The first berberine-based inhibitors of tyrosyl-dna phosphodiesterase 1 (Tdp1), an important dna repair enzyme

Article abstract of DOI:10.3390/ijms21197162

A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure–activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Full text of DOI:10.3390/ijms21197162

International Journal of
Molecular Sciences
Article
The First Berberine-Based Inhibitors of Tyrosyl-DNA
Phosphodiesterase 1 (Tdp1), an Important DNA
Repair Enzyme
Elizaveta D. Gladkova 1,2, Ivan V. Nechepurenko , Roman A. Bredikhin , Arina A. Chepanova ,
1
1
3
3
1
3
3
Alexandra L. Zakharenko , Olga A. Luzina
, Ekaterina S. Ilina , Nadezhda S. Dyrkheeva ,
3
2,3
4
Evgeniya M. Mamontova , Rashid O. Anarbaev , Jóhannes Reynisson
,
Konstantin P. Volcho 1 * , Nariman F. Salakhutdinov
,2,
1,2
and Olga I. Lavrik
2,3
1
N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia; liza95@nioch.nsc.ru (E.D.G.);
niv@nioch.nsc.ru (I.V.N.); bred@nioch.nsc.ru (R.A.B.); luzina@nioch.nsc.ru (O.A.L.);
anvar@nioch.nsc.ru (N.F.S.)
Novosibirsk State University, Pirogova str. 1, Novosibirsk 630090, Russia; anarbaev@niboch.nsc.ru (R.O.A.);
lavrik@niboch.nsc.ru (O.I.L.)
Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, 8, Akademika Lavrentieva Ave., Novosibirsk 630090, Russia;
arinachepanova@mail.ru (A.A.C.); sashaz@niboch.nsc.ru (A.L.Z.); katya.plekhanova@gmail.com (E.S.I.);
elpida80@mail.ru (N.S.D.); evgeniya.mm.94@gmail.com (E.M.M.)
School of Pharmacy and Bioengineering, Keele University, Hornbeam Building, Staffordshire ST5 5BG, UK;
j.reynisson@keele.ac.uk
2
3
4
*
Correspondence: volcho@nioch.nsc.ru
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 31 August 2020; Accepted: 26 September 2020; Published: 28 September 2020
Abstract: A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and
tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine
derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine
based Tdp1 inhibitor. A structure–activity relationship analysis revealed the importance of bromine
substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that
the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to
increased potency, while most of the derivatives containing an alkyl fragment at the same position
were not active. According to the molecular modeling, the bromine atom in position 12 forms a
hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically
important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives
1
1g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be
used for further development to sensitize the action of clinically relevant Topo1 inhibitors.
Keywords: berberine; tetrahydroberberine; Tdp1 inhibitor; cancer; molecular modeling; DNA repair
enzyme; SAR
1
. Introduction
A promising strategy to enhance the efficacy of anticancer therapy is the inhibition of various DNA
repair enzymes, which counteract the effect of many anticancer drugs [1,2]. This is particularly important
where resistance to chemotherapy is observed. An interesting example is the poly (ADP-ribose)
polymerase (PARP), which inhibitors were studied both in combination with chemotherapeutic agents
and as individual drugs. Now olaparib, rucaparib and niraparib are in clinical use for the treatment
Int. J. Mol. Sci. 2020, 21, 7162; doi:10.3390/ijms21197162
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2020, 21, 7162
2 of 16
of ovarian cancers [
attracted considerable interest in the last few years mainly due to its ability to repair DNA lesions caused
topoisomerase 1 (Top1) poisons, a well-established class of anticancer drugs [ ]. The anticancer activity
of Top1 poisons, camptothecin and its clinically important derivatives, topotecan and irinotecan [ ],
3]. Another DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (Tdp1) has
4
5,6
is based on their ability to bind to the covalent intermediate complex Top1/DNA and prevent the
restoring of DNA integrity. This leads to the stabilization of the covalent bond between catalytic
0
tyrosine (Y723) of Top1 and the 3 - end of DNA (Figure 1). The Tdp1 mechanism of action is the
phosphotyrosyl bond hydrolysis [7], resulting in the resumption of DNA replication and cell division.
Figure 1. DNA single-strand cleavage by nucleophilic attack of Tyr723 of Top1, and covalent cleavage
complex formation.
A few classes of Tdp1 inhibitors are known such as furamidines (compound 1, Figure 2),
tetracyclines (compound
types have been found to inhibit Tdp1, including derivatives of bile acids
usnic acid 5a 12 13] and 14], monoterpenoid derivatives 15 18] and oxinitidine
inhibitory activity in the micro- or submicromolar range. Importantly, the hydrazinothiazole derivative
of usnic acid 5a 13 20] and monoterpene-substituted 4-arylcoumarin 7a 21] significantly increased
topotecan efficacy in vivo.
The aim of this study was to establish the potency of a novel structural class of natural
products, the derivatives of berberine (Scheme 1), which like their usnic acid counterparts
2
), aminoglycosides (compound
3) [
8
,
9
]. Also, natural products of various
10 11], of lichen metabolite
19] with
4
[
,
,
b
[
,
6
[
7
[
–
8 [
[
,
[
9
are phenolic compounds. It is known that the isoquinoline plant alkaloid berberines have many
beneficial physiological effects, e.g., they are hypocholesterolemic, antibacterial, hypoglycemic agents,
antioxidants and, finally, they suppress tumor growth [22
of berberines and tetrahydroberberines have been reported as promising hypocholesterolemic
agents [27 28]. Although berberine derivatives never used as Tdp1 inhibitors, a preliminary molecular
modeling study indicated that berberines with 9-sulfonate group would bind to Tdp1. In the present
study, 9-sulfonate-berberine and tetrahydroberberine derivatives 10 12 with aliphatic and aromatic
–26]. Interestingly, the sulfonate derivatives
,
–
substitutes were synthesized and their potency against Tdp1 was tested. To the best of our knowledge,
this has not been done previously. Using the HeLa cervical cancer cell line, derivatives 11g and 12g
were found to be nontoxic and sensitized the cancer cells to topotecan.

Int. J. Mol. Sci. 2020, 21, 7162
3 of 16
Figure 2. Examples of established Tdp1 inhibitors 1–8.
2
. Results and Discussion
2
.1. Chemistry
Sulfonates 10
purpose, berberine
–
9
12 were synthesized in accordance with previously reported methods [27]. For this
was selectively demethylated at 190 C under vacuum as previously described [29].
◦
After treatment with HBr, berberrubine hydrobromide 13 was isolated in 89% yield (Scheme 1).
Compound 13 was reduced with sodium borohydride in methanol according to a procedure described
previously [30] yielding tetrahydroberberrubine 14. The bromination of compound 14 with a bromine
in dioxane solution afforded 12-bromotetrahydroberberrubine 15 in 52% yield. The reaction of
tetrahydroderivatives 14 and 15 with polyfluoroaryl and alkyl sulfonylchlorides as well as with
tosylchloride in dichloromethane in the presence of triethylamine produced tetrahydroberberrubine
9-O-sulfonates 11a
–h (44–84% yields) and 12-bromotetrahydroberberrubine 9-O-sulfonates 12a–h
(
49–93% yields). New berberine type 10 derivatives were synthesized by the reaction of berberrubine
hydrobromide 13 with different alkyl sulfochlorides. The reactions were carried out in dichloromethane
in the presence of triethylamine for 5 h at room temperature. Sulfonates 10 were isolated by precipitation
from the reaction mixtures.

