cells to 1b induced hyperacetylation of nuclear histone
H4 protein, the substrate of HDAC1/2, but it did not hyperacetylate cytoplasmatic α tubulin, a
substrate of HDAC6 (Figure 4). This result suggested that 1b has a preferential nuclear localization
that could be relevant for the exploitation of the dual HDAC/Top1 mechanism, being Top1 a
nuclear enzyme.
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Compound 1b was also tested on MM473 and MM487 cell lines to assess the effect on cell cycle
progression and induction of apoptosis. FACS analysis revealed a steady block of treated-cells in S
or S-G2M phase. Moreover, an effective induction of apoptosis (sub-G0/1 population) was triggered
by 1b on both tumor cell lines, with a higher effect on biphasic MM487 mesotelioma cell line
(Tables 7,8).
Table 7. Flow cytometric evaluation of human tumor cell line MM473 exposed to 1b
G0/1 (%) S (%) G2/M (%) Apo (%)
24 h
Control 54.5
28.0 58.97
29.8
15.7
13.1
2.5
3.8
1b
48 h
Control 71.8
4.5
G0/1 (%) S (%) G2/M (%) Apo (%)
23.9
83.5
4.3
2.2
9.9
12.0
1b
72 h
Control 80.2
6,.2
G0/1 (%) S (%) G2/M (%) Apo (%)
15.1
4.6
1.4
79.3
14.5
11.0
1b
Tumor cells were exposed for 72 h to test items (2xIC50 dose, 4µM), stained with PI, and processed on a FACScan flow
cytometer. The CellQuest software was used to acquire data and assess apoptosis, while cell cycle analysis was
performed with the ModFit software.
Table 8. Flow cytometric evaluation of human tumor cell line MM487 exposed to 1b
G0/1 (%) S (%) G2/M (%) Apo (%)
24 h
Control 57.9
20.6 77.62
25.2
16.9
1.8
0.5
2.6
1b
48 h
Control 73.8
16.5
G0/1 (%) S (%) G2/M (%) Apo (%)
13.7
67.8
12.5
15.7
0.3
13.1
1b
G0/1 (%) S (%) G2/M (%) Apo (%)
72 h
Control 80.0
19.6
14.6
59.2
5.4
0.6
21.2
25.4
1b
Tumor cells were exposed for 72 h to test items (2xIC50 dose, 0.3µM), stained with PI, and processed on a FACScan
flow cytometer. The CellQuest software was used to acquire data and assess apoptosis, while cell cycle analysis was
performed with the ModFit software.
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The efficacy of the new dual inhibitor 1b was evaluated on human mesothelioma primary cell line
MM473 orthotopically xenografted in CD-1 nude mice. After tumor injection and when the tumors
were in growing phase (day 15, from tumor injection), mice were intravenously treated with 1b (90
mg/kg, q4dx3w), irinotecan (20 mg/kg, q7dx3) or vehicle as negative control. The tumor volume
was measured as BLI signal and expressed as average radiance.
The molecule had a strong antitumor activity as shown in Figure 5 and summarized in Table 9.
Additionally, the tumor chemioluminescence of animals treated with 1b indicated that the
compound acted both by reducing the tumor growth progression and, in some cases, by reducing the
tumor size until the disappearance of the bioluminescent signal. Interestingly, the antitumor activity
was observed at well tolerated doses (no reduction in body weight was observed). Conversely,
irinotecan administered at 20 mg/kg had only a marginal antitumor effect (not significant, p>0,5).
