Exploring the Binding Mode of Triflamide Derivatives at the Active

J. Chem. Sci. (2020) 132:50 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12039-020-1750-2 Sadhana(0123456789().,-volV)FT3](0123456789().,-volV) REGULAR ARTICLE

Exploring the binding mode of triﬂamide derivatives at the active site of Topo I and Topo II enzymes: In silico analysis and precise molecular docking

ANDRADE-PAVO´ N DULCEa,*,GO´ MEZ-GARCIÁ OMARb,* and A´ LVAREZ- TOLEDANO CECILIOc aEscuela Nacional de Ciencias Biolo´gicas, Instituto Politećnico Nacional, Prolongacioń de Carpio y Plan de Ayala S/N; Colonia Santo Toma´s, 11340 Mexico City, Mexico bDepartamento Quı´mica Orgańica, Escuela Nacional de Ciencias Biolo´gicas-Instituto Politećnico Nacional, Prolongacioń de Carpio y Plan de Ayala S/N; Colonia Santo Toma´s, 11340 Mexico City, Mexico cInstituto de Quı´mica-UNAM, Circuito Exterior, Ciudad Universitaria, C.P. 04510 Coyoacań, Mexico City, Mexico E-mail: [email protected]; [email protected] In honor of my father Leonardo Andrade Capetillo.

MS received 20 January 2019; revised 19 August 2019; accepted 15 September 2019

Abstract. The DNA topoisomerase enzymes, Topo I and Topo II, have been used as molecular targets for drug design of anticancer agents. The search is for new anticancer therapies that respond to the toxicity of current drug treatments and tumor resistance. The present study provides insights into a likely dual inhibitory effect on Topo I and II of trifluoromethylsulfonamide (triflamide) derivatives by computational docking studies. The physicochemical properties of these compounds were evaluated by Lipinski’s rules. Molecular docking simulations were conducted to determine the possible molecular target, mode and energy binding of the triflamide derivatives. An in silico analysis indicated that the triflamide derivatives interact with amino acid residues at the active site of Topo I and Topo II. The highest binding energy for the Topo I complex was shown by 1g and for the Topo II complex by 1e; these studies were validated by the analysis of decoys. Virtual mutations of Topo I and Topo II were tested, revealing the importance of certain active site residues on the binding mode and binding energy of the test triflamide derivatives. Overall, the results suggest that the compounds 1g and 1e could be drugs promising for the future design and development of anticancer agents.

Keywords. Triﬂamides; anticancer drugs; molecular docking; virtual mutations.

1. Introduction cancers.4 Given the multiple types of cancer as well as their prevalence and grave consequences, a broad range of drugs have been developed and used for treatment, Cancer is among the diseases that results in the highest including synthesized compounds and those from natural mortality rate worldwide. Besides causing great sources (plants and bacteria).1 For example, paclitaxel is human suffering, this disorder generates enormous the active component of the plant called paciﬁc yew,5 and economic losses for both families and hospitals.1–3 It the anthracyclines are obtained from bacterial cultures is characterized by the accelerated growth and (Streptomyces).6 On the other hand, among the many abnormal proliferation of cells, and the ability of these antineoplastic compounds synthesized till date are plat- cells to spread to other tissues. inum-based drugs (e.g., cisplatin), analogues the natural Various types of tumorigenesis exist, such as carcino- product camptothecin (e.g., topotecan and irinotecan),7–9 mas and sarcomas as well as hematopoietic and neural and DNA topoisomerase enzyme inhibitors.

*For correspondence

Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-020-1750-2) contains supplementary material, which is available to authorized users. 50 Page 2 of 20 J. Chem. Sci. (2020) 132:50

