Obtained results of MTT testing of PS substance, its conjugate CPt-PS and description of the experiment are given in Table 1 in the form of numerical values and are presented in the form of diagrams (Fig. 3). As can be seen, death of tumor cells is about 83% at the dose of cisplatin 0.3 g/m2. 10-fold reduction in dose leads to decrease up to 33%, and at 100-fold dilution cisplatin loses its antitumor effect. Individual new isothiazole derivative PS itself in all tested doses did not show any noticeable bioactivity under experimental conditions, but increased cytotoxic effect of cisplatin, although observed effect was lower than for the morpholine MS salt the test results of which were published by us earlier [6]. Thus, addition of 5 µg/ml of adjuvant (0.01 PS) to a 100-fold dilution of cisplatin (0.01 CPt), when it does not exhibit a cytotoxic effect itself, leads to noticeable antiproliferative activity and death of C6 glioma cells – up to 18%. We found that the effect of adjuvant addition to cisplatin taken in 10-fold dilution (0.1 CPt) increases with decreasing dose (quite surprising!), and the cell death for combination 0.1 CPt + 0.1 PS is 38%, and for 0.1 CPt + 0.01 PS it increases up to 48%, which is almost one and a half times higher than for individual cisplatin at such dosage (32%).
Table 1
Optical density of C6 cell culture under the action of cisplatin (CPt), an adjuvant (PS) and their binary mixture at different concentrations and after subtracting the background value (optical density of DMSO)
|
С6
|
С6 + CPt
|
С6 + 0.1 CPt
|
С6 + 0.01 CPt
|
С6 + PS
|
С6 + 0.1 PS
|
С6 + 0.01 PS
|
С6 + 0.1 CPt + 0.1 PS
|
С6 + 0.1 CPt + 0.01 PS
|
С6 + 0.01 CPt + 0.1 PS
|
С6 + 0.01 CPt + 0.01 PS
|
Mean
|
0.34
|
0.06
|
0.23
|
0.35
|
0.33
|
0.33
|
0.33
|
0.21
|
0.18
|
0.28
|
0.31
|
Error of Mean
|
0.01
|
0.01
|
0.02
|
0.02
|
0.03
|
0.03
|
0.02
|
0.02
|
0.01
|
0.02
|
0.02
|
Since the adjuvants (MS and PS) were used in doses when they themselves did not exhibit cytotoxic action, we assumed that synergistic effect appears due to conjugation of cisplatin with isothiazole derivative and subsequent antitumor effect of molecules of resulting conjugate.
In this regard, it would be very informative to determine structural and electronic changes that occur in the system of two conjugated molecules. The information can be obtained as a result of correct quantum chemical calculations. Taking into account chemical and structural features of cisplatin and adjuvants, we assumed that association of their molecules occurs due to non-covalent interactions. To assess structure and strength of cisplatin and adjuvant complexes, as well as their physicochemical characteristics, it is correct to use methods of quantum chemistry, adapted for calculating forces of intermolecular interaction. That, DFT/CAM-B3LYP/cc-pvdz/LanL2DZ(Pt) level of theory was used in our justifications.
We calculated the optimal geometry of molecules, dipole moment, charge distribution, localization and energy characteristics of frontier molecular orbitals (FMO). Calculations were carried out both for individual compounds and their conjugates in two versions: isolated molecules in vacuum and with consideration to aqueous medium, which simulates situation in living cells. Molecular structures (Fig. 4) were obtained as a result of calculations with full optimization of all geometric parameters.
Tribak and colleagues have used data of quantum-chemical calculations of electronic structure of compounds to analyze biological activity of these substances [25, 26]. Biological activity was considered by analogy with chemical activity of molecules. We also tried to apply results of calculations of optimized CPt-MS and CPt-PS structures to interpret the effect of adjuvant and elucidate possible causes of synergism in conjugate “cisplatin – isothiazole derivative”. We calculated distribution of frontier molecular orbitals (FMO): Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) for CPt, CPt-MS and CPt-PS in vacuum and with consideration to aqueous medium. Results of calculating localization of HOMO and LUMO are shown in the form of 3D isosurfaces (Fig. 5–7). Determination of localization of FMO is important for establishing preferred directions and regions of the molecule for attack by nucleophiles and electrophiles. These are molecular fragments of antitumor agent that interact with target sites as applied to interpretation of biological activity.