Int. J. Mol. Sci. 2020, 21, 7162
4 of 16
O
O
O
O
O
O
O
O
1
) 180°C,
Br
2
0-30 mm Hg
NaBH4
MeOH
0°C
2
) HBr, EtOH
Br2
dioxane
N
N
N
N
O
O
O
O
Cl
Br
O
OH
OH
OH
9
13
14
15
RSO2Cl
NEt3
CH2Cl2
RSO2Cl
NEt3
CH2Cl2
RSO2Cl
NEt3
CH2Cl2
O
O
O
O
O
O
Br
N
N
N
O
O
O
O
O
Br
O
O
O
S
O
O
S
S
R
O
R
R
O
11
12
10
10a: R = CH3;
10b: R = C2H5;
10c: R = C3H7;
10d: R = C4H9
11a: R = CH3;
12a: R = CH3;
12b: R = C2H5;
12c: R = C3H7;
12d: R = C4H9;
12e: R = p-C6H5CH3;
12f: R = C6F5;
11b: R = C2H5;
11c: R = C3H7;
11d: R = C4H9;
1
1e: R = p-C6H5CH3;
1
1
1
1f: R = C6F5;
1g: R = p-C6F4H;
1h: R = p-C6F4CF3
12g: R = p-C6F4H;
12h: R = p-C6F4CF3
Scheme 1. The synthetic pathways to sulfonates 10, 11 and 12.
1
13
The structures of the new compounds were confirmed by H-NMR, C-NMR, IR and HRMS
methods; the results are shown in the experimental section and the supplementary information.
2
.2. Effects of the Berberine Sulfonates on Tdp1 Activity
An oligonucleotide real-time biosensor was used based on the ability of Tdp1 to remove fluorophore
0
quenchers from the 3 -end of DNA, as previously described [31]. The hexadecameric oligonucleotide
0
carried 5(6)-carboxyfluorescein (FAM) at the 5 -end and the fluorophore quencher BHQ1 (Black Hole
Quencher-1) at the 3 -end. Tdp1 inhibitors prevent removal of fluorophore quenchers, thus reducing
0
fluorescence intensity. The results of the Tdp1 assay for derivatives 10–12 and cytotoxic effects are
shown in Tables 1 and 2.
Table 1. Tdp1 inhibiting activity (IC50—half maximal inhibitory concentration) of sulfonate berberine
derivatives with aliphatic substituents and their cytotoxicity (CC —half maximal cytotoxic
5
0
concentration) in HeLa cells. Furamidine was used as a positive control (IC50 = 1.2 ± 0.3 µM).
Code,
Structure
1
1a–d
1
10a–d
1
12a–d
R
IC , µM
CC50, µM
ND *
ND
IC50, µM
>15
CC50, µM
ND
IC50, µM
>15
CC50, µM
ND
50
a; Me
b; Et
c; Pr
d; Bu
>15
>15
>15
>15
>15
ND
>15
ND
ND
>15
ND
2.9 ± 1.3
4 ± 1.0
>100
>100
ND
>15
ND
*
ND—not determined.

Int. J. Mol. Sci. 2020, 21, 7162
5 of 16
Table 2. Tdp 1 inhibiting activity (IC ) of sulfonate berberine derivatives with polyfluoroaromatic
50
substituents and their cytotoxicity (CC ) in HeLa cells. Furamidine was used as a positive control
5
0
(IC50 = 1.2 ± 0.3 µM).
Code, structure
1
1
2e–h
1
1e–h
R
IC , µM
CC , µM
IC , µM
50
CC , µM
5
0
50
50
>15
ND
>15
ND
e;
1
.0 ± 0.20
11 ± 2.0
0.53 ± 0.01
9.9 ± 4.5
f;
1
.05 ± 0.05
>100
1.3 ± 0.30
1.4 ± 0.30
95 ± 5.0
2.2 ± 1.5
g;
0
.9 ± 0.20
2.6 ± 0.1
h;
It is clear from the data in Table 1 that both alkyl sulfonates of berberine and tetrahydroberberine
are not very active with the exception of the tetrahydroberberine derivatives containing both
sufficiently long alkyl substituent and bromine in a para-position to the sulfonate substituent,
1
2c and 12d. The favorable substitution pattern of these derivatives is confirmed by the results
of the tetrahydroberberine sulfonates with aromatic and polyfluoroaromatic substituents as shown
in Table 2. Both sulfonates with para-toluenesulfonyl substituent (11e and 12e) are inactive, but all
three fluorinated sulfonates (11f, 11g, 11h) show good inhibitory activity with IC50 values of ~1 µM.
Bromine substitution is not important for 12e–h activity as comparison with their non-brominated
analogues 11e–h shows.
Based on the data shown in Tables 1 and 2 it can be stated that some berberine derivatives inhibit
Tdp1. It was found that the structure of the substituent in the sulfonate affects the inhibitory activity.
Polyfluorinated arylsulfonates 11f–h and 12f–h exhibited inhibitory activity in the low micromolar
range, while their non-fluorinated analogs (11e, 12e) were inactive at these concentrations. In the
series of alkylsulfonates, inhibitory activity was found only for compounds with a sufficiently long
alkyl substituent (propyl-, butyl-) in the sulfonate group and in the presence of bromine substituent
at position 12 (compounds 12c,d). To explain the observed effects, a molecular modeling study was
carried out.

Int. J. Mol. Sci. 2020, 21, 7162
6 of 16
2
.3. Molecular Modeling of the Berberine Derivatives
Twenty-three berberine derivatives were docked into the binding site of Tdp1 (PDB ID: 6DIE,
resolution 1.78 Å) [32] with three water molecules (HOH814, 821 and 1078). It is established that
keeping these crystalline water molecules improves the prediction quality of the docking scaffold
(for further information see the Methodology section) [13]. The binding predictions of the scoring
functions used are given in Table S1, all the ligands show reasonable scores.
Compound 12f is the ligand with the best IC50 value and according to the docking; 12f fits neatly
in the catalytic region as shown in Figure 3A. This region contains the catalytic histidine 263 and 493
amino acid residues thus the ligand blocs any activity of the enzyme. Indeed, 12f forms a weak H-bond
with the His493 imidazole site group via the bromine substituent as shown in Figure 3B as well as with
Asn283’s amide side chain. Finally, the amide group of Asn516 forms an H-bond with one of the oxygen
atoms in the 1.3-benzodioxole moiety of the ligand. It is worth mentioning that the methoxy group
on 12f can potentially form a weak H-bond with the thiol on Cys205, if the flexibility of the protein
would be accounted for, and the same methoxy group has lipophilic contacts with Ile285 aliphatic side
chain, stabilizing the binding mode. Interestingly, the modeling of the 11f ligand, which does not
contain a bromine group, but also with a good IC50 value, did not give consistent results across the
scoring functions used, i.e., different conformations were predicted. This strongly indicates that the
bromine group with its weak H-bonds to Asn283 and His493 is essential for anchoring the ligand in
the catalytic site.
Figure 3. The docked configuration of 12f in the binding site of Tdp1 as predicted by the ChemPLP
scoring function. (
positive charge on the surface; red depicts hydrophobic region with a partial negative charge and
grey shows neutral areas. The ligand occupies the catalytic pocket blocking access to it. ( ) H-bonds
are shown as green lines between 12f and the amino acids Asn283, His493 and Asn516 side chains.
A potential lone pair- stacking interaction is shown as a blue line between the carboxylic backbone
group in Gly458 and the centroid (green ball) of the fluorinated phenyl group (3.5 Å).
A) The protein surface is rendered; blue depicts a hydrophilic region with a partial
B
π
Interestingly, derivatives 11e and 12e are essentially inactive (IC50 >15
analogues of the 11f 12f pair with hydrogens on the phenyl ring as well as a para methyl substitution
instead of fluorine groups. According to the modeling, the 11e/12e pair has a different binding mode
from 11f 12f with the bromine moiety pointing into the aqueous phase for the former pair as shown in
µM); they are structural
/
/
Figure S1 in the Supplementary Information. This indicates the importance of the fluorine substitution
on the phenyl rings; the modeling suggests that the phenyl ring is leaning against the carboxyl moiety in