Representative of the latter compounds are topotecan and herein examined for 8 trifluoromethylsulfonamide deriva- etoposide commonly administered for cancer therapy. tives and two reference compounds (topotecan and eto- Ongoing research continues to seek new topoisomerase poside): H-bond donors, H-bond acceptors, molecular inhibitors because of two important drawbacks of weight and the partition coefficient (log P). topotecan and etoposide: their toxic effect on different types of non-cancerous cells and the capacity of tumor cells to develop mechanisms of resistance to them.15,16 2.2 Protein and ligand structures Topotecan and etoposide act on DNA topology by inhibiting the topoisomerase enzymes.10 Topotecan The crystallographic structures of human Topo I (PDB code: inhibits DNA topoisomerase I (Topo I, EC 5.99.1.2) 1sc7) and II (PDB code: 3qx3) in complex with a 22-base pair DNA duplex were downloaded from the protein data bank and etoposide inhibits DNA topoisomerase II (Topo II, (http://www.rcsb.org/).26 The proteins were optimized with the EC 5.99.1.3), which are a target for the design of new 27 11–14 nanoscale molecular dynamics (NAMD) software program drugs with anticancer activity. and the parameters for docking simulation were obtained with On the order hand, some studies have demonstrated VMD 1.9.1.28 Before docking runs were made, the coordinates that the presence of a triflamide functional group favors of the protein were set, water molecules were removed, cytotoxic action on cancerous cells.17–21 However, hydrogen atoms were added to the polar atoms (considering pH although the cytotoxic action of triflamide derivatives at 7.4), and Kollman charges were assigned. The 3D structures on tumor cells is known, there is no evidence yet of the of topotecan and etoposide were downloaded from the Zinc12 molecular mechanism of topoisomerase inhibition. database.29 The structures of the ligands in two dimensions The aim of the present study is to examine the (2D) were sketched with ChemSketch (https://www.acdlabs. molecular interactions involved in the antitumor effect com/resources/freeware/chemsketch/)andoptimizedwiththe of trifluoromethylsulfonamide derivatives. Hence, Gaussian 16W and Gaussian 6.0 software in order to obtain the minimum energy conformation for the docking studied. The molecular docking simulations were carried out to minimum energies were obtained for each compound (con- explore whether or not these compounds inhibit Topo I vergence) as well as the RMS gradient.30 and II, important therapeutic targets used as a model for testing new chemotherapeutic drugs.22,23 Through this in silico analysis, the binding site and the binding 2.3 Molecular docking studies and virtual energies of the derivatives were established in order to mutations gain insights into the mechanisms of their pharmacological action. Decoys were generated and included in The protein-ligand interaction was observed on Autodock 31 docking simulations to add even greater rigor to the version 4.0 and AutoDock Tools 1.5.6. All the possible current inquiry. Furthermore, the generation of virtual rotatable bonds, torsion angles, atomic partial charges and mutants helped to predict the importance of certain non-polar hydrogens were determined for each ligand. For amino acid residues in the recognition between the tri- Topo I, the grid dimensions in AutoDock Tools were 90 x 90 x 90 A˚ 3 with points separated by 0.375 A˚ , centered at: X= flamide derivatives and the Topo I and II enzymes. 85.385, Y= -10.629 and Z= 10.945. For Topo II, the grid dimensions were 98 x 98 x 98 A˚ 3 with points separated by 0.375 A˚ , centered at: X= 33.82, Y= 96.868 and Z= 43.739. The hybrid Lamarckian Genetic Algorithm was applied for 2. Materials and Methods minimization, utilizing default parameters. A total of one hundred docking runs were conducted and the conformation 2.1 In silico analysis displaying the lowest binding energy (kcal/mol) was adopted of trifluoromethylsulfonamide derivatives for all further simulations. AutoDock Tools 1.5.6 was used to and known inhibitors of Topo I and II prepare the script and files as well as to visualize the docking results, which were edited in Discovery 4.0 Client.32 The The physicochemical and toxicological properties of tri- genetic algorithm parameters used for docking were the fol- fluoromethylsulfonamide derivatives and known inhibitors lowing: number of GA runs: 100, population size: 150, (topotecan and etoposide) were previously determined maximum number of evals: 2500000 and maximum number with the OSIRIS DataWarrior V4.7.2 program (http:// of generations: 27000. The MD (molecular dynamics) sim- www.organic-chemistry.org/prog/peo/),24 as were their ulations were carried out for a time 10 nanosecond scale using pharmacokinetic and drug-likeness properties with the the NAMD program. All simulations performed in this study SwissADME server.25 Physicochemical properties are reached equilibrium, being used for the following analyzes. commonly assessed with Lipinski’s rules, which are based To explore the structural alterations caused by mutations, on the ADME properties (absorption, distribution, meta- virtual mutations were carried out in Modeller 9.10 pro- bolism and excretion). Accordingly, four parameters were gram,33 each involving a change in a single amino acid. J. Chem. Sci. (2020) 132:50 Page 3 of 20 50

