Tyrosyl-DNA Phosphodiesterase 1 Inhibitors: Usnic Acid Enamines Enhance the Cytotoxic Effect of Camptothecin

Article abstract of DOI:10.1021/acs.jnatprod.6b00979

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top1) cleavage complexes and other 3′-end DNA lesions. TDP1 is a perspective target for anticancer therapy based on Top1-poison-mediated DNA damage. Several novel usnic acid derivatives with an enamine moiety have been synthesized and tested as inhibitors of TDP1. The enamines of usnic acid showed IC₅₀ values in the range of 0.16 to 2.0 μM. These compounds revealed moderate cytotoxicity against human tumor MCF-7 cells. These new compounds enhanced the cytotoxicity of the established Top1 poison camptothecin by an order of magnitude.

Full text of DOI:10.1021/acs.jnatprod.6b00979

Article
pubs.acs.org/jnp
Tyrosyl-DNA Phosphodiesterase 1 Inhibitors: Usnic Acid Enamines
Enhance the Cytotoxic Effect of Camptothecin
Alexandra Zakharenko,†,# Olga Luzin⊥a,,∥‡,# Olga Koval,†,§ Dmitry Nilov,⊥ Irina Gushchina,∥
†
Nadezhda Dyrkheeva, Vytas Svedas, Nariman Salakhutdinov,‡,§ and Olga Lavrik*,†,§
̌
†Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences,
Novosibirsk, 630090, Russian Federation
‡N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk,
630090, Russian Federation
§Novosibirsk State University, Novosibirsk, 630090, Russian Federation
∥
⊥Belozersky Institute of Physicochemical Biology and Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State
University, Moscow, 119991, Russian Federation
S
* Supporting Information
ABSTRACT: Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a
repair enzyme for stalled DNA-topoisomerase 1 (Top1)
cleavage complexes and other 3′-end DNA lesions. TDP1 is
a perspective target for anticancer therapy based on Top1-
poison-mediated DNA damage. Several novel usnic acid
derivatives with an enamine moiety have been synthesized
and tested as inhibitors of TDP1. The enamines of usnic acid
showed IC50 values in the range of 0.16 to 2.0 μM. These
compounds revealed moderate cytotoxicity against human
tumor MCF-7 cells. These new compounds enhanced the
cytotoxicity of the established Top1 poison camptothecin by
an order of magnitude.
The currently known structure-to-property relationships
suggest that the UA enamine derivatives have some improved
biological properties in comparison with a native UA. Such UA
derivatives as enamines isolated from Usnea longissima inhibit
the growth of human hepatoma HepG2 cells and reveal high
antifungal and antibacterial activities.6
The main mechanism underlying the moderate cytotoxic
activity of UA is apoptosis via the intrinsic pathway.4 It is
known that UA has specific effects on tumors, which include
suppression of cell colony growth as well as inhibition of
angiogenesis.4 The decreased cytotoxicities of the UA enamine
derivatives recently reported by Bazin et al. indicate high
antitumor potential of the synthetic agents.7
The development of inhibitors of DNA repair pathways is
one of the main goals of medicinal chemistry. The elaboration
of drugs based on inhibition of DNA repair systems can provide
a new effective treatment for cardiovascular, oncological, and
neurodegenerative diseases. These pathologies are often
associated with DNA repair abnormalities.8−10
Tyrosyl-DNA phosphodiesterase I (TDP1) is associated with
the human neurodegenerative disease SCAN111 (spinocer-
ebellar ataxia with axonal neuropathy) and protects cancer cells
snic acid (UA) is the best known lichen secondary
Umetabolite. It is produced by Cladonia (Cladoniaceae),
Usnea (Usneaceae), Lecanora (Lecanoraceae), Ramalina
(Ramalinaceae), Evernia, Parmelia (Parmeliaceae), Alectoria
(Alectoriaceae), and other lichen genera.1 UA has been of
interest to chemists and pharmacologists owing to the broad
range of its biological activity, such as antitumor, antiviral,
antimicrobial, anti-inflammatory, and insecticidal effects.1 It is
one of the few commercially available lichen metabolites and
has been the most extensively investigated. In the 1990s, it was
reported that UA has hepatotoxicity observed following long-
term consumption of large UA doses.2 However, in spite of the
limitations imposed by UA toxicity, its biological properties
continue to be studied, and up-to-date research methods
provide intimate knowledge of biological mechanisms that
mediate its action as well as expand the scope of practical
applications.