Table 9. Antitumor activity of 1b compared to irinotecan on human mesothelioma tumor intrapleurally xenografted in
nude mice
N°
Tumor
cell line
BWV
(%)
TVI
(%)
of Drug/Dose Schedule/route
mice
BLI (p/s/cm2/sr)
Irinotecan
20 mg/kg
1.96x106±2.82x106
(+31)
10
8
q4dx3w/IV
-
-
-
-
37
-
MM473-
Luc
3.49x106±2.2x106
(+40)
Vehicle
7.61x105±9.4x105
(+40)
1b
90 mg/kg
8
q4dx3w/IV
78
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Vehicle
Day 15 Day 40
1b
Irinotecan
Day 15 Day 40
a
Day 15
Day 40
c
b
Fig.5. Efficacy of 1b in an orthotopic xenograft model of human epithelioid mesothelioma. a) bioluminescence signal
of experimental groups observed at day 15 (when treatment started) and at day 40 (10 days after the last treatment); b)
Body weight and c) tumor growth (expressed as Luciferase Average radiance) measurements throughout the
experimental period. Statistical analysis performed using non parametric Mann-Whitney test (U) 1b group versus
Vehicle *p<0,05
3. Conclusions
Synergistic effects have been reported by co-treatment of various cancer cells with topoisomerase I
and HDAC inhibitors. Based on a rational design method, camptothecin-psammaplin A hybrids
were synthesized and evaluated. In particular, compound 1b displayed a broad spectrum of
antiproliferative activity, with IC50 values in the nanomolar range, on a series of human solid tumor,
hematologic and mesothelioma cell lines. Moreover, the compound showed a significant antitumor
activity and very high tolerability in an in vivo human mesothelioma tumor model. Further work to
develop analogues with optimized structure and improved bioactivity is ongoing.
4. Experimental section
4.1. Chemistry
All reagents and solvents were reagent grade or were purified by standard methods before use.
Melting points were determined in open capillaries and are uncorrected. NMR spectra were
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recorded in CDCl3 (where not otherwise stated) at 300 MHz. Chemical shifts (δ values) and
coupling constants (J values) are given in ppm and Hz, respectively. Solvents were routinely
distilled prior to use; anhydrous tetrahydrofuran (THF) and ether (Et2O) were obtained by
distillation from sodium-benzophenone ketyl; dry methylene chloride was obtained by distillation
from phosphorus pentoxide. All reactions requiring anhydrous conditions were performed under a
positive nitrogen flow, and all glassware were oven dried and/or flame dried. Isolation and
purification of the compounds were performed by flash column chromatography on silica gel 60
(230-400 mesh). Analytical thin-layer chromatography (TLC) was conducted on Fluka TLC plates
(silica gel 60 F254, aluminum foil). Analyses indicated by the symbols of the elements or functions
were within ± 0.4 % of the theoretical values.
[29]
Compounds 4a-b,[28] 6a-b,
6-aminooxyhexanoic acid hydrobromide,[26] and 2a-b[25,27] were
prepared according to literature procedures.
4.2. 6-(camptothecin-7-yl(methyleneaminooxy)hexanoic acid (3a). To a solution of 7-
(dimethoxymethyl)camptothecin 2a (422 mg, 1 mmol) in acetic acid (20 ml) 6-aminooxyhexanoic
acid hydrobromide (570 mg, 2.5 mmol) was added and the mixture heated 3 h at 80 °C. The solvent
was evaporated and the residue was purified by flash chromatography with CH2Cl2 : CH3OH 93:7
1
to give 370 mg of pure product (yield : 73%). Mp: 132-133°C. H NMR (300 MHz, DMSO-d6) δ:
9.29 (1H, s); 8.63-8.48 (1H, m); 8.29-8.10 (1H, m); 7.94-7.81 (1H, m); 7.80-7.65 (1H, m); 7.32 (1H,
s); 6.55 (1H, s); 5.43 (2H, s); 5.27 (2H, s); 4.35 (2H, t, J = 7.0 Hz); 2.23-2.11 (2H, m); 1.98-1.72
13
(4H, m); 1.66-1.52 (2H, m); 1.50-1.39 (2H, m); 0.89 (3H, t, J = 7.0 Hz). C NMR (300 MHz,
DMSO-d6) δ: 174.9, 172.9, 157.0, 152.8, 150.3, 149.0, 145.5, 145.1, 131.5, 130.8, 130.2, 128.6,
127.2, 125.4, 124.6, 119.6, 97.1, 75.3, 72.8, 65.7, 52.6, 34.1, 30.7, 28.8, 25.4, 24.8, 8.24. ). Anal.
calcd for C27H27N3O7: C, 64.15; H, 5.38; N, 8.31.Found: C, 63.80; H, 5.40; N, 8.29.