During the docking studies, the three amino acid residues and etoposide. The value of LogS, on the other hand, is selected for these mutations had been found to intervene in the important for evaluating the pharmacokinetic parameters binding mode between the proteins (Topo I and II) and most of drug distribution and absorption.36 For all the test and of the triflamide derivatives. Each mutant was minimized to reference compounds, this value was within the permitted be subjected to docking simulations. limits (-4.5 to -1). Also related to absorption is the polar surface area, calculated to be below 140 square angstrom 2.4 Validation of molecular docking through for all the trifluoromethylsulfonamide derivatives, sug- the generation and analysis of decoys gesting that they are able to adequately cross the plasma membrane.37 Decoys were generated to confirm that the present triflamide The three parameters utilized tocompare theinhibitory derivatives act as dual inhibitors of Topo I and II, and to capacity of the compounds were ligand efficiency, lipo- demonstrate that the protein-ligand recognition results are not philic ligand efficiency and ligand efficiency lipophilic the product of a positive bias. The decoy structures have price.38 According to the value of ligand efficiency, characteristics similar to the ligand from which they were expressed in kcal/mol per non-H atom, the derivatives generated, but with weak bond energies compared to the suggest that a greater effectiveness of the test compounds previously reported triflamide derivatives. The DUD-E ser- 34 that is more effective than topotecan and etoposide. ver was employed to generate the decoys, and 37 of these Lipophilic ligand efficiency refers to the necessary structures for each ligand were subjected to molecular lipophilicity for a drug to be considered for clinical use. docking. For practical purposes, the only valid ligands, according to the computational study, were the two reference The test compounds showed values above 5, within the compounds (topotecan and etoposide) and two of the normal range. Finally, the ligand efficiency lipophilic derivatives (the one with the greatest affinity for each of the price represents the price of thecompound efficiency paid 39 topoisomerase enzymes, 1g and 1e, respectively). in LogP. Its value fell within the desirable range, from 0–7.5, for all the trifluoromethylsulfonamide derivatives. Based on all the parameters herein determined, the test 3. Results and Discussion compounds proved to be good candidates for further research as anticancer drugs. 3.1 Determination of the pharmacokinetics The compounds were evaluated with Lipinski’s rules and toxicological properties to explore the possibility of biological activity after of trifluoromethylsulfamide derivatives and known being administered.40 All the test compounds complied inhibitors of Topo I and II with the four considerations of this rule: not having more than 5 H-bond donors or more than 10 H-bond An in silico analysis on the OSIRIS DataWarrior acceptors, a molecular weight \500 and a LogP value program established the physiochemical properties and \5. The results suggest that these derivatives are not indicators of potential toxicity of the 8 trifluo- only likely to carry out their pharmacological action but romethylsulfonamide derivatives, topotecan and may also serve as lead compounds for the design of new etoposide.24 The properties examined in each of the drugs (Table 2). compounds were: molecular weight, LogP (partition The bioavailability of the triflamide derivatives was coefficient), LogS (solubilization coefficient), polar assessed by using the rules of Ghose, Verber, Egan and surface area, ligand efficiency, lipophilic ligand effi- Muegge. Whereas etoposide was found to violate all ciency, ligand efficiency lipophilic price, and H-ac- the aforementioned rules, the 8 test compounds com- ceptor and H-donor capacity. ply with all of them, with the exception of 1d (the Since the test compounds meet the specifications for bioavailability of which may be reduced by substituent each of the aforementioned parameters (Table 1), they CF3). The bioavailability data are significant for new can be considered as candidates for cancer therapy. molecules with possible anticancer activity. Specifically, the molecular weight of all the triflamide Pharmacokinetics (Table 3), on the other hand, were derivatives was below 450, being even lower than that of examined with several parameters, including the GI and topotecan and etoposide. Hence, the derivatives may be BBB value. While the former estimates the capacity of absorbed more quickly than the reference compounds. a drug to be absorbed in the gastrointestinal tract (GIT), Another parameter indicative of absorption is the LogP the latter probes its ability to cross the blood-brain value,35 which expresses the hydrophobicity of a com- barrier. According to the present results, it is highly pound. The greater the hydrophobicity, the more its probable that topotecan and all of the triflamide ability to penetrate the plasma membrane. Each of the 8 derivatives except 1g and etoposide can be absorbed by derivatives displayed a higher LogP value than topotecan the GIT. The BBB data demonstrate that none of the 50 Page 4 of 20 J. Chem. Sci. (2020) 132:50 compounds can cross the blood-brain barrier (which evidence any risk of mutagenicity, tumorigenicity, ter- could indicate selectivity) and thus would not be ben- atogenicity or irritant effects (Table 4). Compounds 1b- eficial for the treatment of diseases of the nervous g apparently does not represent any serious health risks, system. Another relevant parameter, Log Kp, evaluates thus eliminating this possible impediment for them to the capacity of the compounds to permeate the skin. be considered as candidates for cancer therapy. Since the most negative value represents the greatest skin permeability, 1g showed an advantage over the other compounds, suggesting that the NO2 substituent could have an influence on this absorption mechanism. 3.2 Molecular docking Additionally, an analysis was made of the ability of of the trifluoromethylsulfamide derivatives the compounds to pass through biological membranes and known inhibitor (topotecan) of Topo I via two types of membrane transport proteins: the ATP binding transporters and the superfamily of cyto- Initially, to identify the triflamide binding sites, a blind chrome P450 (CYP). Compound 1g and two reference docking study was carried out, the results showed that drugs (topotecan and etoposide) are probably sub- such derivatives bind to some of the key amino acid strates of the ATP binding transporter, which if true residues that reference compounds (topotecan and would imply their tendency to be eliminated through etoposide). In addition, we carried out a directed the membrane. Regarding the five CYPs considered, docking at recognition site, evidencing that the tri- the derivatives are more likely to be substrates of flamide derivatives studied here bind to the active site CYP2C19 and CYP3A4, while the predilection of of both enzymes (Topo I and Topo II) and identify the topotecan is towards CYP3A4 and CYP2C9. Etopo- amino acid residues that they share. side is a probable substrate of only one CYP. Inter- Docking studies were conducted to explore the estingly, the data reveal that the derivatives are most molecular interaction of the test compounds and ref- likely eliminated through this compartment, and erence drugs at the active site of human Topo I. The therefore can be proposed as anticancer agents. binding mode, binding energy and interaction residues The possible risk of toxicity was also estimated for were evaluated. the test and reference compounds, finding that topote- According to the initial analysis of the binding can, etoposide and most of the 8 derivatives do not mode, the test compounds (1a-1h) bind in a manner

Table 1. Estimated chemical properties of triﬂamide derivatives 1a-h and two known inhibitors of Topo I and II.

