Anticancer potential of some β-diketonates: DNA interactions, protein binding properties, and molecular docking study

Abstract With the goal to discover a new antitumor drug with the better or similar effects to existing, a small series of β-diketonate was tested on a cisplatin-resistant MDA-MB-231 and HeLa tumor cell lines, and nontumor MRC-5 cell line. All compounds showed notable cytotoxicity against both tumor cell lines and good selectivity. Importantly, β-diketonates displayed greater selectivity than cisplatin, which is the crucial factor for a new antitumor drug candidate. Further, investigations with biomacromolecules such as DNA and serum albumin were performed. Investigations showed that tested compounds bind to DNA through intercalation and have appropriate affinity for binding to bovine serum albumin. In addition, the molecular docking study was performed to investigate more specifically the sites and binding mode of tested β-diketonate to DNA or bovine serum albumin. In conclusion, all results indicated the big potential of these compounds for application in clinical practice in future. Graphical Abstract


Introduction
Molecules such as b-diketonates have been widely used as starting substrates in medicinal chemistry and pharmacology to obtain new potential drugs. (Zherebker et al. 2014;Kabirifard et al. 2015;Guo et al. 2016;Huang et al. 2016;Kulakov et al. 2017;Gein et al. 2018;Shehab and El-Bassyouni 2018;Stepanova et al. 2019). This structural fragment can be found in many biologically active compounds and natural products. Recently, a large number of natural products have been reported to show very good anticancer potential. (Mahal et al. 2017;El-ajaily et al. 2019;Mahal et al. 2019;Duan et al. 2021;Mohapatra et al. 2021). Also, many b-diketonate derivatives have been synthesised due to very favorable synthetic conditions. (Liu et al. 2006;Uchil et al. 2007;Jiang et al. 2012;Kormanov et al. 2017;Obydennov et al. 2018). These molecules are at the center of research for many scientists, so there are many studies that point to the importance of b-diketonates in medical chemistry and pharmacology as important precursors for the development and discovery of new drugs. Numerous aspects of the application of b-diketonates, such as the synthesis and isolation of their derivatives, the development of new synthetic methodologies, the evaluation of their biological properties as well as the mechanisms of action of b-diketonate derivatives, are widely studied. (Liu et al. 2006;Uchil et al. 2007;Jiang et al. 2012;Zherebker et al. 2014;Kabirifard et al. 2015;Guo et al. 2016;Huang et al. 2016;Kormanov et al. 2017;Kulakov et al. 2017;Gein et al. 2018;Obydennov et al. 2018;Shehab and El-Bassyouni 2018;Stepanova et al. 2019). In addition to the fact that b-diketonates are important structural segments of a large number of biologically important molecules and potential drugs, they are also widely used for the synthesis of many biologically active molecules and natural products. Bearing in mind previous, and knowing that the tumor is one of the most common causes of death in humans and that no adequate cure has been found yet for this vicious disease, we decided to test the antitumor potential of a small series of b-diketonates that we previously reported. (Petronijevic et al. 2017;Joksimovi c et al. 2019aJoksimovi c et al. , 2019c).

Results and discussion
The molecular structures of tested b-diketonates A-E whose cytotoxic potential was investigated are presented in Figure 1. The antitumor potential and selectivity of b-diketonates A-E was examined on two tumor cell lines (HeLa and MDA-MB 231) and normal MRC-5 cells after 24 and 48 h treatment with a range of concentrations. Based on the obtained results, IC 50 values were calculated and presented in Table 1.
As shown in Table 1, all compounds, except A, showed high cyototoxic activity against cancer cell lines. After 48 h incubation with B-E, their IC 50 values were close to the IC 50 values of cisPt. MDA-MB 231 and HeLa cells displayed diverse sensitivity on tested compounds. Obtained IC 50 values were lower in HeLa cells, indicating a higher sensitivity of these cells comparing to MDA-MB 231.
Based on these results, we selected four b-diketonates (B-E) for further testing. To determine their selectivity, the cytotoxic effects of these compounds were examined on human fetal lung fibroblasts. MRC-5 cells were treated with the same concentrations of b-diketonates for 24 and 48 h. The calculated IC 50 values are presented in Table 1. Significantly, all IC 50 values were higher than 100 lM, indicating good selectivity of compounds B-E. This is very important since good selectivity is one of the crucial factors for a new antitumor drug candidate.
Examining the dependence of the activity on the structure of the compounds, it can be concluded that the best activity of the compounds was when the 3-(4-methoxybenzamido)phenyl (C) or 4-(benzyloxy)-3-methoxystyryl (E) groups were bonded to the b-diketonates scaffold. b-diketonates, which contained a 3-(cyclopropanecarboxamido)phenyl (B) and 4-hydroxy-3-methoxystyryl (D) groups as its residue, also showed good anticancer activity. The worst activity in this series was found in b-diketonate which had attached allyl group (A). The cytostatic effect was dependent on the presence of conjugation in the studied compounds. That the bigger was the delocalised p-system in b-diketonates, the greater the cytotoxic activity was noticed (B-D). The decrease of conjugation degree decreases the activity of the compound (A). The effect of compound conjugation was particularly pronounced on MDA MB 231 cell lines. For example, this effect can be observed even better with both compounds that have the most structural similarities, such as compounds B and C containing 3- (cyclopropanecarboxamido)phenyl and 3-(4-methoxybenzamido)phenyl fragments. In this example, it can be clearly seen that extended delocalised p-system in the case of compound C influenced the increase of activity compared to compound B.