4.3. 6-(10-Hydroxycamptothecin-7-ylmethyleneaminooxy)hexanoic acid (3b). To a solution of 10-
hydroxy-7-formylcamptothecin 2b (294 mg, 0.75 mmol) in ethanol (15 mL) 6-aminooxyhexanoic
acid hydrobromide (342 mg, 1.5 mmol) and pyridine (2.5 ml, 30 mmol) were added and heated 4h
to reflux. The solvent was evaporated and dichloromethane (20 ml) was added. The crude was
heated to reflux for 1 h. After cooling to rt, the solid was filtered. This treatment was repeated using
CH2Cl2: CH3OH 95:5 (20 ml) and CH2Cl2:CH3OH 99:1 (20 ml) to give 287 mg of product (yield:
1
75%). H-NMR (300 MHz, DMSO- d6) δ: 10.53 (1H, brs); 10.38 (1H, s); 9.04 (1H, s); 8.70 (1H,
brs); 8.08 (1H, d, J = 8.5 Hz); 7.74 (1H, d, J = 2.7 Hz); 7.50 (1H, dd, J = 8.5, 2.7 Hz); 7.28 (1H, s);
6.52 (1H, s); 5.43 (2H, s); 5.30 (2H, s); 4.41-4.27 (2H, m); 2.08-1.96 (2H, m); 1.95-1.73 (4H, m);
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13
1.69-1.53 (2H, m); 1.52-1.38 (2H, m); 0.90 (3H, t, J = 7.0 Hz). C-NMR (DMSO-d6): δ 174.8,
172.9, 157.8, 157.0, 150.4, 149.4, 145.9, 144.9, 144.4, 131.9, 128.8, 127.6, 127.0, 123.2, 118.7,
105.7, 96.2, 75.1, 72.8, 65.7, 52.2, 34.1, 30.8, 28.8, 25.5, 24.8, 8.2. Anal. calcd for C27H27N3O8: C,
C, 62.18; H, 5.22; N, 8.06.Found: 61.99; H, 5.23; N, 8.07.
4.4. 6-camptothecin-7-yl(methyleneaminooxy)hexanoic acid (2-{2-[3-(4-hydroxyphenyl)-2-
hydroxyiminopropionylamino]ethyldisulfanyl}ethyl)amide (1a).
To a solution of 3a (50 mg, 0.1 mmol) in dry DMF (5 ml) WSC (25 mg, 0.13 mmol) and HOBt (18
mg, 0.12 mmol) were added, the mixture was stirred 24 h at rt, then 6b (80 mg, 0.18 mmol) was
added. The reaction was stirred 2 h at rt. The solvent was evaporated and the residue was purified
by flash chromatography with CH2Cl2:CH3OH 20:1 to give 60 mg (yield: 74%) of product. Mp:
1
108 °C. H-NMR (300 MHz, DMSO-d6) δ: 9.31 (1H, s); 9.13 (1H, brs); 8.59 (1H, d, J = 8.6 Hz);
8.21 (1H, d, J = 8.6 Hz); 8.05-7.83 (3H, m); 7.78-7.69 (1H, m); 7.34 (1H, s); 6.95 (2H, d, J = 8.4
Hz); 6.59 (2H, d, J = 8.4 Hz); 6.52 (1H, brs); 5.42 (2H, s); 5.33 (2H, s); 4.33 (2H, t, J = 6.6 Hz);
3.33-3.18 (4H., m); 2.84-2.62 (4H, m); 2.10 (2H, t, J = 7.3 Hz); 1.91-1.71 (4H, m); 1.65-1.50 (2H,
m); 1.49-1.33 (2H, m); 0.89 (3H, t, J = 7.1 Hz). 13C-NMR (DMSO-d6): 172.9, 172.6, 163.8, 157.1,
156.0, 152.7, 150.4, 149.1, 145.6, 145.2, 133.2, 131.7, 131.6, 130.9, 130.1(×2), 129.5, 129.2, 128.6,
127.2, 125.4, 119.6, 115.4 (×2), 97.1, 75.3, 72.8, 65.7, 53.3, 38.6, 38.2, 37.8, 37.3, 35.7, 30.7, 28.8,
28.4, 25.5 (×2), 8.2. Anal. calcd for C40H44N6O9S2: C, 58.81; H, 5.43; N, 10.29. Found: 58.95; H,
5.41; N, 10.31.
4.5. Camptothecin-7-yl-methyleneaminooxy)hexanoic acid (2-{2-[3-(3-bromo-4-hydroxyphenyl)-2-
hydroxyiminopropionylamino]-ethyldisulfanyl}ethyl)amide (1b).