H H Compound R2 R4 MW LogP LogS PSA LE LLE LELP A D

1a H H 311.279 3.365 -3.533 80.85 0.446 3.141 7.541 5 1 1b H Br 390.175 4.091 -4.367 80.85 0.418 2.317 9.771 5 1 1c H Cl 345.724 3.971 -4.269 80.85 0.422 9.409 9.409 5 1 1d HCF3 379.276 4.214 -4.311 80.85 0.367 2.206 11.482 5 1 1e H OCH3 341.305 3.295 -3.551 90.08 0.403 3.171 8.173 6 1 1f HCH3 325.306 3.709 -3.877 80.85 0.423 2.777 8.753 5 1 1g HNO2 356.276 2.444 -3.993 126.6 0.384 4.003 6.355 8 1 1h F 329.269 3.466 -3.847 80.85 0.423 3.015 8.186 5 1 Topotecan 421.452 0.461 -1.959 103.2 0.377 8.061 1.223 8 2 Etoposide 588.560 0.672 -3.952 160.83 0.2035 5.5576 3.3051 3 3

MW= molecular weight; LogP= partition coefficient; LogS= solubilization coefficient; PSA= polar surface area; LE= ligand efficiency; LLE= lipophilic ligand efficiency; LELP= ligand efficiency lipophilic price; HA= H-bond acceptors; and HD= H-bond donors. J. Chem. Sci. (2020) 132:50 Page 5 of 20 50 similar to topotecan at the active site of the enzyme bonds. In the reference compound (topotecan), carbon- Topo I. Consequently, they likely act on the same hydrogen bonds (CHÁÁÁO) are present between the molecular target, interacting with important amino dimethylamine group and the amino acid side chains acid side chains at the active site.41 An example of the Asn631 and Asp533, and conventional hydrogen binding mode observed in the docking study is shown bonds are formed with DNA residues (TGP11, DG12 for topotecan and 1g. The conformations of 1g and and the phosphodiester link Ptr723). The existence of topotecan are similar, implying that the former com- similar interactions for 1b (with DG12 and Thr718), pound probably exerts an inhibitory effect on Topo I 1e (with Arg364, TGP11 and DG12), 1f (with DG12), (Figure 1). and 1g (with Arg488, Lys532, PTR723, DT10 and Although the binding mode of all the test com- TGP11) confirm a like interaction mechanism between pounds was similar to that of topotecan, differences these compounds and topotecan. exist in the affinity of each for the enzyme. The A conventional hydrogen bond (OHÁÁÁO, NHÁÁÁO) docking simulations with the triflamide derivatives was formed between Topo I and compounds 1b, 1e (Table 5) gave the binding energies (DG, expressed in and 1h. The oxygen of the carbonyl group of these kcal/mol), the amino acid residues at the active site of derivatives bound with residues Thr718, Arg364 and the enzymes, and the polar and hydrophobic interac- TGP11, respectively. On the other hand, the NH of the tions formed. triflamide moiety in 1c, 1d, 1e, 1g and 1h forms a Of all the test compounds, 1g showed the best hydrogen bond with the DNA fragment (DG12) or the binding energy value (-7.4 kcal/mol), which is cor- amino acid chain (Asp533), depending on the ligand. roborated by a previous study demonstrating that this Another hydrogen bond was encountered between the compound has a low IC50 value (lM) in most of the SO2 group of 1a and 1h and two amino acids of the human cell lines used to test its cytotoxic activity (e.g., active site of Topo I (His632 and Arg488). IC50 = 35.02±4.0 for PC-3 and IC50 = 27.61±1.1 for Hydrophobic and electrostatic interactions were also SKLU-1).17 observed for most of the derivatives herein examined. The docking study revealed that the reference Electrostatic pi-anion interactions were exhibited compound (topotecan) and most of the 8 derivatives between the amino acid Asp533 and the electron-de- bind to certain amino acid residues (Arg364, Arg488, ficient quinoline ring of topotecan, 1g and 1h. Like- Lys532, Ile535, Asp533 and His632) in the active site wise, the benzene ring of 1b and 1c showed a pi-cation of Topo I, as well as to a DNA fragment (DG12 and interaction with the guanidinium moiety of Arg488, as TGP11). These residues are involved in hydrophilic, did the benzene ring of 1d and 1e with Arg364. hydrophobic and electrostatic interactions with most A hydrophobic interaction (pi-sigma) was identi- of the ligands. fied between the DNA residue (DG12) and the aro- The observed hydrophilic interactions entailed car- matic ring in 1a. Other hydrophobic interactions bon-hydrogen bonds and conventional hydrogen include those of the aromatic ring of 1b with the imidazole ring of Hys362 (T-shaped pi-pi) and with the amino acid residues Lys532 and Ile535 (pi-alkyl). Table 2. Estimated druglikeness properties for triflamide Similar interactions are displayed for 1c and 1f. derivatives 1a–h and known inhibitors of Topo I and II. Meanwhile, a pi-alkyl interaction between DG12 and Compound Ghose Veber Egan Muegge the carbon of the CF3 on an aromatic ring was detected for 1d. 1a Yes Yes Yes Yes Halogen bonds were found between the amino acid 1b Yes Yes Yes Yes side chain and the trifluoromethyl group of most 1c Yes Yes Yes Yes 1a 1d No* Yes No* No* derivatives, including (with Arg488, Ile535, 1e Yes Yes Yes Yes Cys630 and Asn631), 1c (with Asp533 and His632), 1f Yes Yes Yes Yes 1d (with Asn631, His632 and Gln633), 1e (with 1g Yes Yes Yes Yes Arg488, Lys532 and Asp533), 1f/1g (with Arg364), 1h Yes Yes Yes Yes and 1h (with Asn631). This type of interaction was Topotecan Yes Yes Yes Yes also evidenced between the trifluoromethyl group in Etoposide No* No* No* No* 1b and DNA fragments (DA113 and DT10), and *1d: Ghose: LogP[5.6; Egan: LogP[5.88; Muegge: between the Br substituent of the aromatic ring of the HA[10. same compound and Ile535. Additionally, such an *Etoposide: Ghose: MW[480; Veber: PSA[140; Egan: interaction was established between the Cl substituent PSA[131.6; Muegge: PSA[150, HA[10. of the aromatic ring of 1c and residues Phe529 and 50 Page 6 of 20 J. Chem. Sci. (2020) 132:50