In recent decades, structure−activity studies designed to
reduce the side effects of UA have been conducted. These
efforts have ultimately led to the discovery of lead compounds
with better pharmacological and toxicity profiles.3,4 The
synthesis of new UA derivatives5 and the investigation of
their biological activity contributes much to the understanding
of the biological action of UA itself and opens up fresh
opportunities for the pharmacological use of this metabolite.
Received: October 25, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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from certain types of chemotherapy.12 TDP1 is implicated in
the processing of different 3′-lesions,13 including stalled Top1−
DNA covalent complexes (Top1cc) processed by proteasomal
degradation,14 and Top1cc are stabilized by Top1 inhibitors,
such as camptothecin and some other anticancer drugs (for
review see ref 11). The inability to reseal Top1cc leads to the
formation of DNA single-strand breaks that can be turned into
more toxic double-strand breaks, thus resulting in cell death or
chromosomal translocations. Inhibiting the activity of TDP1
one can enhance the therapeutic effect of clinical Top1
inhibitors.15,16 This is confirmed by several studies: tdp1−/−
mice and human cell lines with SCAN1 mutation are
hypersensitive to camptothecin.13,17 Furthermore, camptothe-
cin causes less DNA damages in cells with high TDP1
expression.18−21 A positive correlation has been observed
between the percentage of NSCLC (non-small-cell lung
cancer) cells in tumor tissues and TDP1 activity.22
phore donor and a quenching 3′-BHQ1 (black hole quencher
1) moiety. The typical curves for BHQ1 removal catalyzed by
TDP1 in the presence of the UA enamines are shown in Figure
1.
A few TDP1 inhibitors have been described in the literature,
and they all demonstrate moderate activity with IC50 values
(concentration of a compound required to reduce the enzyme
activity by 50%) in the concentration range of 0.4 to 100
μM.13,23−30 Two classes of TDP1 inhibitors have also been
reported by our group: diazaadamantane derivatives (IC50
values from 15 to 68 μM)31 and more effective benzopenta-
thiepine derivatives (IC50 values from 0.2 to 6 μM).32 In this
paper we present efficient inhibitors of TDP1 with a novel
structural motif. Their biological properties have been studied,
and the most potent compound, 8, was shown to inhibit TDP1
Figure 1. Dependence of TDP1 residual activity as a function of agent
concentration. The concentration of TDP1 was 1.3 nM, and TDP1-
biosensor concentration was 50 nM. Red graph designates 8; blue
graph designates (+)5.
with an IC50 value of 0.16
0.04 μM, to possess low
cytotoxicity (CC50 value >50 μM), and to enhance the
cytotoxicity of the Top1 inhibitor camptothecin by 1 order of
magnitude.
The results for the enamines are presented in Table 1. IC50
values ranged from 0.16 to 2.2 μM. The compounds with the
best inhibitory properties, 8 and 7, contained bulky substituent
(a 3,5-di-tert-butyl-4-hydroxyphenyl ring) and had IC50 values
of 0.16 and 0.19 μM, respectively. The length of the linker
between the UA core and the bulky substituent had no
significant effect on the IC50 values. Compound 9, with a
hydroxyphenyl ring, was used to elucidate the role of tert-butyl
groups. The IC50 value for this compound was 0.79 μM. The
comparison of inhibitors with and without tert-butyl groups
demonstrated that introduction of this bulky substituent can
substantially increase inhibitory activity.