Int. J. Mol. Sci. 2020, 21, 7162
7 of 16
the Gly458 backbone, which can form an interaction between the electron deficient fluoride substituted
ring and the lone pairs of the oxygen atom in the carboxylic group as shown in Figure 3B.
To investigate the binding stability of the ligands molecular dynamic (MD) runs were conducted
using the docked conformations of 11f, 12f and 12e for 10 ps at 1000 K. In general, the ligands are
stable within the binding pocket and not ejected; the most stable intramolecular bond being between
the fluorinated phenyl ring and the Gly458 carboxyl group for 12f. In contrast, the phenyl group in 12e
is very mobile. The other H-bonding interactions predicted are often broken to be reestablished during
the MD run.
2
.4. Cytotoxicity
Top1 poisons are used as anticancer drugs for the treatment for various oncological diseases [33
–
35].
Since Tdp1 is involved in the removal of DNA damage caused by Top1 poisons, the activity of Tdp1 can
lead to the development of drug resistance [36]. Thus, it is believed that Tdp1 inhibition can enhance
the efficacy of Top1 poisons [37]. Tdp1 inhibitors should have the lowest possible intrinsic toxicity
to minimize potential side effects. Therefore, we studied the intrinsic cytotoxicity of the compounds
against HeLa cells (cervical carcinoma). EZ4U cell proliferation and cytotoxicity assay results are
shown in Figure 4 for the ligands with Tdp1 inhibitory activity.
Figure 4. The berberine derivatives’ cytotoxicity according to the EZ4U test. Error bars show
standard deviations.
The results show that 9-O-sulfonates of 12-bromotetrahydoberberine with aliphatic substituents
1
9
2c and 12d, (Table 1 and Figure 4, black and red traces) are non-toxic up to 100
-O-sulfonates strongly depends on the structure of the polyfluorinated fragment. Compounds with a
CF -group in the para-position were the most toxic; CC values of 2.6 M and 2.2 M for 11h and 12h,
respectively (Table 2 and Figure 4, green and brown traces). The replacement of the trifluoromethyl
group with a fluorine atom in the para-position reduced toxicity with CC50 values of 10 M for 11f and
2f (Table 2 and Figure 4, blue and magenta traces, respectively). The compounds with a hydrogen
atom in the para-position were non-toxic (11g, Table 2 and Figure 4, violet trace) or moderately toxic
12g, Table 2 and Figure 4, orange trace).
µM. The toxicity of
µ
µ
3
50
µ
1
(
2
.5. Sensitizing Effects
The sensitizing effect of the berberine inhibitors on topotecan’s cytotoxic potential was investigated.
In order to determine the optimal concentration for the inhibitors to provide the maximum sensitizing
effect, but remaining non-toxic, their concentrations were varied with topotecan concentration of 2 M,
its CC50 for HeLa cells. Topotecan significantly increased the cytotoxicity of compounds 11g 12g
µ
,
,

Int. J. Mol. Sci. 2020, 21, 7162
8 of 16
and 11f, the reliability was confirmed by the Mann–Whitney U-test, p = 0.05 and the results are shown
in Figure 5. The original data are given in Table S2.
Figure 5. The influence of 2
bars denote cell viability in the presence of a Tdp1 inhibitor. The hatched histogram bars indicate cell
viability in the presence of a combination of a Tdp1 inhibitor with 2 M of topotecan. The concentration
of the Tdp1 inhibitor in µM is given under each pair of bars. Error bars show standard deviations.
µM topotecan on Tdp1 inhibitors’ cytotoxicity. The unshaded histogram
µ
1
1g is non-toxic in the concentration range used; 100% of living cells with a 100
concentration. In the presence of topotecan, a significant decrease in cell survival is observed (~30%) at
all concentrations. Compound 12g has a low toxicity potential of 95 M (CC ). Topotecan weakly,
but significantly reduces this value to 62 M. Compound 11f is inherently toxic, and 90% of the cells die
at 20 M. At lower concentrations, the effect of topotecan is significant. CC50 value for 11f decreases
three fold, from 11 to 3.7 M, in the presence of topotecan. For other ligands (11h 12c ), the effect
µM maximum
µ
5
0
µ
µ
µ
,
, d, f, h
of topotecan was negligible or unreliable.
Non-toxic concentrations of the berberine derivatives (5
µM) were then tested at different
concentrations of topotecan. The most toxic compound 11f caused 20% cell death at this concentration;
the rest of the compounds were not toxic. In general, our Tdp1 inhibitors doubled the cytotoxic
potential of topotecan as can be seen in Figure 6 and Table 3.
Table 3. The influence of the Tdp1 inhibitors at 5 µM on the cytotoxic potential of topotecan (Tpc).
Compounds
Tpc
CC50, µM–5 µM Tdp1 inhibitor
CC , µM–20 µM Tdp1 inhibitor
50
6.8 ± 1.1
Tpc + 11g
Tpc + 12g
Tpc + 11f
3.5 ± 0.6
2.9 ± 0.4
1.7 ± 0.3
2.3 ± 0.5
3.3 ± 1.1
not determined