First of all, it should be noted that the results of calculations for isolated molecules in vacuum and in an aqueous medium differ significantly. It follows from calculation data that cisplatin molecules (CPt) and isothiazole derivatives (MS and PS) form conjugates with shortened interatomic distances due to non-covalent interactions caused by hydrogen and van der Waals bonds (Fig. 4). Hydrogen bonds are clearly expressed between oxygen atom of isothiazole carboxylate residue and amine fragment of cisplatin C=ОMS/PS···H-NCPt with interatomic distances of 1.79–1.93 Å in vacuum in both conjugates. These connections are absent in aquatic environment. There are some other interplays in structures of conjugates that differ in aqueous medium and vacuum. For example, for both complexes, shortened contacts are observed between chlorine atoms of cisplatin and СН fragments of morpholine and piperazine (ClCPt···H-Cmorph, ClCPt···H-Cpip), which values in aqueous medium (2.29, 3.22, 3.43 Å) are less than in vacuum (3.50, 3.87 Å), and corresponding values for CPt-PS conjugate are higher than for CPt-MS conjugate. However, there is no common single trend in the change in interatomic distances between the corresponding molecular fragments during the transition from vacuum to an aqueous medium. In particular, for CPt-PS conjugate, the Clisoth···H-NCPt and ClCPt···H-Npip values in vacuum are lower than in aqueous medium, in contrast to ClCPt···H-Cpip values. It can only be stated with confidence that mutual arrangement of molecules of heterocycles (MS and PS) and cisplatin (CPt) and their relative orientation in vacuum and in aqueous medium differ significantly. The differences found are of great importance for interpretation and modeling of antitumor effect of substances, since the agent-target interaction in reality takes place in aquatic environment.
Both in vacuum and aqueous medium HOMO is localized on Pt and Cl atoms in individual cisplatin molecule CPt (Fig. 5a,c). LUMO is localized on NH3 groups in vacuum (Fig. 5b) and delocalized throughout the molecule in aqueous medium (Fig. 5d). It is important to know what happens to its molecule when conjugated with adjuvant. It turns out that LUMO is localized on isothiazole heterocycle in both CPt-MS and CPt-PS conjugates in vacuum and aqueous medium. HOMO is localized in CPt-MS conjugate, both in vacuum and aqueous medium, on cisplatin molecule with maximum density on Cl-Pt-Cl fragment, same as in individual cisplatin molecule, but the contribution of Pt atom in aqueous medium in CPt-MS is greater than in individual cisplatin. HOMO is also located on Cl-Pt-Cl fragment in CPt-PS conjugate in vacuum. The picture fundamentally changes in aquatic medium – HOMO is localized on piperazine fragment.
For explanation of results obtained the mechanism of cytotoxic action of cisplatin was taken into account. Despite the differences in interpretation of some precise details, it is believed that cytotoxic effect of cisplatin is due to disruption of DNA functions via binding to purine bases in its molecule. This leads to intra- and inter-stranded DNA cross-linking, which causes DNA damage, impaired replication and transcription, and subsequently apoptosis of cancer cells [27–29].
Localization of HOMO and LUMO determines which fragments of the molecule are preferred for attack by electrophiles and nucleophiles, respectively. The following conclusions can be drawn from the analysis of FMO distribution. Formation of CPt-MS and CPt-PS conjugates leads to displacement of LUMO from cisplatin molecule and its localization on isothiazole heterocycles both in vacuum and aqueous medium. Since LUMO determines interaction with target nucleophilic sites, this type of binding will be realized through isothiazole heterocycle and not through cisplatin molecule. With regard to generally accepted mechanism, we can say that binding to purine bases in CPt-MS and CPt-PS conjugates will proceed with direct participation of adjuvant, which explains its role in manifestation of synergistic effect.