To a solution of 6-(camptothecin-7-ylmethyleneaminooxy)-hexanoic acid 3a (50 mg, 0.1 mmol) in
dry DMF (5 ml) WSC (25 mg, 0.13 mmol) and HOBt (18 mg, 0.12 mmol) were added and the
reaction was stirred 4 h at rt, then 6a (85 mg, 0.16 mmol) was added and the mixture was stirred 2 h
at rt. The solvent was evaporated and the residue was purified by flash chromatography with
1
CH2Cl2:CH3OH 10:1 to give 50 mg (yield: 89%) of compound 1b. Mp: 132 °C. H-NMR (300
MHz, DMSO-d6) δ: 11.84 (1H, s); 10.02 (1H, s); 9.33 (1H, s); 8.61 (1H, d, J = 8.2 Hz); 8.23 (1H, d,
J = 8.2 Hz); 8.13-8.03 (1H, m); 8.01-7.90 (2H, m); 7.82-7.71 (1H, m); 7.36 (1H, s); 7.26 (1H, s);
6.99 (1H, d, J = 8.2 Hz); 6.81 (1H, d, J = 8.2 Hz); 6.54 (1H, s); 5.44 (2H, s); 5.35 (2H, s); 4.40-4.30
(2H, m); 3.66 (2H, s); 2.85-2.64 (4H, m); 2.18-2.03 (2H, m); 1.96-1.72 (4H, m); 1.67-1.52 (2H, m);
13
1.51-1.35 (2H, m); 1.30-1.14 (2H, m); 1.14-1.00 (2H, m); 0.88 (3H, t, J = 7.1 Hz). C NMR (75
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MHz, DMSO- d6) δ: 172.9, 172.6, 163.7 (×2), 157.0, 152.7, 152.2, 150.4, 149.0, 145.5, 145.0,
133.2, 131.5, 130.8, 130.2, 129.5, 129.2, 128.6, 127.1, 124.5, 125.3, 119.6, 116.5, 109.3, 97.1, 75.3,
72.8, 65.7, 52.6, 38.6, 38.2, 37.9, 37.3, 35.7, 30.8, 28.8, 28.1, 25.5 (×2), 8.2. Anal. calcd for
C40H43BrN6O9S2: C, 53.63; H, 4.84; N, 9.38;. Found: 53.75; H, 4.85; N, 9.36.
4.6. 10-Hydroxy-camptothecin-7-yl-methyleneaminooxy)-hexanoic acid (2-{2-[3-(3-bromo-4-
hydroxyphenyl)-2-hydroxyiminopropionylamino]ethyldisulfanyl}ethyl)amide (1c).
To a solution of 3b (30 mg, 0.06 mmol) in dry DMF (5 ml) WSC (15 mg, 0.08 mmol) and HOBt
(10 mg, 0.07 mmol) were added and the reaction was stirred 3 h at rt, then compound 6a (47 mg,
0.09 mmol) was added the mixture was stirred 2 h at rt. The solvent was evaporated and the residue
was purified by flash chromatography with CH2Cl2:CH3OH 10:1 give 30 mg (yield: 57%) of
1
product. Mp: 138 °C. H-NMR (300 MHz, DMSO-d6) δ: 11.84 (1H, s); 10.48 (1H, s); 10.02 (1H,
s); 9.03 (1H, s); 8.15-7.82 (3H, m); 7.74 (1H, s); 7.54-7.40 (1H, m); 7.36-7.17 (2H, m); 7.04-6.90
(1H, m); 6.90-6.75 (1H, m); 6.50 (1H, s); 5.42 (2H, s); 5.31 (2H, s); 4.40-4.22 (2H, m); 3.66 (2H,
s); 2.97-2.65 (4H, m); 2.20-2.02 (2H, m); 1.94-1.68 (4H, m); 1.67-1.50 (2H, m); 1.50-1.36 (2H, m);
1.31-1.16 (2H, m); 0.87 (3H, t, J = 7.0 Hz). 13C NMR (150 MHz, DMSO- d6) δ: 172.5, 172.2, 163.2,
157.5, 156.7, 152.3, 151.8, 150.1, 149.2, 145.6, 144.8, 144.1, 132.8, 131.6, 129.1, 128.8, 128.6,
127.4, 126.7, 122.9, 188.2, 116.1, 108.8, 105.5, 95.8, 74.7, 72.4, 65.2, 51.9, 37.4, 36.8, 36.5, 35.2,
33.3, 30.2, 28.3, 27.7, 25.0 (×2), 7.8. Anal. calcd for C40H43BrN6O10S2: C, 52.69; H, 4.75; N, 9.22;
Found: 52.55; H, 4.76; N, 9.20.