RESULTS AND DISCUSSION
■
Chemistry. Compounds 1−11 were synthesized according
to Scheme 1. The enamines 1−11 were prepared by interaction
of (+)- or (−)-usnic acid with the appropriate amines or
anilines33 (Scheme 1).
Scheme 1. Synthesis of Compounds
The introduction of a halogen substituent in the phenyl ring
increased inhibitory properties and led to IC50 values ranging
from 0.26 μM (p-bromophenyl derivative (+)5) to 1.8 μM (p-
chlorophenyl derivative (+)4). The bromophenyl group
containing compounds (−)5, (+)5, and 6 were the most
effective inhibitors among halogen derivatives. No substantial
influence of angular methyl group orientation was observed
(compare enantiomer pairs 1, 2, 3, and 5). The position of the
halogen atom on the phenyl ring also had no effect on IC50
values (pairs (−)2 and 3; (+)5 and 6).
The presence of an aromatic substituent was crucial for
enzyme inhibition since compounds 10 (acyclic substituent)
and 11 (morpholine substituent) did not inhibit TDP1 in the
investigated concentration range.
To validate the data obtained by the fluorescence assay, we
performed polyacrylamide gel electrophoretic separation of the
Tdp1 reaction products in the presence of compound (+)5
(Figure 2A). We used the oligonucleotide of the same sequence
radiolabeled at the 5′-end, containing the same quencher
BHQ1 at the 3′-end. In the absence of the inhibitor, the
enzyme removed the BHQ1 residue (lane 1), and the amount
A 1.1 mmol amount of the appropriate amine was added to 1
mmol of (+)- or (−)-usnic acid, and the mixture was dissolved
in 12 mL of ethanol. The reaction mixture was refluxed in a
water bath for 3 h and cooled, and then 10 mL of distilled water
was added. A light precipitate was formed, which was collected
on a filter, washed with water, and dried in air. The precipitate
was chromatographed on a silica gel column (fractions 60−200
μM, Merck) by chloroform with a methanol gradient from 0 to
5%.
The structures of the compounds were confirmed by NMR
spectroscopy and mass spectrometry.
Biology. We recently designed a new simple fluorophore-
quencher-coupled DNA-biosensor for real-time measurement
of TDP1 cleavage activity.32 The substrate is a 16-mer single-
stranded oligonucleotide containing both a 5′-FAM fluoro-
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Table 1. Influence of UA Enamines on Activity of Purified TDP1 and MCF-7 Viability in a Single Treatment and in a
Combination with Camptothecin
*
**
n/d, not determined. CC50 for pure camptothecin/CC50 in the presence of the UA enamines ratio. The values for the most effective sensitizers
are shown in boldface.
C
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Figure 2. BHQ1 excision from the 3′-end of [32P]-labeled oligonucleotide catalyzed by Tdp1. (A) Electrophoretic separation of the reaction
products. Arrows indicate positions of the starting substrate and the reaction product. (B) Typical dependence of the reaction rate on the inhibitor
concentration.
of the product decreased with increasing concentration of the
inhibitor (lanes 2−8). Figure 2B shows the typical dependence
of the reaction rate on the inhibitor concentration. The IC50
value for compound (+)5 was 4.6 μM.
cells. The substantial reduction of CC50 values in the presence
of the TDP1 inhibitors is presented in Figure 3 and Table 1.
As the usnic acid and its derivatives are known to possess
antitumor activity,4 the UA enamines were tested against the
human breast adenocarcinoma cell line MCF-7. The MCF-7
cell line was chosen because of the higher level of Tdp1 gene
expression compared to the average according to the BioGPS
database (http://biogps.org/#goto=welcome).
The cells were exposed to 0.1 to 100 μM of the compounds.
The 50% cytotoxic concentration (CC50) was defined as the
concentration required to reduce the cell number by 50%
compared to that for the untreated controls. The average CC50
values for the compounds shown in Table 1 were derived from
three independent experiments. All compounds demonstrated
mild cytotoxicity (CC50 values were not less than 10 μM).
