3.2.2. Expression and Purification of Human Recombinant Tyrosyl-DNA Phosphodiesterase 2 (TDP2)

The recombinant N-terminally His-tagged TDP2 was expressed in *E. coli* BL21 (DE3) cells. The plasmid pLATE-31-TDP2 expression vector was constructed as follows. cDNA encoding the TDP2 protein was cloned in the pLATE-31 expression vector using a LICator LIC Cloning and Expression System (Thermo Scientific, Waltham, MA, USA). Using specific primers and total cDNA of HeLa cells, the TDP2 coding sequence was amplified by PCR and annealed with a linearized pLATE-31 vector. The sequence of the cloned cDNA was confirmed at the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia). Plasmids were transformed into BL21 cells by electroporation, and the cells were grown in LB medium at pH 7.5 with 100 mg/mL ampicillin at 30 ◦C. Two hours after induction with 1 mM IPTG, cells were harvested. Cell pellets were thawed on ice, resuspended in binding buffer (0.5 M NaCl, 5% glycerol, 50 mM Tris-HCl, pH 8.0, and a mixture of protease inhibitors), and broken by sonication. After centrifugation, 10 mM imidazole was added to the supernatant. The Ni sepharose column (GE Healthcare, UK) was washed with binding buffer (0.5 M NaCl, 10 mM imidazole, 50 mM Tris-HCl, and pH 8.0). Elution of the proteins was carried out with an elution buffer (0.5 M NaCl, 500 mM imidazole, 50 mM Tris-HCl, pH 8.0, and protease inhibitors), and the eluate was loaded into the heparin sepharose column (GE Healthcare, Pollards Wood, UK). Elution of the proteins was carried out with a NaCl gradient of 0.1–1 M in 50 mM Tris-HCl, pH 8.0, with protease inhibitors. The protein TDP2 was stored in 50 mM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 mM DTT, and 50% glycerol at −20 ◦C. The enzyme samples were estimated to be more than 95% pure. Enzyme concentrations were estimated by Bradford assay. The Coomassie-stained protein gel is shown in Supplementary Material (Figure S29).

## 3.2.3. Real-Time Detection of TDP1 Activity

The biosensor (50 -[FAM] AAC GTC AGGGTC TTC C [BHQ]-30 ) was synthesized in the Laboratory of Nucleic Acid Chemistry at the Institute of Chemical Biology and Fundamental Medicine (Novosibirsk, Russia) and was used for TDP1 enzyme activity real-time fluorescence detection [43]. The reaction mixture (200 µL) contained a TDP1 reaction buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 7 mM β-mercaptoethanol), 50 nM oligonucleotide, varied concentrations of the tested compounds, and purified TDP1 in a final concentration 1.5 nM. The reaction mixtures were incubated at a constant temperature of 26 ◦C in a POLARstar OPTIMA fluorimeter (BMG LABTECH, GmbH, Ortenberg, Germany). Fluorescence intensity was measured (Ex485/Em520 nm) every 1 min for 10 min. The average values of the half maximal inhibitory concentration (IC50) were determined using an eleven-point concentration response curve and calculated using MARS Data Analysis 2.0 (BMG LABTECH). The 50% inhibitory concentration (IC50) was defined as the concentration of the compound that inhibited 50% of the enzyme activity when compared to the untreated controls. At least three independent experiments were carried out to obtain the IC<sup>50</sup> values. To determine the kinetic parameters of the TDP1 enzymatic reaction, the apparent maximum rate of enzymatic reaction (Vmax), Michaelis constant (KM), possible inhibition mechanism, and steady-state kinetic experiments were carried out at 5 fixed concentrations of the substrate, with variation in the inhibitor concentrations [54]. The standard reaction mixtures (200 µL) contained reaction buffer components; 50 nM, 100 nM, 200 nM, 500 nM, or 1000 nM substrate; an inhibitor; and 1.5 nM recombinant human TDP1. The initial kinetic curves were obtained in three independent experiments and statistically processed in OriginPro 8.6.0 (OriginLab, Northampton, MA, USA).

## 3.2.4. Gel-Based TDP2 Activity Assay

Oligonucleotide 50 - tyrosine -AAC GTC AGG GTC TTC C- FAM -30 was synthesized as described above and used for the indication of TDP2 enzyme activity in polyacrylamide gel. TDP2 gel-based assays were performed to a final volume 20 µL using 100 nM substrate incubated with 200 nM recombinant human TDP2 in the absence or

presence of an inhibitor for 10 min at 37 ◦C in a buffer containing 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 7 mM β-mercaptoethanol, and BSA. Reactions were terminated by the addition of a gel loading buffer (TBE, 10% formamide, 7 M carbamide, 0.1% xylene cyanol, and 0.1% bromophenol blue, and 20 mM EDTA). The samples were heated before loading at 90 ◦C for 7 min. The reaction products were separated by electrophoresis in a 20% denaturing PAGE with 7 M carbamide at a ratio of acrylamide to bisacrylamide of 19:1. A Typhoon FLA 9500 phosphorimager (GE Healthcare, Boston, MA, USA) was used for gel scanning and imaging, and the data were analyzed with QuantityOne 4.6.7 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