Int. J. Mol. Sci. 2020, 21, 7162
9 of 16
1
1
20
00
Tpc + DMSO
Tpc + 11g
Tpc + 12g
Tpc + 11f
8
6
4
2
0
0
0
0
0
0
2
4
6
8
10
Topotecan, μM
Figure 6. The influence of the Tdp1 inhibitors at 5 µM on topotecan cytotoxicity. Error bars show
standard deviations.
For comparison, the concentration of the inhibitors was increased to 20
µM. Compound 11f was
not used due to its high toxicity. Again, compounds 12g and 11g had a significant effect, p < 0.05
,
confirmed by the Mann–Whitney U-test (Figure 7 and Table 3). It is interesting to note that the
sensitizing effect of Tdp1 inhibitors was practically the same at 5 and 20 µM.
Figure 7. Influence of Tdp1 inhibitors at 20
µM on topotecan cytotoxicity. Error bars show
standard deviations.
Derivatives 11g and 12g can be considered to be the lead compounds since they are, unlike 11f
non-toxic (11g) or moderately toxic (12g) and have a pronounced sensitizing effect on topotecan.
,
2
.6. Chemical Space
The calculated molecular descriptors MW (molecular weight), log P (water-octanol partition
coefficient), HD (hydrogen bond donors), HA (hydrogen bond acceptors), PSA (polar surface area) and
−1
RB (rotatable bonds)) are given in Table S3. The MW of the ligands lies between 325.4 and 684.4 g mol
and falls into drug-like and Known Drug Space (KDS) regions. Log P spans from 1.6 to 5.4, i.e., over the

Int. J. Mol. Sci. 2020, 21, 7162
10 of 16
three defined volumes in chemical space, with most in lead-like chemical space and only one ligand
2h in KDS. The HD, HA, RB and PSA values are within the lead- and drug-like definitions (for the
1
definition of lead-like, drug-like and Known Drug Space regions see ref. [38] and Table S4).
The Known Drug Indexes (KDIs) for the ligands were calculated to gauge the balance of the
molecular descriptors (MW, log P, HD, HA, PSA and RB). This method is based on the analysis of drugs
in clinical use, i.e., the statistical distribution of each descriptor is fitted to a Gaussian function and
normalized to 1 resulting in a weighted index. Both the summation of the indexes (KDI —Equation (1))
2a
and multiplication (KDI —Equation (2)) methods were used [39]. The numerical results are given in
2b
Table S3 in the supplementary information.
KDI2a = IMW + Ilog P + IHD+ IHA + IRB + IPSA
KDI2b = IMW × Ilog P × IHD× IHA × IRB × IPSA
(1)
(2)
The KDI2a values range from 4.35 to 5.70 with a theoretical maximum of 6 and the average of
.08 for known drugs. KDI2b range from 0.06 to 0.73, with a theoretical maximum of 1 and with
4
KDS average of 0.18. The berberine ligands can be considered reasonably well balanced in terms
of their molecular descriptors and therefore biocompatible. The most active compound 12f, has a
KDI2a value of 4.58 and KDI2b of 0.12; the relatively low KDI2b value can be explained by its high
MW, which is compensated by the favorable values for the other descriptors resulting in a good KDI2a
value. The KDI2b index is sensitive to any outliers since multiplication of small numbers leads to
small numbers.
3
. Materials and Methods
3
.1. Chemistry
Berberine chloride was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan).
Methanesulfochloride and ethanesulfochloride were purchased from Acros Organics (Belgium),
-propanesulfochloride and 1-buthanesulfochloride were purchased from Alfa Aesar (Karlsruhe,
1
Germany). 48% aqueous HBr solution was purchased from Acros Organics (the Netherlands).
All solvents used in the reactions were purified and dried. Bromine, sodium borohydride and
triethylamine were purchased from Sigma-Aldrich. Column chromatography was carried out on
neutral alumina LL40/250. All reactions were monitored by TLC analysis using Merck Aluminium
oxide 60 F2 plastic sheets (Darmstadt, Germany), eluent CH Cl -MeOH.
54
2
2
1
13
The H and C NMR spectra were recorded on a Bruker AM-400 spectrometer (400.13 and
00.61 MHz) for 5–10% solutions of the compounds in CDCl or DMSO-d using the signal of the
1
3
6
solvent CDCl as the standard (δ 7.24 for 1H and δ 76.90 for 13C). The IR spectra were measured
3
on a Vector 22 FTIR spectrometer in KBr pellets. High-resolution electrospray ionization (HRESI)
mass spectra were carried out using a time-of-flight high-resolution mass spectrometer micrOTOF-Q
(Bruker Daltonics, Germany) with an Agilent 1200 liquid chromatograph (Agilent Technologies,
USA/Germany). Positive ion scanning in the range m/z = 100–3000. Drying gas (nitrogen) flow rate of
4
L/min; temperature—190 °C; sprayer pressure—1.0 bar.
The spectroscopic and analytical measurements were carried out at the Multi-access Chemical
Service Center of the Siberian Branch of the Russian Academy of Sciences.
3
.1.1. General Synthesis of Compounds 11a–g and 12a–g
Desired compounds 11a–d and 12a–g were synthesized according to the general procedure
, f, g
described previously [27], compound 11e was also synthesized according to the procedure described
previously [30].