4.7. Cell lines
Human primary epithelioid MM288 (ME-C001), MM317 (ME-C011), MM404 (ME-C021),
MM473 (ME-C051), MM481 (ME-C061); biphasic MM487 (MB-C071) and MM491 (MB-C081);
sarcomatoid MM432 (MS-C031) and MM472 (MS-C041) mesothelioma cell lines.
Human K562 erythromyeloblastoid leukemia (ATCC # CCL-243), ARH-77 plasma leukemia
(ATCC # CRL-1621), THP1 and MV4-11 monocytic leukemia (ATCC # TIB-202, CRL-9591),
Jurkat acute T-cell leukemia (ECACC # 90112119), U937 histiocytic lymphoma (ATCC # CRL-
1593), RAJI, DG-75, and RAMOS Burkitt lymphoma (DSMZ # ACC-319, ACC-83, ACC-603);
MAVER, MINO, REC-1 (DSMZ # ACC-717, ACC-687, ACC-584), JECO-1, Z-138 (ATCC #
CRL-3006, CRL-3001) mantle cell lymphoma; KM-H2 and L-428 Hodgkin lymphoma (DSMZ #
ACC-8, ACC-197), OCI-LY3 and U-2932 diffuse large B-cell lymphoma (DSMZ # ACC-731,
ACC-633), RPMI-8226 and NCI-H929 multiple myeloma (ATCC # CCL-155, CRL-9068).
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Human NCI-H460 NSCLC (ATCC # HTB-177), Capan-1 pancreatic carcinoma (ATCC # HTB-
79), A431 epidermoid carcinoma (ATCC # CRL-1555), HeLa cervix carcinoma (ATCC # CCL-2),
A2780 and A2780-Dx multidrug-resistant ovarian carcinoma (ECACC # 93112519, 93112520),
HT29 colorectal carcinoma (ECACC # 91072201), HepG2 hepatocellular carcinoma (ATCC # HB-
8065), DU145 prostate carcinoma (ATCC # HTB-81).
4.8. Cell cultures
All mesothelioma cell lines were cultured in F-10 Nutrient Mixture medium supplemented with
10 % Foetal Bovine Serum (FBS), 2 mM L-glutamine and gentamicin sulfate.
NCI-H60, A2780, A2780-Dx, HeLa, U937, RPMI-8226, ARH-77, Jurkat, RAJI, REC-1, DG-75, U-
2932, KM-H2 and L-428 cells were cultured in RPMI-1640 medium supplemented with 10 % FBS,
2 mM L-glutamine and gentamicin sulfate. RAMOS, OCI-LY3, JECO-1, MAVER-2, MINO cells
were cultured in RPMI-1640 medium supplemented with 20 % FBS, 2 mM L-glutamine and
gentamicin sulfate. THP1 and H929 cells were cultured in RPMI-1640 medium supplemented with
10 % FBS, 0.05 mM mercaptoethanol, L-glutamine and gentamicin sulfate. K562, MV4-11, Z-138
cells were cultured in IMDM medium supplemented with 10 % FBS, L-glutamine and gentamicin
sulfate. Capan-1 cells were cultured in IMDM medium supplemented with 10 % FBS, 2 mM L-
glutamine and gentamicin sulfate. A431, DU145, and HepG2 cells were cultured in EMEM medium
supplemented with 10 % FBS, 2 mM L-glutamine and gentamicin sulfate. HT29 cells were cultured
in McCoy’s medium supplemented with 10 % FBS, 2 mM L-glutamine and gentamicin sulfate.
Cells were maintained in an incubator at 37°C with saturated humidity and an atmosphere of 95%
air and 5% CO2, and were sub-cultured every 2-3 days.