Cross-linking of DNA strands can cause local denaturation and damage to DNA and appearance of new sites, including electrophilic ones. Binding to electrophilic DNA sites determines localization of HOMO in conjugate. Calculations for conjugate molecules in vacuum showed that HOMO in both conjugates is completely localized on cisplatin molecule. Calculations with consideration to aquatic medium revealed differences in localization of HOMO in conjugates. It still remains on cisplatin molecule in CPt-MS, while in CPt-PS it is predominantly localized on PS piperazine residue. This may be one of the reasons for lower activity of CPt-PS compared to CPt-MS.
The energies of frontier molecular orbitals (FMO) are also used as one of characteristics of biological activity of a molecule. The key characteristics of a molecule in FMO theory are difference between the energies of HOMO and LUMO (ΔE), global hardness and softness of the system (η and S) which are treated as descriptors. We performed calculation of descriptors as well as dipole moments for optimized structures CPt, CPt-MS and CPt-PS in vacuum and with consideration to aqueous medium. Obtained values are shown in Table 2.
Table 2
Calculated DFT method values of descriptors ΔE, η and S and dipole moments in vacuum and in aqueous medium for CPt, CPt-MS and CPT-PS
|
In vacuum
|
In aqueous medium
|
Descriptors
|
CPt
|
CPt-MS
|
CPt-PS
|
CPt
|
CPt-MS
|
CPt-PS
|
LUMO (eV)
|
-0.7946
|
-0.7215
|
-0.4042
|
-0.3053
|
-0.5667
|
-0.5550
|
HOMO (eV)
|
-7.8982
|
-8.0759
|
-8.0815
|
-8.2420
|
-8.2638
|
-8.0346
|
ΔE (eV)
|
7.1035
|
7.3544
|
7.6773
|
7.9367
|
7.6971
|
7.4796
|
η
|
3.5518
|
3.6772
|
3.8386
|
3.9683
|
3.8486
|
3.7398
|
S
|
0.1408
|
0.1360
|
0.1303
|
0.1260
|
0.1299
|
0.1337
|
Dipole Moment (Debye)
|
10.954
|
3.271
|
2.502
|
16.438
|
19.032
|
18.990
|
The differences in calculated descriptors values for CPt, CPt-MS and CPt-PS are small. However, there is tendency to decrease in ΔE when going from CPt to conjugates in calculations with consideration to aqueous medium. This indicates an increase in reactivity of the system, which in our case means more active binding with DNA. There is a slight decrease in global hardness and a small increase in global softness in CPt – CPt-MS – CPt-PS series. It should be noted that the opposite situation is observed for calculations in vacuum.
The difference in values of dipole moments is very significant, and in vacuum dipole moment of conjugates is greatly reduced in comparison with cisplatin (3.27, 2.50 and 10.95 D respectively), but in aqueous medium, on the contrary, dipole moment of conjugates is higher than that of cisplatin (19.03, 18.99 and 16.44 respectively), which indicates a greater polarity of conjugate molecules. We have noted such differences in the data for isolated molecules in vacuum and aqueous medium during FMO analysis. This represents great influence of environment (water) on structure and properties of the objects under consideration.
We used one more descriptor for the analysis of “cisplatin – isothiazole derivative” system – molecular electrostatic potential (MEP) [30]. It allows evaluating electrostatic component of the energy of intermolecular interactions and is clearly presented in the form of color diagrams. The MEP chart can be used to predict reactivity and active sites for interactions. Negative electrostatic potential corresponds to proton attraction by total electron density in a molecule, that is, protonation of a molecule (shades of red), and positive electrostatic potential corresponds to proton repulsion by atoms (shades of blue). Potential increases in the following order: red < orange < yellow < green < blue. The MEP distribution diagrams in different formats calculated with consideration of aquatic environment are shown in Fig. 8. Calculation results show that regions with negative potential are located on oxygen atoms of carboxyl group (–O–C = O) of isothiazole derivative. Areas with positive potential are localized around amino groups of cisplatin. Thus, conjugation of adjuvant with cisplatin results in formation of molecular substrate in which types of electrostatic interactions are shared between cisplatin and isothiazole ligand.