Int. J. Mol. Sci. 2020, 21, 7162
11 of 16
3
.1.2. General Procedure for the Synthesis of Sulfonates 10a–d
.5 mmol of triethylamine was added to the suspension of 1 mmol of berberrubine hydrobromide
2
in 25 mL of methylene chloride. 1.5 mmol of sulfochloride in 3 mL of methylene chloride was added
under stirring at room temperature. After 5 hours the precipitate was filtered, washed with methylene
chloride and the pure product 10a–d was isolated.
3
.1.3. 10-Methoxy-9-methanesulfonyloxy-2,3-methylenedioxyprotoberberine chloride (10a)
Yield: 7%
Spectra NMR H (400 MHz, DMSO-d ,
1
δ, ppm, J/Hz): 3.22 (2H, t, J = 6.07 Hz, H-5), 3.73 (3H, s,
6
SCH ), 4.12 (3H, s, OCH ), 5.00 (2H, t, J = 5.72 Hz, H-6), 6.19 (2H, s, OCH O), 7.11 (1H, s, H-4), 7.82
3
3
2
(
1H, s, H-1), 8.27 (1H, d, J = 9.06 Hz, H-12), 8.34 (1H, d, J = 9.06 Hz, H-11), 9.08 (1H, s, H-13), 9.72 (1H, s,
H-8). Spectra NMR 13C (100 MHz, DMSO-d6,
δ
, ppm): 26.18 (C-5), 40.02 (SO CH ), 55.70 (C-6), 57.44
2
3
(
OCH ), 102.23 (OCH O), 105.60 (C-1), 108.49 (C-4), 120.20 (C-13b), 120.86 (C-13), 121.79 (C-8a), 126.47
3 2
(
C-12), 128.03 (C-11), 131.05 (C-4a), 131.44 (C-12a), 133.31 (C-13a), 138.59 (C-9), 144.04 (C-8), 147.77
−1
(C-2), 150.17 (C-3), 151.89 (C-10). Spectra IR (cm ): 804.21, 925.71, 1033.71, 1097.35, 1224.63, 1263.20,
+
+
1
365.42, 1506.20, 1610.34, 1623.84, 3039.40, 3320.97, 3635.33. MS (ESI): m/z (M ) calcd for C H NO S ,
20
18
6
4
00.085 found: 400.081.
3
.1.4. 9-Ethanesulfonyloxy-10-methoxy-2,3-methylenedioxyprotoberberine chloride (10b)
Yield: 25%
Spectra NMR H (400 MHz, DMSO-d ,
1
δ
, ppm, J/Hz): 1.52 (3H, t, J = 7.32 Hz, CH CH ), 3.22 (2H,
2 3
6
t, J = 6.05 Hz, H-5), 3.89 (2H, q, J = 3.50 Hz, J = 10.96 Hz, CH CH ), 4.12 (3H, s, OCH ), 5.01 (2H,
1
2
2
3
3
t, J = 5.91 Hz, H-6), 6.18 (2H, s, OCH O), 7.11 (1H, s, H-4), 7.82 (1H, s, H–1), 8.27 (1H, d, J = 9.48 Hz
,
2
13
H-12), 8.34 (1H, d, J = 9.48 Hz, H-11), 9.10 (1H, s, H-13), 9.64 (1H, s, H–8). Spectra NMR C (100 MHz,
DMSO-d6, , ppm): 8.18 (CH CH ), 26.19 (C-5), 47.00 (SCH ), 55.83 (C–6), 57.44 (OCH ), 102.20
δ
2
3
2
3
(
OCH O), 105.61 (C-1), 108.45 (C–4), 120.18 (C-13b), 120.90 (C-13), 121.98 (C-8a), 126.42 (C-12), 128.02
2
(
1
1
C-11), 131.02 (C-4a), 131.27 (C-12a), 133.36 (C-13a), 138.63 (C-9), 143.91 (C-8), 147.75 (C-2), 150.17 (C-3),
−1
51.71 (C-10). Spectra IR (cm ): 806.14, 923.78, 1033.71, 1097.35, 1164.85, 1222.70, 1278.2063, 1363.49,
+
+
506.20, 1608.42, 1621.92, 3023.98, 3423.19, 3629.54. MS (ESI): m/z (M ) calcd for C H NO S , 414.101
21 20 6
found: 414.096.
3
.1.5. 10-Methoxy-2,3-methylenedioxy-9-((propane-1-sulfonyl)oxy)protoberberine chloride (10c)
Yield: 34%
Spectra NMR H (400 MHz, DMSO-d ,
1
δ
, ppm, J/Hz): 1.11 (3H, t, J = 7.45 Hz, CH CH ), 1.94–2.03
2 3
6
(
2H, m, CH CH ), 3.22 (2H, t, J = 5.72 Hz, H-5), 3.87 (2H, t, J = 7.58 Hz, SCH CH ), 4.12 (3H, s, OCH ),
2 3 2 2 3
5
.01 (2H, t, J = 6.02 Hz, H-6), 6.19 (2H, s, OCH O), 7.11 (1H, s, H-4), 7.82 (1H, s, H-1), 8.27 (1H, d,
2
J = 9.33 Hz, H-12), 8.34 (1H, d, J = 9.33 Hz, H-11), 9.10 (1H, s, H-13), 9.64 (1H, s, H-8). Spectra NMR
1
3C (100 MHz, DMSO-d6,
δ, ppm): 12.49 (CH CH ), 17.17 (CH CH CH ), 26.16 (C-5), 53.27 (SCH ),
2 3 2 2 3 2
5
5.80 (C-6), 57.46 (OCH ), 102.18 (OCH O), 105.60 (C-1), 108.42 (C-4), 120.14 (C-13b), 120.86 (C-13),
3 2
1
1
1
21.93 (C-8a), 126.37 (C-12), 127.98 (C-11), 130.96 (C-4a), 131.23 (C-12a), 133.33 (C-13a), 138.55 (C-9),
−
1
43.91 (C-8), 147.70 (C-2), 150.12 (C-3), 151.69 (C-10). Spectra IR (cm ): 817.71, 925.71, 1037.56, 1095.42,
+
162.92, 1222.70, 1263.20, 1363.49, 1506.20, 1610.34, 1621.92, 3023.98, 3427.04. MS (ESI): m/z (M ) calcd
+
for C H NO S , 428.116 found: 428.121.
22
22
6

Int. J. Mol. Sci. 2020, 21, 7162
12 of 16
3
.1.6. 9-(Butane-1-sulfonyl)oxy-10-methoxy-2,3-methylenedioxy protoberberine chloride (10d)
Yield: 50%
Spectra NMR H (400 MHz, DMSO-d ,
1
δ
, ppm, J/Hz): 0.97 (3H, t, J = 7.34 Hz, CH CH ), 1.48–1.58
6
2 3
(
2H, m, CH CH ), 1.89–1.97 (2H, m, SCH CH ), 3.22 (2H, t, J = 5.96 Hz, H-5), 3.91 (2H, t, J = 7.68 Hz,
2 3 2 2
SCH ), 4.11 (3H, s, OCH ), 5.02 (2H, t, J = 6.04 Hz), 6.18 (2H, c, OCH O), 7.10 (1H, s, H-4), 7.82 (1H, s,
2
3
2
H-1), 8.27 (1H, d, J = 9.21 Hz, H-12), 8.34 (1H, d, J = 9.21 Hz, H-11), 9.13 (1H, s, H-13), 9.67 (1H, s, H-8).
Spectra NMR 13C (100 MHz, DMSO-d6,
, ppm): 13.44 (CH CH ), 20.74 (CH CH ), 25.29 (SCH CH ),
6.19 (C-5), 51.75 (SCH ), 55.80 (C-6), 57.46 (OCH ), 102.20 (OCH O), 105.62 (C-1), 108.45 (C-4), 120.19
δ
3
2
3
2
2
2
2
2
3
2
(
(
9
C-13b), 120.91 (C-13), 121.98 (C-8a), 126.42 (C-12), 128.01 (C-11), 131.02 (C-4a), 131.30 (C-12a), 133.36
−
1
C-13a), 138.62 (C-9), 143.97 (C-8), 147.75 (C-2), 150.16 (C-3), 151.72 (C-10). Spectra IR (cm ): 811.92,
25.71, 1035.63, 1099.28, 1222.70, 1265.13, 1365.42, 1504.27, 1610.34, 1619.99, 2960.33, 3425.11. MS (ESI):
+
+
m/z (M ) calcd for C H NO S , 442.132 found: 442.133.
23
24
6
3
.2. Biology
3
.2.1. Detection of Tdp1 Activity
The methodology has been reported in our previous work [30] and consists of fluorescence
intensity measurement in a reaction of quencher removal from a fluorophore quencher-coupled DNA
oligonucleotide catalyzed by Tdp1. The reaction was carried out at different concentrations of inhibitors
(
the control samples contained 1% of DMSO, Sigma, St. Louis, MO, USA). The reaction mixtures
contained Tdp1 buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 7 mM -mercaptoethanol), 50 nM
biosensor, and an inhibitor being tested. Purified Tdp1 (1.5 nM) triggered the reaction. The biosensor
β
0 0
5 -[FAM] AAC GTC AGGGTC TTC C [BHQ]-3 ) was synthesized in the Laboratory of Biomedical
(
Chemistry at the Institute of Chemical Biology and Fundamental Medicine (Novosibirsk, Russia).
The reactions were incubated on a POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH,
Ortenberg, Germany) to measure fluorescence every 55 s (ex. 485/em. 520 nm) during the linear
phase (here, data from minute 0 to minute 8). The values of IC50 were determined using a six-point
concentration response curve in minimum three independent experiments and were calculated using
MARS Data Analysis 2.0 (BMG LABTECH, GmbH, Ortenberg, Germany).
3
.2.2. Cytotoxicity Assays
Cytotoxicity of the compounds to HeLa (human cervical cancer) cell line was examined using
the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria), according to the
manufacturer’s protocols. The cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) with
40 µg/mL gentamicin, 50 IU/mL penicillin, 50 µg/mL streptomycin (MP Biomedicals, Santa Ana, CA,
USA), and 10% of fetal bovine serum (Biolot, St. Petersburg, Russia) in a 5% CO atmosphere. After
2
formation of a 30–50%-monolayer, the tested compounds were added to the medium. The volume of
the added reagents was 1/100 of the total volume of the culture medium, and the amount of DMSO
(Sigma, St. Louis, MO, USA) was 1% of the final volume. Control cells were grown in the presence
of 1% DMSO. The cell culture was monitored for 3 days. To assess the influence of the inhibitors on
the cytotoxic effect of topotecan (ACTAVIS GROUP PTC ehf., Bucharest, Romania), 50% cytotoxic
concentrations of topotecan and of each inhibitor were determined to attain a defined single-agent
effect. Then, minimum two independent tests were performed with each inhibitor in combination with
topotecan. When using a combination of drugs, Tdp1 inhibitors were first added, then topotecan was
added immediately (within 10–15 min).
3
.3. Molecular Modeling
The compounds were docked against the crystal structure of TDP1 Tdp1 (PDB ID: 6DIE, resolution
1.78 Å) [32] which was obtained from the Protein Data Bank (PDB) [40,41]. The Scigress version