4.9. Cytotoxicity assay
Human tumor cells were seeded in 96-wells plastic plates, allowed to attach, and exposed to scalar
concentrations of the drug compounds. Cell survival was evaluated upon 72 h by the
sulphorhodamine B (SRB) or MTT assay and the IC50 value (drug concentration inhibiting 50% of
cell growth) calculated by the ALLFIT program.
4.10. HDAC’s profiling
The experiments were carried out by HDAC Fluorescent Activity Assay in RBC facility
(http://www.reactionbiology.com/), using in triplicate 10 concentrations of each compound starting
from 10 µM.
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Assay description: The HDAC Fluorescent Activity Assay is based on the unique Fluorogenic
Substrate and Developer combination. The assay procedure has two steps. First, the Fluorogenic
Substrate, which comprises an acetylated lysine side chain, is incubated with a purified HDAC
enzyme. Deacetylation of the substrate sensitizes the substrate so that, in the second step, treatment
with the Developer produces a fluorophore.
Testing compounds were dissolved in 100% DMSO to specific concentration. The serial dilution
was conducted by epMotion 5070 in DMSO.
HDAC reaction buffer: 50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2.
Add fresh: 1 mg/ml BSA, 1% DMSO.
Substrate HDAC 1, 2, 6, 10: Fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)AMC);
HDAC4: Fluorogenic HDAC Class 2a Substrate (Trifluoroacetyl Lysine).
General Reaction Procedure: (Standard IC50 determination): Deacetylation Step: 1. Deliver 2X
enzyme in wells of reaction plate except No Enzyme control wells. Add buffer in No En wells. 2.
Deliver compounds in 100% DMSO into the enzyme mixture by Acoustic technology (Echo550;
nanoliter range). Spin down and pre-incubation. 3. Deliver 2X Substrate Mixture (Fluorogenic
HDAC Substrate and co-factor if applicable) in all reaction wells to initiate the reaction. Spin and
shake. 4. Incubate for 1-2 h at 30 °C with seal. Development Step: 5. Add Developer with
Trichostatin A (or TMP269) to stop the reaction and to generate fluorescent color. 6. Fluorescence
was read (excitatory, 360; emission, 460) using the EnVision Multilabel Plate Reader (Perkin
Elmer). 7. Take endpoint reading for analysis after the development reaches plateau.
Data Analysis: The percentages of enzyme activity (relative to DMSO controls) and IC50 values
were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation.
4.11. Protein analysis
Exponentially growing mesothelioma cells (MM473) were collected in Dulbecco’s 1x PBS buffer
and lysed in RIPA lysis buffer (50mM Tris-HCl, 150 mM NaCl, 0,5% Na-Deoxycholate, 1% NP-40,
0.15% SDS) containing protease and phosphatase inhibitors and then centrifuged at 12000 rpm for
10 minutes at 4 °C. The supernatants, containing the total proteins extracts, were transferred in new
vials. The protein concentration was measured by Bradford Protein Assay. Equal amounts of
proteins for each sample were then resolved by SDS-PAGE and loaded on 10-12% polyacrilamide
gels. The proteins were then transferred on nitrocellulose membranes. Molecular weights were
estimated based upon the relative migration with molecular weight protein markers. The non-
specific binding were blocked by incubation of membranes in TBS-tween (25 mM Tris, 150 mM
NaCl, 0.1% Tween 20) with 5% nonfat dry milk (BIORAD, Milan) for 60 minutes. Subsequently,
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the membranes were incubated overnight with primary antibodies: anti-Histone H4 (Millipore,
1:500), anti-acetyl-H4 (Santa Cruz, 1:1000) anti-p21 (Santa Cruz, 1:1000), anti-acetyl-tubulin
(abcam, 1:1000), anti-α-tubulin (Sigma, 1:5000), anti β-actin (Sigma, 1:5000) and anti-
Topoisomerase 1 (Santa Cruz, 1:500). After three washes with TBST blots were incubated with
HRP-conjugated secondary anti-mouse and anti-rabbit antibodies (Santa Cruz, 1:5000) 1 hour at
room temperature. After three washes with TBST the blots were developed with ECL substrate (GE
Healthcare) and chemiluminescence detected by ChemiDoc Imaging System (Biorad). The optical
density of the protein bands detected by western blotting was normalized on α-tubulin levels and
analyzed by ChemiDoc software (Biorad).