#### 3.2.5. PARP1 and PARP2 Enzyme Assay

The radioactive labeled [32P]-NAD+ was synthesized from α–[32P]-ATP according to [55]. The reaction of autopoly(ADP-ribosyl)ation was carried out as follows: for PARP1, 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 150 mM NaCl, and 7 mM β-mercaptoethanol, as well as activated DNA 2 oe/mL, 0.3 mM [32P]-NAD+ at 37 ◦C. The reaction was initiated by adding PARP1 to 200 nM and the reaction mixtures were incubated for 2 min. For PARP2: 50 mM Tris-HCl, pH 8.0, 3 mM spermin, 150 mM NaCl, and 7 mM β-mercaptoethanol, as well as activated DNA 2 oe/mL, 0.6 mM [32P] NAD+ at 37 ◦C. The reaction was initiated by adding PARP2 to 800 nM and the reaction mixtures were incubated for 5 min. The tested compounds were added at a final concentration 500 nM for reactions with PARP1 and 1 µM for reactions with PARP2. The reaction was stopped by placing 10 µL aliquots onto Whatman 1 paper filters soaked with 5% TCA. The filters were washed with 5% TCA four times and dried in the air after the removal of TCA with 90% ethanol. The incorporation of radioactivity into the product was calculated using a Typhoon FLA 9500 scanner (GE Healthcare, Chicago, IL, USA). Measurements were done in at least two independent experiments.

### 3.2.6. Cell Culture Cytotoxicity Assay

Cytotoxicity of the compounds was examined against human cell lines HEK293A (human embryonic kidney)—WT, TDP1 deficient (*Tdp1*-/-), PARP1 deficient (*PARP1*-/-), and HeLa (cervical cancer) using an EZ4U colorimetric test (Biomedica, Vienna, Austria). The HEK293A cell line was obtained from Thermo Fisher Scientific (Waltham, MA, USA), and the HeLa cell line was obtained from the Russian Cell Culture Collection (RCCC) Institute of Cytology RAS, St. Petersburg, Russia. The cells were grown in DMEM/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), with 1x GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 50 IU/mL penicillin, 50 µg/mL streptomycin (MP Biomedicals), and in the presence of 10% fetal bovine serum (Biolot, Saint-Petersburg, Russia) in a 5% CO<sup>2</sup> atmosphere. Cells were grown in the presence of 1% DMSO in the control wells. After the formation of a 30–50% monolayer, the tested compounds were added to the medium, and the cell culture was monitored for 3 days. The values were normalized to their own control in each case. At least three independent experiments were carried out. The 50% cytotoxic concentration (CC50) was defined as the compound concentration that reduced the cell viability by 50% when compared to the untreated controls. The compound concentration that caused 50% cell growth inhibition was determined using OriginPro 8.6.0 software (OriginLab, Northampton, MA, USA). The measurements were carried out in three independent experiments.

### 3.2.7. Plasmid Construction for Human *PARP1* Gene Knockout

sgRNAs design was performed using the Benchling CRISPR tool (https://www. benchling.com/, accessed on 9 November 2019). Two protospacers (PAM sequences in brackets) were selected for the DNA sequence deletion that includes 3–5 exons of the *PARP1* gene: PARP1-gRNA1 CTAGAACCTCCAATACCATG (TGG) and PARP1-gRNA2 GCAAGTGACCACAAAGGTGC (AGG). Corresponding oligonucleotides were cloned in plasmid pSpCas9(BB)-2A-GFP (PX458) (the plasmid was a gift from Feng Zhang (Addgene

plasmid #48138; http://n2t.net/addgene:48138; RRID:Addgene\_48138)) as previously described [48]. Transfection-grade plasmid DNA was isolated using the Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany).

#### 3.2.8. Knockout HEK293A Clones' Generation

<sup>5</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> HEK293A cells were plated into each well of a 12-well plate and co-transfected with the constructed plasmids PARP1-gRNA1 and PARP1-gRNA2 (0.25 µg of each) using a Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The growth medium contained DMEM/F12 (Gibco) 1:1, 10% FBS (Gibco), 100 U/mL penicillin– streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1× GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). A total of 48 h after transfection the cells were detached using TrypLE Express (TrypLE, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and the GFP-positive cell population was enriched by cell sorting using the BD FACSAria III Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA). Transfected cells were plated onto a 96-well plate, one cell per well. Single-cell clones grew for two weeks before they were replicated to another 96-well plate, so we obtained two equal 96-well plates with cell clones: one plate was used for PCR analysis of the deletion in the *PARP1* gene, and the other plate was used for the mutant cell clone multiplying.