Int. J. Mol. Sci. 2020, 21, 7162
13 of 16
FJ 2.6 program [42] was used to prepare the crystal structure for docking, i.e., the hydrogen atoms
were added, the co-crystallized ligand benzene-1,2,4-tricarboxylic acid was removed as well as
crystallographic water molecules except HOH 814, 821 and 1078. The waters were set on toggle—bound
or displaced by the ligand during docking—and spin—automatic optimization of the orientation of the
hydrogen atoms. The Scigress software suite was also used to build the inhibitors and the MM2 [43
]
force field was used to optimize the structures. Furthermore, Scigress was used for the 10 ps MD runs
at 1000 K; the MM2 force filed was used, 5 Å radius was defined around the ligand and allowed to be
flexible whereas the rest of the protein structure was held ridged (locked). First the binding pocket
with the ligand was structurally optimized followed by the MD run. The docking center was defined as
the position of a carbon on the ring of the co-crystallized benzene-1, 2, 4-tricarboxylic acid (x = −6.052,
y = 14.428, z = 33.998) with 10 Å radius. Fifty docking runs were allowed for each ligand with
−
default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated.
Furthermore, aspartic and glutamic acids were assumed deprotonated. The GoldScore (GS) [44
and ChemScore (CS) [45 46] ChemPLP (Piecewise Linear Potential) [47] and ASP (Astex Statistical
]
,
Potential) [48] scoring functions were implemented to predict the binding modes and relative energies
of the ligands using the GOLD v5.4.1 software suite.
The QikProp 3.2 [49] software package was used to calculate the molecular descriptors of the
molecules. The reliability of it QikProp established for the calculated descriptors. [50] The Known
Drug Indexes (KDI) were calculated from the molecular descriptors as described by Eurtivong and
Reynisson [40]. Five of the compounds (
9 and 10a–d) carry a positive charge and QikProp does not
compute charged molecules. The MW, Log P, HD and HA were derived using the Scigress version FJ
2
.6 program [42] software, PSA and RB are not available in this software suite.
4
. Conclusions
The berberine and tetrahydroberberine sulfonates and their brominated analogues were tested
to evaluate their Tdp1 inhibitory activity. To our knowledge, this is the first report that documents
berberine-based compounds as inhibitors of this enzyme. The IC50 values are in the 0.53 to 4 M range.
µ
Of the alkyl sulfonates, only derivatives with bromine substitution on site 12 of tetrahydroberberrubine
are active. While both tetrahydroberberrubine and 12-bromtetrahydroberberrubine derivatives
containing polyfluoroaromatic substituents all have inhibitory activity with IC50 values of ~1 µM.
According to the inhibitory activity, toxicity data and ability to sensitize topotecan against the HeLa
cancer cell line the two most promising derivatives were identified—11g and 12g. These results indicate
that sulfonates of tetrahydroberberine have potential to be developed as new agents for anticancer
therapy due to their inhibitory activity and lack of toxicity.
Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/19/
7
162/s1.
Author Contributions: Chemistry investigation, E.D.G., I.V.N., R.A.B. and O.A.L.; in vitro investigation, A.L.Z.,
A.A.C., E.S.I., N.S.D., E.M.M. and R.O.A.; modeling, J.R.; methodology, N.F.S. and O.I.L.; project administration,
K.P.V.; supervision, K.P.V.; writing—original draft, E.D.G., A.L.Z. and O.A.L.; writing—review and editing, K.P.V.,
J.R., N.F.S. and O.I.L. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded by the Russian Science Foundation grant № 19-13-00040.
Acknowledgments: Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for
spectral and analytical measurements.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
Tdp1
PARP
Tyrosyl-DNA phosphodiesterase 1
Poly (ADP-ribose) polymerase