4.12. In vivo antitumor activity
MM473 cells were cultured in Ham’s F-10 Nutrient Mixture supplemented with 10 % FBS, 2 mM
L-glutamine, and G418 antibiotic and expanded in a 37 °C incubator with saturated humidity and an
atmosphere of 95% air and 5% CO2, and were sub-cultured every 2-3 days. Before the injection in
mice, cells were checked with MycoAlert® Mycoplasma Detection Kit (Lonza) to exclude
mycoplasma contamination, counted by trypan blue dye exclusion, evaluated for cell viability (>
95%). Human epithelioid mesothelioma cell lines (MM473) were expanded and re-suspended at the
concentration of 1x107/ml in M199. Tumor cells were orthotopically inoculated intrapleurally (i.pl.)
in CD-1 nude mice (Charles River) at concentration of 1x106/200µl M199/mouse. Compound 1b
was diluted in 10% 1:1 Absolute Ethanol-Cremophor solution in saline. 1b was administered i.v at
90 mg/kg using a q4dx3w schedule. Irinotecan was administered iv at 20mg/kg, using the schedule
q4dx3w. Tumor growth was evaluated weekly by using the IVIS Spectrum (PerkinElmer) in vivo
technology system, through intraperitoneal injection of D-Luciferin (100µL/10gr, PerkinElmer). For
each animal the specific average radiance signal was evaluated. Ethics approval was obtained from
the Italian Ministry of Health and all experiments were in accordance with the European guidelines
for the care and use of laboratory animals.
4.13. Statistical analysis
Statistical analysis was performed by using U-Test (GraphPad Prism 6). Outliers were removed by
using the Rout test (Q = 10%, GraphPad Prism 6).
4.14. Molecular Modeling
The ligand molecules were refined using a systematic conformer search followed by geometry
optimisation of the lowest energy structure with MOPAC7 (PM3 Method, RMS gradient 0.0100).
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The DNA-Topoisomerase-I and HDAC-II models were derived from the deposited X-ray structures
(Protein Data Bank entry 1T8I and 5IWG, respectively).
Energy minimisations and molecular modeling calculations were performed by using the CUDA®
version of the GROMACS package [32] and the AMBER-03 [33] force field, a variant of the
AMBER-99 [-34] potential in which charges and main-chain torsion potentials have been derived
based on QM+continuum solvent calculations and each amino acid is allowed unique main-chain
charges (nucleic acids have not been modified from AMBER-99).
Molecular docking experiments were performed with Autodock 4.0 [35]. We used the Lamarckian
Genetic Algorithm which combines global search (Genetic Algorithm alone) to local search (Solis
and Wets algorithm) [36]. Ligands and receptors were further processed using the Autodock Tool
Kit (ADT) [37]. Gasteiger–Marsili charges [38] were loaded on the ligands in ADT and Cornell
parameters were used for the phosphorous atoms in the DNA. Solvation parameters were added to
the final structure using the Addsol utility of Autodock. Each docking consisted of an initial
population of 100 randomly placed individuals, a maximum number of 200 energy evaluations, a
mutation rate of 0.02, a crossover rate of 0.80, and an elitism value of 1. For the local search, the so-
called pseudo-Solis and Wets algorithm was applied using a maximum of 250 iterations per local
search. 250 independent docking runs were carried out for each ligand. The grid maps representing
the system in the actual docking process were calculated with Autogrid. The dimensions of the grids
were 80 × 80 × 80, with a spacing of 0.1 Å between the grid points and the center close to the cavity
left by the ligand after its removal. The simpler inter-molecular energy function based on the
Weiner force field in Autodock was used to score the docking results. Results differing by less than
1.0 Å in positional root-mean-square deviation (rmsd) were clustered together and were represented
by the result with the most favourable free energy of binding. The poses were equilibrated by a 5.0
ns molecular dynamics simulation using the CUDA® version of the GROMACS package.
Acknowledgments
The authors gratefully acknowledge Prof. Lucio Merlini and Dr. Alberto Bargiotti for helpful
suggestions and discussions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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Highlights
A new dual HDAC-topoisomerase I inhibitor was investigated.
Molecular docking was used to predict the optimal conformation in Top1/HDAC sites.
The compound showed antiproliferative activity with IC50 values in the nanamolar range.
The compound exhibited an appreciable in vivo antitumor activity.