#### 3.2.9. Analysis of CRISPR/Cas9-Mediated Deletions in *PARP1* Gene

Genome DNA was extracted from cells on one of two 96-well plates using 50 µL of QuickExtract™ DNA Extraction Solution (Lucigen, Madison, WI, USA) per well. The DNA extracts were diluted with 200 µL of mQ water. Two microliters of the diluted DNA extract were used to PCR amplify the target region with primers to detect the presence of deletions (PARP1-Del-F 50 -AGTGTGCCCTGCGTATTTGC-30 and PARP1-Del-R 50 - CACAGGGATGAA-TCTTTCTGGTC-30 ) and wild-type alleles (PARP1-In-F 50 - CGCTCCCTTGGTACCACATATG -30 and PARP1-In-R 50 -GGCTTACTGACAGTCAGCGAAG-30 ). Both reactions were run on an S1000 Thermal Cycler (Bio-Rad, Singapore) using BioMaster HS-Taq PCR-Color (2×) (Biolabmix, Novosibirsk, Russia) with the following program: 95 ◦C for 3 min; 35 cycles: 95 ◦C for 30 s; 60 ◦C for 30 s; 72 ◦C for 30 s; and 72 ◦C for 3 min. The products of the reactions were resolved in 1% agarose gel stained with ethidium bromide.

## **4. Conclusions**

TDP1 promotes the cleavage of the stable DNA–TOP1 complexes with the clinically used anticancer drug topotecan (Tpc), which is a TOP1 inhibitor. TDP1 activity may be a possible cause of tumor resistance to TOP1 inhibitors. A series of new UA thioether and sulfoxide derivatives were synthesized.

The usnic acid thioether and sulfoxide derivatives efficiently suppressed TDP1 activity with the IC<sup>50</sup> values in the 1.4–25.2 µM range. The structure of the heterocyclic substituent affects the TDP1 inhibitory efficiency of these compounds. Derivatives containing a fivemembered heterocyclic fragment itself or fused to a benzene ring (**7b**,**c**,**e**–**g** and **10a**) inhibit TDP1 in the low micromolar concentration range (IC<sup>50</sup> of 1.4–4.4 µM). Compounds containing a six-membered heterocycle (**7i**–**k**) inhibit TDP1 at higher concentrations (IC<sup>50</sup> > 11 µM). The presence of a halogen in the *para*-position of the benzene substituent enhances the inhibitory properties of the compounds. For the most effective inhibitors of TDP1 **7g**,**h** and their sulfoxide analogs **10a**,**b**, we observed the uncompetitive type of inhibition. The uncompetitive inhibitors prevent the second step of the reaction stabilizing the enzyme–DNA covalent complex. Thus, uncompetitive TDP1 inhibitors could lead to the accumulation of the single-strand breaks in cancer cells.

The anticancer effect of the TOP1 inhibitors can be significantly enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. We tested the ability of the synthesized compounds to inhibit the TDP1, TDP2, and PARP1 activities. We found the compounds act as dual or triple inhibitors of TDP1, TDP2, and PARP1. Some of the compounds inhibited not only TDP1 but also TDP2 and PARP1, but at significantly higher concentration ranges than TDP1.

Interestingly, the sulfoxide analogs **10a**,**b** were less cytotoxic than their thioester analogs **7g**,**h**. Compound **10b** was one of the less toxic among the ten tested compounds. One of the most effective TDP1 inhibitors, **10a** (IC<sup>50</sup> 2.1 µM, Table 1), also moderately inhibit TDP2, and showed promising synergy on HeLa cells in conjunction with topotecan. That is of great importance for further development of sensitizers to topotecan and other clinically used TOP1 inhibitors.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms222111336/s1.

**Author Contributions:** Chemistry investigation, A.S.F., O.A.L., E.A.B., K.V.K. and D.A.S.; in vitro investigation, N.S.D., A.L.Z., E.S.I., K.A.O., I.A.C. and T.E.K.; recombinant proteins purification: R.O.A. and K.N.N.; *PARP1* knockout clones' generation, A.A.M., S.P.M. and S.M.Z.; methodology, N.F.S. and O.I.L.; writing—original draft, N.S.D., A.L.Z., A.S.F. and O.A.L.; writing—review and editing, O.A.L., N.F.S. and O.I.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant from the Ministry of Science and Higher Education Russian Federation (agreement no. 075-15-2020-773).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS, Novosibirsk, Russia, for their assistance with the analytical and spectroscopic measurements. Cell lines were obtained from the Russian Cell Culture Collection (RCCC) Institute of Cytology RAS, St. Petersburg, Russia.

**Conflicts of Interest:** The authors declare no conflict of interest.

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