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 β-mercaptoethanol), 50 nM biosensor, and an inhibitor being tested. Purified Tdp1 (1.5 nM) triggered the reaction. The biosensor (50 -[FAM] AAC GTC AGGGTC TTC C [BHQ]-30 ) 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 IC<sup>50</sup> 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<sup>2</sup> atmosphere. After 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 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.
