DNA binding, and apoptosis-inducing activities of a β-ionone-derived ester in human myeloid leukemia cells: multispectral and molecular dynamic simulation analyses

Abstract

β-Ionone is the end-ring counterpart of β-carotenoids, which are widely found in fruits and vegetables. Recent studies have illustrated the antimetastatic, anti-proliferative, and apoptosis-inducing activities of β-ionone both in vitro and in vivo. We aimed to explore the anti-cancer potency of β-Ionone-derived ester, (E)-4-(2,6,6-trimethylcyclohex-1-enyl) but-3-en-2-ylpyrazine-2-carboxylate (4-TM.P). The cytotoxic effects of the compound on K562 cells were evaluated by MTT assay. The mechanisms of apoptosis induction were investigated by acridine orange/ethidium bromide (AO/EtBr) double staining, cell cycle analysis, and Annexin V/PI staining. Furthermore, the 4-TM.P-DNA interactions have been thoroughly elucidated by various methods, such as ultraviolet–visible spectroscopy, fluorescence assays, viscosity measurements, molecular docking, and dynamic simulation. The MTT cytotoxicity assay revealed that the growth of K562 cells was inhibited by treatment with β-ionone-derived ester, with an IC50 of 25 ± 5.0 µM at 72 h. Morphological studies revealed the occurrence of apoptosis in treated cells, and G0/G1 cell cycle arrest was observed after treatment of the cells with the IC50 value of the compound. Analyses of multi-spectroscopy and viscosity assays revealed that 4-TM.P binds to DNA in the minor groove mode, which was supported by molecular docking studies. The dynamic stability of the complex was also confirmed using molecular dynamic simulation analyses.

Introduction

Cancer is a major topic of controversy for the international community. However, there is currently no known therapy for cancer, and progress in cancer treatment is still in the interest of researchers¹. Chronic myeloid leukemia (CML), a type of blood cancer that is distinguished by the unbridled growth of myeloid cells at various stages of development². CML is an infrequent hematologic malignancy with an annual incidence of 1.0–1.5/100,000³. In the last two decades, clinical translational biomedical studies have demonstrated the importance of CML as a subject of transformational therapeutic advances. Typically, patients present three stages of pathologies referred to as the chronic phase (CP), accelerated phase (AP) and blast phase (BP) or crisis (BC) with more un-differentiated lineage blasts developing in myeloid, lymphoid and mixed-type. According to the World Health Organization (WHO) criteria, AP is defined by 10–19% peripheral blood or bone marrow blasts and BC is characterized when approximately 25% blasts are present, while the majority of patients in CP would remain undiagnosed or untreated and will progress into AP and BC^4,5.

Translational studies into the genomic landscape of myeloid leukemia and an elaborate understanding of the mechanisms underlying resistance to typical therapies have rapidly developed the ameliorative armamentarium of this disorder in recent years⁶. However, drug resistance is still an issue of concern for most patients. Research into the processes of opposition to these new agents is illuminating the development of the following types of anti-leukemia drugs⁷. Furthermore, the organization of combination regimens aimed at optimally utilizing therapeutic vulnerabilities, with the final goal of eliminating all sub-clones of the illness and improving treatment rates in leukemia, has been planned. The intake of dietary carotenoids has been associated with a lower frequency of cancer. Carotenoids are involved in a wide range of biological functions, such as antioxidant activity, anti-proliferative and pro-apoptotic activity, as well as reinforcing the immune system⁸. Carotenoids, especially β-carotene, are of interest not only due to their antioxidant activity but also for their metabolites, such as vitamin A, which has anti-cancer activity. These metabolites are involved in different crucial mechanisms, including the induction of apoptosis cell death and cellular differentiation⁹. β-carotene has attracted the most attention due to its pro-vitamin A activity and its rampant activity in many foods^10,11. β-Ionone, a carotenoid precursor, is one of the most important groups of natural pigments. The cyclic sesquiterpene β-ionone (4-[2,6,6-trimethyl-1-cyclohexen-1-yl]-3-buten-2-one), a type of carotenoid, is an end-ring analog and an oxidative cleavage product of β-carotene¹². Previous studies have shown that β-ionone possesses a wide range of biological activities, such as antioxidant and antimutagenic properties. Furthermore, dietary studies have shown that this compound has anti-proliferative and chemopreventive effects. Recent studies have shown that β-ionone might protect against free radical-induced DNA damage by a method other than direct free radical scavenging¹³. In this regard, using β-ionone and related derivatives with a trimethylcyclohexane scaffold, which can serve as key building blocks in designing and making ionone-based anti-cancer drugs, is imperative. In particular, β-ionone has attracted much attention due to its ability to inhibit tumor cells, suppress metastasis and induce apoptosis both in vitro and in vivo¹⁴. In addition to β-ionone, pyrazinoic acid (PA) and its derivatives have been used as anti-cancer agents due to their nitrogenous heteroaromatic ring¹⁵. Recently, the anti-proliferative and apoptosis-inducing impacts of pyrazoline analogs on various human cancer cell lines have been reported^16,17. Moreover, numerous studies have verified the potential of pyrazinoic acid derivatives to interact with DNA, leading to the antitumor activity of these compounds^17,18.