Compound (−)5 was the most toxic for MCF-7 cells.
Compounds (+)1, 7, and 8 demonstrated an unspecific effect
on MCF-7 cells: cell survival was reduced compared with the
control but was independent of the inhibitor concentration.
Low cytotoxicity of UA enamines (CC50 > 10 μM) is crucial
for presupposed application in combination with clinically
established anticancer drugs. We expect that the use of TDP1
inhibitors would increase the sensitivity of tumor cells to
clinical Top1 inhibitors (sensitize tumor cells to the action of
Top1 inhibitors). This is expected to reduce the dosage of
clinical anticancer drugs and consequently reduce their toxic
effects, so the toxicity of TDP1 inhibitors should be minimized.
Camptothecin (CPT) is a natural quinoline alkaloid that
inhibits Top1 and possesses anticancer activity.16 Its bioavail-
able derivative irinotecan is a key chemotherapeutic drug for
metastatic colorectal cancer and other types of tumors.34
Another CPT derivative, topotecan, is used to treat ovarian
cancer when other treatments have failed and to treat certain
types of lung cancer (small-cell lung cancer).35 Since TDP1
removes DNA damage caused by Top1 inhibitors, TDP1 is
believed to be responsible for drug resistance of some
cancers.13 For instance, positive correlation between the
percentage of non-small-cell lung cancer tumor cells in
human tissues and TDP1 activity was observed.22 Thus, a
combination of Top1 and TDP1 inhibitors is expected to
improve the efficiency of treatment or allow dose reduction of
traditional drugs.
Figure 3. CPT concentration dependence of MCF-7 viability in the
presence of UA enamines. MTT assay data.
The presented data show that the most effective sensitizers of
MCF-7 cells to CPT (reducing CC50 values more than 10
times) were compounds 3, 6, 8, and 9.
There was no correlation among the level of inhibition of
TDP1, toxicity of compounds, and their ability to enhance the
effect of CPT (see Table 1). However, one of the promising
compounds, 8, that showed maximum inhibitory activity against
TDP1 was nontoxic and enhanced the effect of CPT by 1 order
of magnitude.
Since bioavailable derivatives of camptothecin are used for
the treatment a lung cancer, we examined the effect of UA
enamines against A-549 cells (adenocarcinomic human alveolar
basal epithelial cells). Four of the most effective sensitizers of
MCF-7 cells to CPT were chosen: 3, 6, 8, and 9. Cytotoxicity
of these compounds against A-549 cells and their influence on
CPT cytotoxic effect are shown in Table 2.
The UA enamines also sensitized A-549 cells to camptothe-
cin, though not as efficiently as MCF-7 cells. Compound 8
enhanced CPT cytotoxic effects 3.5 times (Figure 4). Most
likely, this difference can be explained by different levels of
Tdp1 gene expression in these cell lines. The level of protein
expression in MCF-7 cells is 10-fold higher than that in A-549
cells according to the data of Proteomics DB (https://www.
proteomicsdb.org). In addition, the ratio of protein to mRNA
of MCF-7 cells is significantly higher than in A-549 cells.
To test this hypothesis, we estimated the cytotoxic effect of
CPT in the presence of TDP1 inhibitors. We used a nontoxic
concentration of UA enamines (0.5 μM) and different
concentrations of CPT to estimate CC50 values for MCF-7
D
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enzyme−substrate complex, and the covalent phosphohistidine
intermediate (Figure 5A−C). Models A and B were designed to
Table 2. Influence of UA Enamines on A-549 Viability as a
Monopreparation or in Combination with Camptothecin
*
CC50 for pure camptothecin/CC50 in the presence of the UA
enamines ratio. The values for the most effective sensitizers are shown
in boldface.
Figure 5. Molecular models of the TDP1 structure. (A) The apo form.