Table 3. Estimated pharmacokinetic properties of triﬂamide derivatives 1a-h and known inhibitors of Topo I and II. Inhibitor GIT BBB per- P-gp Log Kp (skin Compound absorption meant substrate permeation) CYP1A2 CYP2C19 CYP2C9 CYP2D6 CYP3A4

1a High No No -6.15 cm/s No Yes No No No 1b High No No -6.40 cm/s No Yes No No Yes 1c High No No -6.17 cm/s No Yes No No No 1d High No No -6.19 cm/s No Yes No No Yes 1e High No No -6.61 cm/s No Yes No No No 1f High No No -6.23 cm/s No Yes No No No 1g Low No Yes -7.14 cm/s No No No No No 1h High No No -6.23 cm/s No Yes No No No Topotecan High No Yes -8.0 cm/s No No Yes No Yes Etoposide Low No Yes -9.46 cm/s No No No Yes No

Table 4. Estimated toxicological parameters of the 1-(Triﬂuoromethylsulfonamido)-propan- 2-yl esters 1a-h and known inhibitors of Topo I and II. Toxicity Risk Compound Mutagenicity Tumorigenicity Reproductive effects Irritant effects

1a 11 1 3 1b 11 1 1 1c 11 1 1 1d 11 1 1 1e 11 1 1 1f 11 1 1 1g 11 1 1 1h 11 2 1 Topotecan 11 1 1 Etoposide 11 1 1

Values assigned to indicate the risk of toxicity: 1=none, 2=low and 3=high.

Figure 1. Binding mode of topotecan (red) and compound 1g (purple) with DNA topoisomerase I (Topo I), illustrated in complex with DNA. Topo I is portrayed by a solid ribbon, the DNA backbone by tubes and the DNA base pairs by the ladder formation. J. Chem. Sci. (2020) 132:50 Page 7 of 20 50

Table 5. Docking results of topotecan and the 1-(Triﬂuoromethylsulfonamido)-propan-2-yl esters 1a-h at the active site of DNA topoisomerase I. Interactions Binding energy Compound DG (kcal/mol) Interacting residues Polar Hydrophobic

1a -6.28 Arg364, Arg488, Gly531, Asp533, Ile535, Arg590, His632, – Cys630, Asn631, His632, Arg488, DG12 Gln633, Thr718, PTR723 Lys532 TGP11, DG12 – 1b -7.19 Arg364, Arg488, Gly531, Lys 532, Asp533, Ile535, Thr718, Ile535 Asn631, His632, Gln633, Arg364 Arg488 Thr718 His632 TGP11, DT10, DG12, DA113 Lys532 DG12, DA113 1c -7.25 Arg488, Phe529, Gly531, – Lys532, Asp533, Ile535, Arg590, Cys630, Asn631, His632, Gln633, – Ile535, Phe529, Tyr537 Thr718, PTR723 TGP11, DT10, DG12, DA113 – 1d -6.58 Arg364, Arg488, Lys532, Arg364, – Asp533, Ile535, Asn631, Asp533, His632, Gln633, Thr718, His632, PTR723 Gln633 TGP11, DT10, DG12, DA13 DG12 – 1e -6.50 Arg364, Arg488, Lys532, – Asp533, Ile535, Asn631, Arg364, – His632, Thr718, PTR723 Arg488, Asp533 – TGP11, DG12 TGP11, DT10, DG12, DA113 – TGP11, DG12 1f -6.80 Arg364, Arg488, Lys532, Asp533, Ile535, Asn631, – Lys532, Ile535 His632, Thr718, Ala715 TGP11, DG12 TGP11, DG12 – 1g -7.40 Arg364, Arg488, Lys532, Arg364, – Ile535, Thr718, PTR723 Arg488, Lys532, TGP11, DT10, DG12 PTR723, – TGP11, DT10, DG12 1h -6.91 Arg364, Arg488, Arg364, – Asp533, Asn631, His632, Arg488, PTR723 Asp533, His632, PTR723, TGP11, DT10, DG12 – TGP11 Topotecan -9.21 Arg364, Arg488, Lys532, Asp533, Ile535, Asn631, Asp533, – 50 Page 8 of 20 J. Chem. Sci. (2020) 132:50

Table 5. (contd) Interactions Compound Binding energy DG (kcal/mol) Interacting residues Polar Hydrophobic