Int. J. Mol. Sci. 2020, 21, 7162
14 of 16
References
1.
2.
3.
4.
Zakharenko, A.L.; Lebedeva, N.A.; Lavrik, O.I. DNA Repair Enzymes as Promising Targets in Oncotherapy.
Russ. J. Org. Chem. 2018, 44, 1–18. [CrossRef]
Ferri, A.; Stagni, V.; Baril , D. Targeting the DNA Damage Response to Overcome Cancer Drug Resistance in
Glioblastoma. Int. J. Mol. Sci. 2020, 21, 4910. [CrossRef]
Jiang, X.; Li, W.; Li, X.; Bai, H.; Zhang, Z. Current status and future prospects of PARP inhibitor clinical trials
in ovarian cancer. Cancer Manag. Res. 2019, 1, 4371–4390. [CrossRef] [PubMed]
à
Comeaux, E.Q.; Waardenburg, R.C. Tyrosyl-DNA phosphodiesterase I resolves both naturally and chemically
induced DNA adducts and its potential as a therapeutic target. Drug Metab. Rev. 2014, 46, 494–507. [CrossRef]
[PubMed]
5
6
7
8
9
1
.
.
.
.
.
Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. DNA topoisomerases and their poisoning by anticancer and
antibacterial drugs. Chem. Biol. 2010, 17, 421–433. [CrossRef] [PubMed]
Kciuk, M.; Marciniakand, B.; Kontek, R. Irinotecan—Still an Important Player in Cancer Chemotherapy:
A Comprehensive Overview. Int. J. Mol. Sci. 2020, 21, 4919. [CrossRef]
Pommier, Y.; Huang, S.-y.N.; Gao, R.; Das, B.B.; Murai, J.; Marchand, C. Tyrosyl-DNA-phosphodiesterases
(TDP1 and TDP2). DNA Repair 2014, 19, 114–129. [CrossRef] [PubMed]
Laev, S.S.; Salakhutdinov, N.F.; Lavrik, O.I. Tyrosyl-DNA phosphodiesterase inhibitors: Progress and
potential. Bioorg. Med. Chem. 2016, 24, 5017–5027. [CrossRef] [PubMed]
Huang, S.N.; Pommier, Y.; Marchand, C. Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors. Expert Opin.
Ther. Pat. 2011, 21, 1285–1292. [CrossRef] [PubMed]
0. Salomatina, O.V.; Popadyuk, I.I.; Zakharenko, A.L.; Zakharova, O.D.; Fadeev, D.S.; Komarova, N.I.;
Reynisson, J.; Arabshahi, H.I.; Chand, R.; Volcho, K.P.; et al. Novel Semisynthetic Derivatives of Bile Acids as
Effective Tyrosyl-DNA Phosphodiesterase 1 Inhibitors. Molecules 2018, 23, 679. [CrossRef]
1
1
1
1
1
1. Xiao, L.-G.; Zhang, Y.; Zhang, H.-L.; Li, D.; Gu, Q.; Tang, G.-H.; Yu, Q.; An, L.-K. Spiroconyone A, a new
phytosterol with a spiro [5,6] ring system from Conyza japonica. Org. Biomol. Chem. 2020, 18, 5130–5136.
[CrossRef] [PubMed]
2. Zakharenko, A.; Luzina, O.; Koval, O.; Nilov, D.; Gushchina, I.; Dyrkheeva, N.; Švedas, V.; Salakhutdinov, N.;
Lavrik, O. Tyrosyl-DNA Phosphodiesterase 1 Inhibitors: Usnic Acid Enamines Enhance the Cytotoxic Effect
of Camptothecin. J. Nat. Prod. 2016, 79, 2961–2967. [CrossRef] [PubMed]
3. Filimonov, A.S.; Chepanova, A.A.; Luzina, O.A.; Zakharenko, A.L.; Zakharova, O.D.; Ilina, E.S.;
Dyrkheeva, N.S.; Kuprushkin, M.S.; Kolotaev, A.V.; Khachatryan, D.S.; et al. New Hydrazinothiazole
Derivatives of Usnic Acid as Potent Tdp1 Inhibitors. Molecules 2019, 24, 3711. [CrossRef] [PubMed]
4. Zakharenko, A.L.; Luzina, O.A.; Sokolov, D.N.; Zakharova, O.D.; Rakhmanova, M.E.; Chepanova, A.A.;
Dyrkheeva, N.S.; Lavrik, O.I.; Salakhutdinov, N.F. Usnic acid derivatives are effective inhibitors of
tyrosyl-DNA phosphodiesterase 1. Russ. J. Bioorg Chem. 2017, 43, 84–90. [CrossRef]
5. Khomenko, T.; Zakharenko, A.; Odarchenko, T.; Arabshahi, H.J.; Sannikova, V.; Zakharova, O.; Korchagina, D.;
Reynisson, J.; Volcho, K.; Salakhutdinov, N.; et al. New inhibitors of tyrosyl-DNA phosphodiesterase I
(Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties. Bioorg. Med. Chem. 2016, 24, 5573–5581.
[CrossRef]
1
1
6. Ponomarev, K.Y.; Suslov, E.V.; Zakharenko, A.L.; Zakharova, O.D.; Rogachev, A.D.; Korchagina, D.V.; Zafar, A.;
Reynisson, J.; Nefedov, A.A.; Volcho, K.P.; et al. Aminoadamantanes containing monoterpene-derived
fragments as potent tyrosyl-DNA phosphodiesterase 1 inhibitors. Bioorg. Chem. 2018, 76, 392–399. [CrossRef]
7. Chepanova, A.A.; Li-Zhulanov, N.S.; Sukhikh, A.S.; Zafar, A.; Reynisson, J.; Zakharenko, A.L.;
Zakharova, O.D.; Korchagina, D.V.; Volcho, K.P.; Salakhutdinov, N.F.; et al. Effective Inhibitors of Tyrosy
l-DNA Phosphodiesterase 1 Based on Monoterpenoids as Potential Agents for Antitumor Therapy. Russ. J.
Bioorg. Chem. 2019, 45, 647–655. [CrossRef]
1
8. Il’ina, I.V.; Dyrkheeva, N.S.; Zakharenko, A.L.; Sidorenko, A.Y.; Li-Zhulanov, N.S.; Korchagina, D.V.;
Chand, R.; Ayine-Tora, D.M.; Chepanova, A.A.; Zakharova, O.D.; et al. Design, Synthesis, and Biological
Investigation of Novel Classes of 3-Carene-Derived Potent Inhibitors of TDP1. Molecules 2020, 25, 3496.
[CrossRef]