Catalytic residues His263 and His493 are shown in green. (B) The
enzyme−substrate complex. The oligonucleotide is shown in red, 3′-
phosphotyrosine is shown in green, and the rest of the peptide is
shown in yellow. (C) The covalent intermediate. 3′-Phosphohistidine
is shown in green. (D) The ternary complex with an inhibitor, (+)5.
The inhibitor molecule is colored by atom type.
simulate interaction with inhibitors of the first step of the
reaction, while model C represents a target for intermediate-
stabilizing inhibitors of the second step. Enamines of usnic acid
were found to bind preferentially to the phosphohistidine
intermediate occupying a cavity adjacent to the active site of
TDP1 (Figure 5D). This cavity is formed by residues Tyr204,
Cys205, Asp230, Lys231, Leu255 Ala258, Phe259, and Thr261,
which are able to mediate hydrophobic and hydrogen-bonding
interactions with a low-molecular-weight ligand. We hypothe-
size that enamine inhibitors in a ternary complex with TDP1
affect the conformation of the Tyr204, Cys205, Phe259, and
Thr261 residues located at the boundary between the active site
and the inhibitor’s cavity, thus disrupting the reactive
orientation of the phosphohistidine intermediate and nucleo-
philic water molecule.
Conclusions. Our research has revealed a new class of UA
enamine derivatives as TDP1 inhibitors. Compounds 7 and 8,
containing 3,5-di-tert-butyl-4-hydroxyphenyl substituents, dem-
onstrated inhibitory activity in the high nanomolar concen-
tration range and to our knowledge were the most potent
TDP1 suppressors.
Figure 4. CPT concentration dependence of A-549 viability in the
presence of UA enamines. MTT assay data.
Molecular Modeling. In the proposed two-step catalytic
mechanism of TDP1, conserved residues His263 and His493
act as a nucleophile and general acid/base, respectively.36−38 In
the first step of the reaction, His263 attacks the 3′-
phosphotyrosyl bond of the substrate. The covalent 3′-
phosphohistidine intermediate is formed, and the leaving
group is protonated by His493. In the second step, the water
molecule activated by His493 acts as a nucleophile, and the 3′-
phosphohistidine intermediate is hydrolyzed to produce 3′-
phosphate DNA. TDP1’s catalytic cycle offers two potential
therapeutic strategies: (1) the inhibition of the first step to
prevent the phosphoryl transfer and (2) the inhibition of the
second step to prevent the hydrolysis of the DNA−protein
intermediate.11 The second approach could somehow be
similar to the stabilization of Top1−DNA covalent complexes
by camptothecins.15,39
All compounds possessed low toxicity against human MCF-7
and A-549 tumor cells, and some of the UA enamines were able
to enhance the cytotoxicity of camptothecin. The most
promising compound, 8, had maximum inhibitory activity
against TDP1, was nontoxic in the investigated concentration
We have modeled three forms of TDP1 enzyme that
represent targets for potential inhibitors: the apo form, the
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wells. IC50 values were determined using an 11-point concentration
response curve. The data were imported into the MARS Data Analysis
2.0 program (BMG LABTECH), and the slope during the linear phase
(here data from 0 to 7 min) was calculated.
range, and was able to improve the cytotoxic effect of
camptothecin by 10-fold.
According to the modeling results, UA enamines bind
preferentially to the covalent DNA−protein intermediate at the
second step of the reaction.
Gel-Based Enzyme Assay. The [32P]-label at the 5′-end of the
oligonucleotide was carried out using T4 polynucleotide kinase, and
the labeled oligonucleotide was purified by electrophoresis in a
denaturing polyacrylamide gel in the presence of 7 M urea. The
reaction mixture contained 50 nM DNA substrate, 5 nM Tdp1, 50
mM Tris-HCl, 50 mM NaCl, 7 mM β-mercaptoethanol, and the
inhibitor (+)5 in a concentration 0−60 μM. The reaction was
conducted at 26 °C for 20 min. The reaction products were separated
by electrophoresis on 20% denaturing polyacrylamide gel with 7 M
urea. A Typhoon FLA 9500 phosphorimager (GE Healthcare) was
used for gel scanning and imaging, and the data were analyzed with
QuantityOne 4.6.7 software. IC50 values were determined using
OriginPro 8.6.0 software.