His632, Thr718, PTR723 PTR723, TGP11, DT10, DG12, Asn631 – DA13, DC112, DA113 –

Tyr537. All these interactions between the test com- binding site. The docking simulations allowed for the pounds and Topo I are schematized in Figure 2. identification of the amino acids and type of interac- Two equivalent interactions are depicted (Figure 3), tions involved in the binding between Topo II and the comparing the reference compound (topotecan) and triflamide derivatives, and for the calculation of the the best triflamide derivative (1g): a) a conventional DG values (Table 6). Because of binding to the active hydrogen bond between the phosphodiester link site of this enzyme, the triflamide derivatives should Ptr723 and the hydroxyl substituent in topotecan is certainly be a useful scaffold to design and develop similar to that found with the NO2 substituent in 1g;b) possible anticancer agents. a pi-anion interaction between amino acid Asp533 and In most of the compounds herein analyzed, hydro- the amino substituent of the quinoline in topotecan is philic, hydrophobic and electrostatic interactions were similar to that detected with the phenyl ring of 1g formed (Figure 4). The substituents of the rings in (Figure 3). etoposide formed conventional hydrogen bonds and The interactions revealed in the current molecular C-H bonds with DNA fragments (DT9, DG10, DC11 docking simulations coincide with previous reports on and DA12) and with the Asp479 residue of the Topo II topotecan and its derivatives,42–44 suggesting that tri- complex. Additionally, there is a pi-cation electrostatic flamide derivatives act on the active site of Topo I. interaction of Arg503 with etoposide and 1b. Similar This emphasizes the importance of in silico analyses, hydrophilic interactions with the DNA fragments which have helped to predict the target of new com- (DT9 and DA12) of Topo II have been reported for 1b, pounds proposed as inhibitors of Topo I.45,46 1d and 1g.49 In the current study, one of the most important hydrophilic interactions is between the amino acid 3.3 Molecular docking residue Arg503 in the active site of the enzyme and of the trifluoromethylsulfamide derivatives most of the triflamide derivatives. Therefore, this and known inhibitor (etoposide) of Topo II interaction may be involved in the mechanism of action of the derivatives on Topo II, as previously Additionally, the binding mechanism of triflamide described for etoposide.48 Another amino acid in the derivatives on Topo II was evaluated and compared to active site of this enzyme, Gln778, forms hydrogen that of etoposide, a selective Topo II inhibitor.47 bonds with 1d, 1f and 1g.In1h, hydrophilic interac- According to the protein-ligand interactions observed, tions are also observed with Glu477, Gly478, Ser480 the test compounds and etoposide bind similarly at the and Ala481. Other types of hydrophobic interactions active site of this enzyme. Some amino acid residues detected for the test compounds include pi-sigma, pi- (e.g., Glu477, Gly478, Asp479, Arg503 and Gln778) alkyl and T-shaped pi-pi, being mostly formed with in the active site of the enzyme48 interact with sub- DNA fragments of the Topo II complex. The same stituents in most of the derivatives, implying that they pattern was found for halogen bonds. likely participate in the mechanism of action. Since the The present results demonstrate that etoposide and DG values were similar for the binding of the tri- all the triflamide derivatives form interactions with flamide derivatives with both Topo I and II, these DNA fragments and with amino acid residues in the compounds could be dual inhibitors acting on the two active site of Topo II.48 Since the test compounds enzymes. interact with both Topo I and II, they could be con- Given that 1e displayed the best binding energy sidered dual inhibitors. According to the docking value (-7.28), the mesomeric effect of the methoxy simulations, the substituent in the para position of the substituent (OCH3) may greatly influence the Topo II aromatic ring of the triflamide derivatives has a great J. Chem. Sci. (2020) 132:50 Page 9 of 20 50

Figure 2. Prediction of interactions of the triﬂamide derivatives and topotecan with the active site of DNA topoisomerase I. The 3D model shows the hydrophilic bonds as well as the main amino acid residues that are part of the active site of the enzyme. The non-hydrogen bonds are omitted for clarity. In the 2D model, the following interactions are portrayed with dotted lines: conventional hydrogen bonds (light green), carbon-hydrogen (dark green), pi-anion (red), pi-cation (orange), pi-sigma (purple), pi-Alkyl (light pink), T-shaped pi-pi (dark pink), pi-sulfur (yellow) and halogen (cyan). The solvent- accessible surface is illustrated for the amino acid residues and ligands. The amino acids are denoted in pink (basic), yellow (acid) and cyan (polar). 50 Page 10 of 20 J. Chem. Sci. (2020) 132:50

