*3.2. Biology Section*

*Real-Time Detection of Tdp1 Activity.* The Tdp1 activity measurements were carried out as described [64]. Briefly, Tdp1-biosensor with a final concentration of 50 nM was incubated in a volume of 200 µL containing 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 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 the compound to that of DMSO control wells. IC<sup>50</sup> values were determined using a 6-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.

*Cell Culture Assays*. Tumor cells from human mammary adenocarcinoma cell line MCF-7 and cervical cancer cell line HeLa (~2000 cells per well) were incubated for 24 h at 37 ◦C in IMDM medium (5% CO2), and then treated with the synthesized derivatives. After 72 h of cell incubation, the relative amount of alive cells was determined using standard colorimetric MTT test [74] or EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Austria), as per the manufacturer's protocols.

*Binding Assay***.** Synthetic DNA encoding human Tdp1 (residues 149-608) was cloned into pET-28a (+) (GenScript), which was then transformed into *Escherichia coli* BL21 (DE3) for recombinant protein production. Protein production was induced with 1 mM IPTG at 28 ◦C with overnight incubation. Purification of Tdp1 was performed using affinity and size exclusion chromatography. Intrinsic protein fluorescence was measured using PerkinElmer EnSpire Multimode Reader. The Tdp1 concentration was 10 µM, and the compound concentrations were 25 µM, 50 µM, 75 µM, 100 µM, 150 µM, and 250 µM. The buffer was composed of 20 mM Tris and 250 mM NaCl, pH 8. The excitation wavelength was 280 nm and the intrinsic fluorescence was measured at 350 nm. Compound control was performed using the buffer and compound only. The total volume per well was 30 µL. Dissociation constants (*K*D) were calculated using the following formula, that takes nonspecific binding into account.

$$I = \frac{I \text{max} \times [L\_{\text{T}}]}{K\_{\text{D}} + [L\_{\text{T}}]} + \text{Ns} [L\_{\text{T}}]$$

In this formula, I indicates changes in fluorescence intensity from the titration, Imax indicates the maximum fluorescence intensity change, [*L*T] is the titration ligand concentration, and Ns is the non-specific term. Non-linear curve fitting was conducted using SigmaPlot 13.0 (Systat Software, San Jose, CA, USA). Experiments were conducted in triplicate and the errors shown are standard derivations.

*Lab animals.* Thre-e to four-month-old male and female C57Bl/6 mice from the breeding colony of the Institute of Cytology and Genetics, SB RAS, were used in the study. The animals were kept on sawdust in plastic cages with 5–7 mice per cage, with free access to ground food ("Laboratorkorm", Moskow, Russia) and tap water. All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of the Institute of Cytology and Genetics. Also, all experimental procedures were performed in accordance with the Directive 2010/63/EU for animal experiments.

*Tumor models.* The experimental tumor used was Lewis Lung Carcinoma (LLC) and Krebs-2. The animals were treated with tpc and the Tdp1 inhibitor **3ba** two days after tumor transplantation. The tumor was transplanted into the muscles of the thigh by 0.2 million cells per mouse. Tpc (Sindan Pharma SRL, Romania) was administered intraperitoneally at a single dose of 0.5 mg/kg; Tdp1 inhibitor **3ba** was administrated intraperitoneally at a single dose of 20, 40, or 80 mg/kg (for Krebs-2), or 120 mg/kg (for LLC) in 15% dimethyl sulfoxide (DMSO)–10% Tween-80 suspension in water (0.2 mL of suspension per mouse) simultaneously with tpc. Control mice were injected with a DMSO-Tween-80 mixture into the stomach.

The antitumor effect was assessed by the size and weight of the solid tumors at 18 days after transplantation. For estimations of daily gain in volume, the tumor nodules were periodically measured with a caliper.

*Statistical analysis.* The experimenter measuring and calculating the primary animal data (tumor size, lifespan) was blinded. After unblinding, the animal data were statistically processed using oneway ANOVA. Post-hoc testing was completed using Turkey's Honestly Significant Difference (HSD). *p* < 0.05 was considered to be statistically significant. The statistical package STATISTICA version 12.5 was used for analysis. All results are expressed as mean ± SEM.

## *3.3. Modeling Section*

*Molecular modeling and chemical space.* The compounds were docked against the crystal structure of Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [75], which was obtained from the Protein Data Bank (PDB) [76,77]. The Scigress version FJ 2.6 program [78] was used to prepare the crystal structure for docking, i.e., the hydrogen atoms were added, and the cocrystallized ligand benzene-1,2,4-tricarboxylic

acid was removed, as well as crystallographic water molecules, except HOH 814, 821, and 1078. The Scigress software suite was also used to build the inhibitors, and the MM2 [77] force field was used to optimize the structures. The docking centre was defined as the position of a carbon on the ring of 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 the default search efficiency (100%). The basic amino acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be deprotonated. The GoldScore(GS) [71] and ChemScore (CS) [69,70], ChemPLP (Piecewise Linear Potential) [68], and ASP (AstexStatistical Potential) [67] scoring functions were implemented to validate the predicted binding modes and relative energies of the ligands using the GOLD v5.4.1 software suite (The Cambridge Crystallographic Data Centre, Cambridge, UK). The QikProp 3.2 [79] software package (Schrödinger, New York, USA) was used to calculate the molecular descriptors of the molecules; the reliability of this method has been established for the calculated descriptors [80].