EXPERIMENTAL SECTION
Chemistry. The analytical and spectral studies were conducted in
■
the Chemical Service Center for the collective use of SB RAS.
1
The H and 13C NMR spectra for solutions of the compounds in
CDCl3 were recorded on a Bruker AV-400 spectrometer (400.13 and
100.61 MHz, respectively). The residual signals of the solvent were
used as references (δH 2.48, δC 39.52). The mass spectra (70 eV) were
recorded on a DFS Thermo Scientific high-resolution mass
spectrometer. The melting points were measured using a Kofler
heating stage. The specific rotation was determined on a PolAAr 3005
and is provided in (deg × mL) × (g × dm)−1, where the concentration
of the solutions is shown in g × (100 × mL)−1. Merck silica gel (63−
200 μm) was used for the column chromatography. Thin-layer
chromatography was performed on Silufol plates (UV-254).
Cell Culture Assays. Tumor cells from human mammary
adenocarcinoma cell lines MCF-7 (∼2000 cells per well) were
incubated for 24 h at 37 °C in IMDM medium (5% CO2), and then
they were treated with the UA enamines. After 72 h of cell incubation
the relative amount of alive cells was determined using 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (standard
colorimetric MTT test),45 and the drug concentration that causes
50% cell growth inhibition (CC50) was determined.
The atom numbering in the compounds was provided for the
assignment of signals in the NMR spectra and is different from the
atom numbering in the nomenclature name. The target compounds
reported here have a purity of at least 97% (HPLC).
Molecular Modeling. Three TDP1 models were used in docking
studies: (A) “apo form”, (B) “enzyme−substrate complex”, and (C)
“covalent intermediate”. Model B was built on the basis of the 1nop
crystal structure with a substrate analogue46 and energy minimized
using a QM/MM implementation in Amber 12.47 The 3′-terminal
nucleotide with a covalently attached tyrosyl moiety and the side
chains of His263 (with protonated Nδ1-atom) and His493 (with
protonated both Nδ1 and Nε2 atoms) were treated quantum
mechanically at the RM1 level of theory.48 Model A was obtained
by removing the substrate molecule from model B. Model C was
constructed by applying a harmonic restraint on the distance between
3′-phosphate and His263 in model B to drive the covalent bond
formation. The PO4:P···His263:Nε2 distance was reduced by the
restrained QM/MM minimization, and then the unrestrained energy
minimization was carried out to obtain the relaxed intermediate
structure. Finally, the tyrosyl-containing peptide, the first reaction
product, was removed from the active site.
Compounds 10 and 11 were synthesized as described by Luzina et
al.40 Compounds (+)1, (−)1, (+)4, (−)4, (+)5, and (−)5 were
synthesized as described previously.41 Compounds 7 and 8 were
synthesized as described by Tazetdinova et al.33 Compounds (+)2,
(−)2, 3, and 6 were synthesized as described by Sokolov et al.42
Compound 9. Compound (+)12 (1 mmol) was treated with 4-
hydroxyphenethylamine (1.1 mmol). The mixture was dissolved in
EtOH (12 mL), refluxed on a water bath for 3 h, cooled, and treated
with distilled H2O (10 mL). The light precipitate was filtered off,
rinsed with H2O, and dried in air. Compound 9 was isolated by
column chromatography over silica gel with elution by a gradient of
MeOH in CH2Cl2 (from 0 to 10%).