Figure 2. continued J. Chem. Sci. (2020) 132:50 Page 11 of 20 50

Figure 2. continued

Figure 3. 2D model of the interactions of amino acid residues with topotecan and compound 1g. The hydrophilic and electrostatic interactions are represented with green and red dotted lines, respectively. influence on its interaction with Topo I and II, evi- the topology of DNA topoisomerase. Also, in a paper denced by the similar amino acids involved. that is being prepared by our research group, the It is noteworthy that both topotecan and all deriva- compounds present here and other triflamide derivatives of triflamides have structural features in com- tives were subjected to a QSAR study and evaluated mon, such as the ester functionality which is present in by relaxation tests in the presence of the human all compounds 1a-g and topotecan (ring E) and the recombinant enzymes Topo I and Topo II. In this aromatic functionality, which is the quinoline ring in work, the inhibition on these two enzymes was topotecan, while in the triflamides it is a benzene ring. demonstrated, as it has been previously demonstrated With respect to etoposide and triflamides, it is evident in sulfonamide derivatives, which are isostere of that both topotecan and triflamides have an ester triflamides.51,52 group; another structural similarity is the aromatic In addition, several reports show that sulfonamide benzene ring present in triflamides and in etoposide. derivatives interact with key residues in the recogni- It is important to mention that triflamides are iso- tion of DNA toposiomerase enzymes,53–55 such as stere of sulfonamide, which have been reported in Gly478, Asp479, Arg503, DT9, DG10 and DA12 various works,50 which act as inhibitors of DNA present in Topo II.50 toposiomerases, being demonstrated by molecular On the other hand, all conformations obtained docking studies. For this reason, we have shown that using the semi-empirical method Austin model 1 triflamides recognize the same interaction site as the (AM1) were optimized. In Table S3 (Supplemen- reference compounds. Additionally, there is experi- tary Information) the minimum energy data of the mental evidence that sulfonamide derivatives act on optimized structure (convergence), of the triflamide 50 Page 12 of 20 J. Chem. Sci. (2020) 132:50 derivatives as well as of the reference compounds the series of triflamides, the compound 1g presented (topotecan and etoposide) are reported. The RMS less energy. gradient for conformational analysis is also reported. At the end of the geometric optimization process, 3.4 Molecular docking validation through decoys taking the numerical results of the computational analysis calculation of the set of AM1 bases contained in Table S3 (Supplementary Information) we can observe To confirm that the 8 triflamide derivatives herein that the molecule with the lowest total energy value examined can actually act as dual inhibitors of human during the geometric optimization is etoposide being Topo I and II, 37 decoys were generated for each cataloged as the most stable structure and therefore compound. For greater clarity, the only results inclu- possibly with the least reactivity. While the molecule ded in Tables S1 and S2 (Supplementary Information) with the highest total energy was the etoposide. From are those related to the binding energies of the 37

Table 6. Docking results of etoposide and 1-(Triﬂuoromethylsulfonamido)-propan-2-yl esters 1a–h at the active site of DNA topoisomerase II. Type of interactions Binding energy Compound DG (kcal/mol) Interacting residues Polar Hydrophobic

1a -6.48 Gly478, Asp479, Ser480, Arg503 – Arg503, Gly504 DC8, DT9, DG10, DA12, DG13 – DT9, DG13 1b -7.1 Asp479, Arg503 Arg503 – DC8, DT9, DG10, DA12, DG13 DA12 DT9, DA12 1c -7.26 Glu477, Gly478, Asp479, Arg503 – Ser480, Ala481, Arg503 DC8, DT9, DG10, DA12, DG13 DA12 – 1d -5.8 Asp479, Arg503, Gly504, Gln778, Met782 Arg503, Gln778 Met782 DC7, DC8, DT9, DA12, DG13, DC14 DT9 DT9 1e -7.28 Arg503, Gln778 Arg503 – DC8, DT9, DA12, DG13 DC8 DC8, DT9 1f -6.42 Gly478, Asp479, Leu502, Arg503, Gly504, Gln778 Gln778 – DC8, DT9, DG13 DC8 DC8 1g -7.22 Arg503, Gly776, Gln778 Arg503, Gln778 – DC8, DT9, DA12, DG13 – DT9 1h -7.08 Glu477, Gly478, Asp479, Glu477, Ser480, Ala481, Arg503, Gly478, Arg503 Gly504, Tyr821 Arg503, Asp479, DC8, DT9, DG10 Ser480, Ala481 DT9 DT9 Etoposide -9.76 Lys456, Asp479, Arg503 Asp479 – DT9, DG10, DC11, DA12, DG13 DT9, DG10, DG10, DC11 DC11, DA12 J. Chem. Sci. (2020) 132:50 Page 13 of 20 50

Figure 4. Prediction of interactions of triﬂamide derivatives and etoposide with the active site of DNA topoisomerase II. The 3D model depicts the hydrophilic bonds formed, as well as the main amino acid residues in the active site of the enzyme. The non-hydrogen bonds are omitted for simplicity. In the 2D model, the following interactions are depicted with dotted lines: conventional hydrogen bond (light green), carbon-hydrogen bond (dark green), pi-anion (red), pi-cation (orange), pi-sigma (purple), pi-alkyl (light pink), T-shaped pi-pi (dark pink) and halogen (cyan). The solvent accessible surface is illustrated for the amino acid residues and the ligand. The amino acids are denoted in pink (basic), yellow (acid) and cyan (polar). 50 Page 14 of 20 J. Chem. Sci. (2020) 132:50

Figure 4. continued J. Chem. Sci. (2020) 132:50 Page 15 of 20 50

Figure 4. continued

Figure 5. Comparison of the binding mode of the reference drugs (topotecan and etoposide), compounds 1g and 1e, and the selected decoys. Topo I (A) and II (B), depicted by a solid ribbon, are illustrated in complex with the DNA. In the red oval are portrayed topotecan and 1g as well as etoposide and 1e. The decoy selected for topotecan and 1g as well as the one chosen for etoposide and 1e are indicated by the green oval. decoys generated for the two selective inhibitors of each derivative (green ovals) (Figure 5A and B). Topo I and II (topotecan and etoposide, respectively) Compared to the test and reference compounds, the and for the triflamide derivatives with the best inter- binding mode of the selected decoys is clearly distinct, action energy for Topo I and II (1g and 1e, respec- evidenced by their lack of interaction at the active site tively). The corresponding decoys exhibited much of Topo I or II. These findings demonstrate that the use lower interaction energies and a different binding of decoys helps to verify in silico studies.34,56,57 mode than the test compounds and two reference drugs. Consequently, the decoy analysis provides further evidence that the 8 derivatives could indeed 3.5 Generation of Topo I and Topo II virtual have two molecular targets, interacting at the active mutants site of the two topoisomerase enzymes proposed in this study. The binding mode of the ligands with Topo 3.5a Analysis of binding energy between Topo I I and II is shown for the reference drugs, selected and Topo II and triflamide derivatives:Oncethe derivatives (red oval), and the best decoy generated for interactions between the triflamide derivatives and 50 Page 16 of 20 J. Chem. Sci. (2020) 132:50