Interactions of compounds C and E with DNA and BSA
DNA and BSA fluorescence binding studies were done according to the previously described method with minor corrections (Joksimovi c et. al. 2019.b). The consequence of strong intercalation between the base pairs of DNA and ethidium bromide (EB) is fluorescence emission. If some compound (in this case, compound C and E) intercalates into DNA molecule, the binding sites of DNA available for EB decrease the quenching of the fluorescence of the EB-DNA system. (Olmsted and Kearns 1977;Jankovi c et al. 2016). Fluorescence quenching spectra ( Figure S1a) of titration EB-DNA with C and E, were recorded in the range of 550-700 nm. These spectra showed a decreasing trend with the increasing concentrations of compounds C or E at 610 nm, signifying that EB was replaced by the compounds. The quenching constants (Kq) were calculated using Sterne-Volmer equation (Lakowicz and Weber 1973). The quenching constants for C and E, presented in Table S1, specify that both compounds have the capacity to displaced EB from the EB-DNA complex by binding to DNA through intercalation (Petronijevi c et al. 2018).
Based on the fact that the effectiveness of potential drugs depends on their capacity to bind to transport proteins, we investigated the binding affinity of two compounds that showed the best biological potential (C and E) for bovine serum albumin (BSA). Fluorimetric titration method was used to investigate the binding properties of these compounds with the protein. The spectra were obtained in the wavelength range of 300-500 nm ( Figure S1b). The obtained K a values (Strekowski and Wilson 2007) are given in Table S2 and are indicating that C and E have a strong binding affinity to BSA. The number of binding sites for C or E (n 1.5, Table S2) indicates that this compound binds to BSA in the molar ratio 1.5: 1.

Simulated docking of compounds C and E with DNA and BSA
For the purpose of the docking studies, structures of examined molecules were generated in DS Visualizer and optimized using MOPAC PM7 method (Stewart 1990). Structures of target DNA and BSA molecules were obtained from crystal structures downloaded from the rscb.org site (Berman et al. 2000). Used structures are 1Z3F (Canals et al. 2005), structure of 6 base pair DNA in complex with ellipticine, 4F5S, (Bujacz 2012) structure of BSA, and 4OR0, (Bujacz et al. 2014) structure of BSA in complex with naproxen. Structures were further prepared using AutoDockTools 1.5.6 (Morris et al. 2009) by removing co-crystalized substrates and water molecules, calculating Gasteiger charges, and merging non-polar hydrogens. Simulations of binding of compounds C and E to DNA were performed using molecular docking tools in order to further confirm results obtained from study of interactions with DNA and to examine possible modes of binding. Results of the docking experiments are given in the Table S3. Calculated energy of binding for compound C is lower when compared to the calculated energy for compound E which indicates stronger binding of compound C. This result comes from the fact that the structure in the best scored result for compound C intercalates between bases with both of its aromatic rings and actualises interactions with all four nearby residues, while in the case of compound E, best result seems to fit only one of its aromatic rings between bases while the other ring hangs outside of the cavity ( Figure S2a). Ester part of both molecules is outside of the cavity, and in the case of compound E, carbonyl oxygen builds hydrogen bond with hydrogen from guanosine amino group.
Binding of compounds C and E to BSA was simulated using molecular docking tools to confirm results obtained from study of interactions with BSA and to examine possible modes of binding. Binding energies of examined compounds were compared with results gained from re-docking of naproxen. Results of the docking experiments for each compound with every target are given in the Table S4. Because lower calculated energies indicate stronger binding, we can conclude that both compounds C and E may have stronger affinity towards TRP213 than naproxen. Naproxen fails to bind in the vicinity of TRP134 in vitro, and much higher affinity towards TRP213 may be the reason. In the case of compounds C and E, based on the similar energies for both binding spots, there is no reason to suspect that these compounds would not bind in the vicinity of TRP134. Both compounds C and D can directly interact with TRP213 when bound near, but when bound in the vicinity of TRP134, there is no direct interaction with that residue. Position of the bound compound C in both positions, and interactions of that compound with residues in the vicinity of TRP213 can be seen in Figure S2b.

Conclusion
In order to investigate the anticancer potential, a small series of b-diketonate was tested on MDA-MB-231 and HeLa cancer cell lines. All tested compounds, except compound A, showed high cytotoxic potential, being more efficient against HeLa cells. In addition, compounds C and E showed better cytotoxicity than cisplatin. Further, investigations of interactions with biomacromolecules such as DNA and BSA are done. Calculated K sv values for the interactions of compounds C and E with DNA implied that our compounds interact with the DNA molecule through intercalation, while the calculated values of the constant Ka indicate that the mentioned compounds can be transported and distributed in an adequate manner. A molecular docking study was performed to investigate in more detail the mode of binding to DNA and BSA. Finally, all results indicate the great potential for the prospective application of these compounds in clinical practice in the future.