( R , E ) - 6 - A c e t y l - 7 , 9 - d i h y d r o x y - 2 - ( 1 - ( 4 -
hydroxyphenethylamino)ethylidene)-8,9b-dimethyldibenzo-
[b,d]furan-1,3(2H,9bH)-dione, 9: yellow, amorphous powder. Yield:
78%; mp 116 °C; [α]28D +254 (c 0.22, CHCl3); 1H NMR (CDCl3, δ, J
Hz) 1.70 (3H, s, H-15), 2.11 (3H, s, H-10), 2.57 (3H, s, H-12), 2.67
(3H, s, H-14), 2.96 (2H, t, J = 6.7, H-17), 3.73 (2H, dt, J = 6.7 and 5.5,
H-16), 5.81 (1H, s, H-4), 6.81 (2H, d, J = 8.3, H-3′ and H-5′), 7.11
(2H, d, J = 8.3, H-2′ and H-6′), 11.89 (1H, s, OH-9), 13.22 (1H, bs,
NH), 13.59 (1H, s, OH-7); 13C NMR (CDCl3, δ) 7.27 (C-10), 18.15
(C-12), 31.04 (C-14), 31.76 (C-15), 34.11 (C-17), 45.54 (C-16),
57.01 (C-9b), 101.15 (C-6), 102.03 (C-4), 102.60 (C-2), 104.81 (C-
9a), 107.84 (C-8), 115.70 (C-3′ and C-5′), 129.68 (C-1′), 129.68 (C-
2′ and C-6′), 155.11 (C-4′), 155.64 (C-5a), 158.00 (C-9), 163.25 (C-
7), 174.80 (C-11), 174.7 (C-4a), 189.9 (C-3), 198.14 (C-1), 200.53
(C-13); MS m/z found 463.1620 [M]+ C26H25O7N, calcd M =
463.1626.
The structures of TDP1 inhibitors were optimized by ACD/
ChemSketch.49 Molecular docking of compounds into models A−C
was performed with the Lead Finder 1.1.16 software.50 In docking
experiments, an energy grid box with edges of 35 Å was centered on
the Nε2 atom of His493. VMD 1.9.251 was used to visualize the
obtained models of TDP1-inhibitor complexes.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00979.
Biology. The recombinant TDP1 was purified to homogeneity by
chromatography on Ni-chelating resin and phosphocellulose P11 as
described43,44 using plasmid pET 16B-Tdp1 kindly provided by Dr. K.
W. Caldecott (University of Sussex, United Kingdom).
1
NMR H, 13C, and mass spectra of compound 9 (PDF)
TDP1-biosensor 5′-(5,6 FAM-aac gtc agg gtc ttc c-BHQ1)-3′ was
synthesized in the Laboratory of Medicinal Chemistry, Institute of
Chemical Biology and Fundamental Medicine, Novosibirsk, Russia.
Real-Time Detection of TDP1 Activity. The TDP1 activity
measurements were carried out as described.32 Briefly, TDP1-
biosensor with a final concentration of 50 nM was incubated in a
volume of 200 μL containing TDP1 buffer (50 mM Tris-HCl pH8,0,
50 mM NaCl, 7 mM β-mercaptoethanol) supplemented with purified
1.3 nM TDP1. The reactions were incubated at a constant temperature
of 26 °C in a POLARstar OPTIMA fluorimeter, BMG LABTECH,
GmbH, to measure fluorescence every 1 min (Ex485/Em520 nm). TDP1
inhibition was calculated by comparing the rate of increase in
fluorescence in the presence of compound to that of DMSO control
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lavrik@niboch.nsc.ru. Tel: +7(383)3635196.
Author Contributions
#A. Zakharenko and O. Luzina contributed equally to this
research.
Notes
The authors declare no competing financial interest.
F
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ACKNOWLEDGMENTS
■
This work was supported by the Ministry of Education and
Science of Russian Federation, State Agreement RFME-
FI60414X0018.
(29) Davies, D. R.; Interthal, H.; Champoux, J. J.; Hol, W. G. J. J. Mol.
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DOI: 10.1021/acs.jnatprod.6b00979
J. Nat. Prod. XXXX, XXX, XXX−XXX