0 -1 -2 -3 -4 -5 -6 -7 ΔG (Kcal/mol) Binding Energy -8 -9 -10 123456789 Wild-type -9.21 -6.28 -7.19 -7.25 -6.58 -6.5 -6.8 -7.4 -6.91 D533A -8.74 -5.33 -6.04 -6.13 -5.05 -5.43 -5.58 -5.51 -5.33 N631L -9.08 -6.19 -5.43 -6.45 -5.82 -5.19 -5.24 -6.45 -5.06 R488D -8.8 -5.78 -5.8 -5.86 -5.59 -5.81 -4.84 -6.48 -5.9

Topotecan 1a 1b 1c 1d 1e 1f 1g 1h

Figure 6. In silico prediction of the binding energies of the reference and test compounds with three mutations of Topo I.

0 -2 -4 -6 -8 ΔG (Kcal/mol) Binding Energy -10 -12 123456789 Wild-type -9.76 -6.48 -7.1 -7.26 -5.8 -7.28 -6.42 -7.22 -7.08 R503A -8.13 -6.39 -6.89 -6.29 -5.24 -6.91 -6.08 -6.53 -6.67 D479V -7.83 -5.84 -6.95 -6.45 -5.42 -6.51 -6.31 -6.38 -5.98 Q778P -8.22 -6.13 -6.37 -6.63 -5.63 -6.82 -6.24 -6.02 -6.31

Etoposide 1a 1b 1c 1d 1e 1f 1g 1h

Figure 7. In silico prediction of the binding energies of the reference and test compounds with three mutations of Topo II.

two molecular targets (Topo I and II) were 3.5b Analysis of binding mode between Topo I examined and verified, three virtual mutants of and Topo II and triflamide derivatives: For clarity, Topo I (D533A, N631L and R488D) and three of interactions are only depicted between a single mutant Topo II (R503A, D479V and Q778P) were and some of the ligands, the latter including the generated. A coupling analysis was carried out for specific inhibitors (topotecan and etoposide) and one each of the triflamide derivatives with the six triflamide derivative (with the best binding energy) for mutantstoevaluatewhetherachangeinanamino each topoisomerase enzyme (Topo I and II). In the acid residue affected the binding energy. There was Topo I protein, the change from aspartate (D) to a lower binding energy for the interactions of the alanine (A) at position 533 significantly affected the derivatives with the mutants versus the wild-type binding energy for all test and reference compounds. enzymes (Figures 6 and 7), suggesting that the The interaction of the corresponding mutant with mutated amino acids likely affected the stability of topotecan and 1g (the latter being the best derivative the enzymes and the formation of bonds with the for this enzyme) revealed the lack of a pi-anion reference and test compounds. interaction, which did indeed take place between these J. Chem. Sci. (2020) 132:50 Page 17 of 20 50

Figure 8. Prediction of the binding mode of the virtual mutant D533A of Topo I with topotecan and compound 1g. These two ligands are portrayed in stick representation and Topo II as line ribbon. The mutation is highlighted in a rectangle. For greater clarity, only hydrophilic interactions are illustrated (green).

Figure 9. Prediction of the binding mode of the virtual mutant R503A of Topo II with etoposide and compound 1e. These ligands are depicted in stick representation and Topo II as line ribbon. The mutation is highlighted in a rectangle. For greater clarity, only hydrophilic interactions are included (green). ligands and the wild-type enzyme (Figure 8). Hence, Overall, the substitution of a single amino acid in the D533 appears to be indispensable for the aromatic ring active site appears to cause a change in the of each inhibitor to strongly bind to the enzyme. A conformation of the protein that leads to a decrease similar effect was observed upon docking topotecan in molecular coupling. 42 with the D533G mutation, indicating the importance For the mutations in Topo II, there was a similar of the D533 amino acid for the interaction between the tendency to a decrease in the stability of the binding protein and 1g. Likewise, similar events were detected energy when the protein was coupled to the triflamide for all derivatives in their interaction with mutants derivatives. Thus, amino acids R503, D479 and Q778P N631L and R488D. For example, the change from seem to have a key role in the stability and confor- arginine (R) to aspartic acid (D) at position 488 of mation of Topo II upon interacting with the reference Topo I produced the loss of the conventional hydrogen and test compounds. This is exemplified by the inter- bond in the interaction with 1g (data not shown). actions of the mutant Topo II R503A with etoposide 50 Page 18 of 20 J. Chem. Sci. (2020) 132:50 and 1e (Figure 9). The docking simulations evidenced Conflict of Interest The authors declare that they have no a lack of a pi-cation interaction with etoposide and no conflict of interest. hydrogen and halogen bonds with 1e. 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