Int. J. Mol. Sci. 2020, 21, 7162
15 of 16
1
2
2
9. Zhang, X.-R.; Wang, H.-W.; Tang, W.-L.; Zhang, Y.; Yang, H.; Hu, D.-X.; Ravji, A.; Marchand, C.; Kiselev, E.;
Ofori-Atta, K.; et al. Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of
DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor
Agents. J. Med. Chem. 2018, 61, 9908–9930. [CrossRef]
0. Zakharenko, A.L.; Luzina, O.A.; Sokolov, D.N.; Kaledin, V.I.; Nikolin, V.P.; Popova, N.A.; Patel, J.;
Zakharova, O.D.; Chepanova, A.A.; Zafar, A.; et al. Novel tyrosyl-DNA phosphodiesterase 1 inhibitors
enhance the therapeutic impact of topotecan on in vivo tumor models. Eur. J. Med. Chem. 2019, 161, 581–593.
[CrossRef]
1. Khomenko, T.M.; Zakharenko, A.L.; Chepanova, A.A.; Ilina, E.S.; Zakharova, O.D.; Kaledin, V.I.; Nikolin, V.P.;
Popova, N.A.; Korchagina, D.V.; Reynisson, J.; et al. Promising New Inhibitors of Tyrosyl-DNA
Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of
Complex Antitumor Therapy. Int. J. Mol. Sci. 2020, 21, 126. [CrossRef] [PubMed]
2
2. Pang, B.; Zhao, L.-H.; Zhou, Q.; Zhao, T.-Y.; Wang, H.; Gu, C.-J.; Tong, X.-L. Application of Berberine on
Treating Type 2 Diabetes Mellitus. Int. J. Endocrinol. 2015, 2015, 905749:1–905749:13. [CrossRef] [PubMed]
3. Chu, M.; Zhang, M.-B.; Liu, Y.-C.; Kang, J.-R.; Chu, Z.-Y.; Yin, K.-L.; Ding, L.-Y.; Ding, R.; Xiao, R.-X.; Yin, Y.-N.;
et al. Role of Berberine in the Treatment of Methicillin-Resistant Staphylococcus aureus Infections. Sci. Rep.
2
2
016, 6, 24748:1–24748:9. [CrossRef] [PubMed]
2
4. Yu, H.H.; Kim, K.J.; Cha, J.D.; Kim, H.K.; Lee, Y.E.; Choi, N.Y.; You, Y.O. Antimicrobial activity of berberine
alone and in combination with ampicillin or oxacillin against methicillin-resistant Staphylococcus aureus.
J. Med. Food. 2005, 8, 454–461. [CrossRef]
2
2
2
5. Peng, L.; Kang, S.; Yin, Z.; Jia, R.; Song, X.; Li, L.; Li, Z.; Zou, Y.; Liang, X.; Li, L.; et al. Antibacterial activity
and mechanism of berberine against Streptococcus agalactiae. Int. J. Clin. Exp. Pathol. 2015, 8, 5217–5223.
6. Tan, W.; Li, Y.; Chen, M.; Wang, Y. Berberine hydrochloride: Anticancer activity and nanoparticulate delivery
system. Int. J. Nanomed. 2011, 6, 1773–1777. [CrossRef]
7. Nechepurenko, I.V.; Shirokova, E.D.; Khvostov, M.V.; Frolova, T.S.; Sinitsyna, O.I.; Maksimov, A.M.;
Bredikhin, R.A.; Komarova, N.I.; Fadeev, D.S.; Luzina, O.A.; et al. Synthesis, hypolipidemic and antifungal
activity of tetrahydroberberrubine sulfonates. Russ. Chem. Bull. Int. Ed. 2019, 68, 1052–1060. [CrossRef]
8. Liu, H.; Wang, J.; Zhang, R.; Cairns, N.; Liu, J. Compounds, Compositions and Methods for Reducing Lipid
Levels. International Patent Application No. PCT/US2008/067762, 20 June 2008.
2
2
3
9. Iwasa, K.; Kamigauchi, M. 13-hydroxylation of tetrahydroberberine in cell suspension cultures of some
Corydalis species. Phytochemistry 1996, 41, 1511–1515. [CrossRef]
0. Nechepurenko, I.V.; Boyarskikh, U.A.; Khvostov, M.V.; Baev, D.S.; Komarova, N.I.; Filipenko, M.L.;
Tolstikova, T.G.; Salakhutdinov, N.F. Hypolipidemic Berberine Derivatives with a Reduced Aromatic
Ring C. Chem. Nat. Comp. 2015, 51, 916–922. [CrossRef]
3
3
3
1. Zakharenko, A.L.; Khomenko, T.M.; Zhukova, S.V.; Koval, O.A.; Zakharova, O.D.; Anarbaev, R.O.;
Lebedeva, N.A.; Korchagina, D.V.; Komarova, N.I.; Vasiliev, V.G.; et al. Synthesis and biological evaluation
of novel tyrosyl-DNA phosphodiesterase 1 inhibitors with a benzopentathiepine moiety. Bioorg. Med. Chem.
2
015, 23, 2044–2052. [CrossRef]
2. Lountos, G.T.; Zhao, X.Z.; Kiselev, E.; Tropea, J.E.; Needle, D.; Pommier, Y.; Burke, T.R.; Waugh, D.S.
Identification of a Ligand Binding Hot Spot and Structural Motifs Replicating Aspects of Tyrosyl-DNA
Phosphodiesterase I (TDP1) Phosphoryl Recognition by Crystallographic Fragment Cocktail Screening.
Nucleic Acids Res. 2019, 47, 10134–10150. [CrossRef] [PubMed]
3. Martino, E.; Della Volpe, S.; Terribile, E.; Benetti, E.; Sakaj, M.; Centamore, A.; Sala, A.; Collina, S. The long
story of camptothecin: From traditional medicine to drugs. Bioorg. Med. Chem. Lett. 2017, 27, 701–707.
[CrossRef]
3
3
3
4. Bjornsti, M.A.; Kaufmann, S.H. Topoisomerases and cancer chemotherapy: Recent advances and unanswered
questions. F1000Research 2019, 8, 1704. [CrossRef] [PubMed]
5. Bailly, C. Irinotecan: 25 years of cancer treatment. Pharmacol. Res. 2019, 148, 104398:1–104398:11. [CrossRef]
[PubMed]
6. Perego, P.; Cossa, G.; Tinelli, S.; Corna, E.; Carenini, N.; Gatti, L.; De Cesare, M.; Ciusani, E.; Zunino, F.;
Luison, E.; et al. Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell
sensitivity to topoisomerase I inhibition. Biochem. Pharmacol. 2012, 83, 27–36. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2020, 21, 7162
16 of 16
3
3
3
4
4
7. Brettrager, E.J.; van Waardenburg, R.C.A.M. Targeting Tyrosyl-DNA phosphodiesterase I to enhance toxicity
of phosphodiester linked DNA-adducts. Cancer Drug Resist. 2019, 2, 1153–1163. [CrossRef] [PubMed]
8. Zhu, F.; Logan, G.; Reynisson, J. Wine Compounds as a Source for HTS Screening Collections. A Feasibility
Study. Mol. Inf. 2012, 31, 847–855. [CrossRef]
9. Eurtivong, C.; Reynisson, J. The Development of a Weighted Index to Optimise Compound Libraries for
High Throughput Screening. Mol. Inf. 2018, 37, 1800068:1–1800068:10. [CrossRef] [PubMed]
0. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E.
The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235–242. [CrossRef]
1. Berman, H.; Henrick, K.; Nakamura, H. Announcing the Worldwide Protein Data Bank. Nat. Struct. Biol.
2
003, 10, 980. [CrossRef]
4
4
2. Scigress Ultra V. F.J 2.6. (EU 3.1.7) Fujitsu Limited 2008–2016.
3. Allinger, N.L. Conformational Analysis. 130. MM2. A Hydrocarbon Force Field Utilizing V1 and V2
Torsional Terms. J. Am. Chem. Soc. 1977, 99, 8127–8134. [CrossRef]
4
4. Jones, G.; Willet, P.; Glen, R.C.; Leach, A.R.; Taylor, R. Development and Validation of a Genetic Algorithm
for Flexible Docking. J. Mol. Biol. 1997, 267, 727–748. [CrossRef] [PubMed]
4
5. Eldridge, M.D.; Murray, C.; Auton, T.R.; Paolini, G.V.; Mee, P.M. Empirical Scoring Functions: I.
The Development of a Fast Empirical Scoring Function to Estimate the Binding Affinity of Ligands in
Receptor Complexes. J. Comput. Aided Mol. Des. 1997, 11, 425–445. [CrossRef]
4
4
4
6. Verdonk, M.L.; Cole, J.C.; Hartshorn, M.J.; Murray, C.W.; Taylor, R.D. Improved Protein-Ligand Docking
using GOLD. Proteins 2003, 52, 609–623. [CrossRef] [PubMed]
7. Korb, O.; Stutzle, T.; Exner, T.E. Empirical Scoring Functions for Advanced Protein Ligand Docking with
−
PLANTS. J. Chem. Inf. Model. 2009, 49, 84–96. [CrossRef]
8. Mooij, W.T.M.; Verdonk, M.L. General and Targeted Statistical Potentials for Protein–ligand Interactions.
Proteins 2005, 61, 272–287. [CrossRef]
4
5
9. QikProp; Version 3.2; Schrödinger: New York, NY, USA, 2009.
0. Ioakimidis, L.; Thoukydidis, L.; Naeem, S.; Mirza, A.; Reynisson, J. Benchmarking the Reliability of QikProp.
Correlation between Experimental and Predicted Values. QSAR Comb. Sci. 2008, 27, 445–456. [CrossRef]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(