In the present study, the multispectral, molecular docking and dynamic simulation of DNA binding, as well as the cytotoxicity and apoptosis-inducing activities of a β-ionone-derived ester, (E)-4-(2,6,6-trimethylcyclohex-1-enyl)but-3-en-2-ylpyrazine-2-carboxylate (4-TM.P), against human myeloid leukemia K562 cells, was investigated. This study provides valuable information about the molecular mechanism and apoptosis-inducing activity of 4-TM.P in the K562 leukemia cells.

Materials and methods

Materials

Calf thymus DNA (Ct-DNA), dicyclohexylcarbodiimide (DCC), and beta-ionol were obtained from Sigma-Aldrich (Germany). Fetal bovine serum (FBS), penicillin–streptomycin, and cell culture medium (RPMI-1640) were obtained from Gibco BRL Life Technologies (Paisley, Scotland). Dimethylsulfoxide (DMSO), MTT reagents [3 0-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], and acridine orange and ethidium bromide (AO/EtBr) were purchased from Sigma Chemical Company (Germany). An Annexin-V/PI (Propidium iodide) staining kit was acquired from Roche Corporation (Germany). The culture plates were acquired from SPL (Korea). K562 cells were obtained from the Pasture Research Institute (Iran).

General experimental procedure for the preparation of the compound

Beta-ionolpyrazinoate has been synthesized previously by our group in the organic chemistry and biochemistry laboratory, University of Tabriz. This compound was synthesized according to the literature by esterification of 2-pyrazinoic acid (2-PA) with beta-ionol (β-I) in the presence of DCC as a coupling reagent catalyzed by N, N´-dimethylaminopyridine (DMAP) (Fig. 1A). The reaction proceeds via an O-acylisourea intermediate, resulting in the activation of 2-PA by DCC through nucleophilic attack of the hydroxyl group of beta-ionol 1 on this intermediate. The resulting ester was identified and characterized by spectral data (¹HNMR, ¹³CNMR, FT-IR), as well as elemental analyses¹⁹.

Fig. 1.

Effect of 4-TM.P on the leukemia K562 cell line. (A) Synthesis and chemical structure of 4-TM.P. The cytotoxicity of 4-TM.P (B) and Dox (C) as a positive control on the viability K562 cell line. The cells were subjected to the indicated concentrations of 4-TM.P for 24, 48 and 72 h. Cell viability was assessed by the MTT test and is presented as a percentage of the corresponding control. The results are the means of three independent experiments. (*) P < 0.05, (**) P < 0.01.

Cell culture and cell viability assay

The leukemia K562 cells were cultured in RPMI 1640 medium containing 15% v/v deactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin (1000 U/ml penicillin and 1000 µg/ml streptomycin), placed in a CO2-conditioned incubator at 37 °C. The MTT assay was employed to assess the compound’s cytotoxicity. Following seeding on the 96-well plates at cell density of 4 × 10⁴ cells/well, they were incubated with different concentrations (10–100 µM) of the compound for 24–72 h. In each well, MTT (20 µM; 5 mg/ml in PBS) was added and then allowed to stand for about three to four hours at 37 °C. Thereafter, the supernatant was eliminated, while the purple-blue formazan precipitate formed was dissolved in DMSO. A multiwell plate reader (Bio-Tek, USA) was used to measure the optical density (OD) of the solution at 570 nm^20,21.

Apoptotic cell morphology

K562 cells (5 × 10⁵) were seeded in 12-well plates and treated with the compound at the IC50 for 72 h. The cells were collected and centrifuged. Then, the cells were stained with 1 µM AO/EtBr solution (100 mg/ml) and viewed under a fluorescence microscope (EUM-5000 FLCD, Labex Instrument).

DNA fragmentation assay

After cell treatment with the IC50 value of the compound (4-TM.P) for 72 h, the cells were collected and washed with PBS. Next, the samples resuspended the samples in 1 mL lysis buffer with 20 mg/mL protease K for 2 h at 56 °C. For DNA extraction, we used a solution of phenol/chloroform/isoamyl alcohol (25:24:1) to remove lipids and proteins from DNA. Then, the precipitated samples were resuspended in Tris–EDTA (TE) buffer mixed with DNA loading buffer, and loaded onto a 1.5% agarose gel. The samples were then electrophoresed for 90 min at 90 V²².

Cell cycle analysis by flow cytometry

This technique allocates cells in four phases of the cell cycle (sub-G1, G1, S and G2/M) based on the amount of DNA stained with propidium iodide (PI) in the cell. Increasing the sub-G1 population of treated cells enhances the number of apoptotic cells. The cells (1 × 10⁵ cells/well) were seeded in 6-well plates and treated with 25 μM (IC50 value) of the compound (4-TM.P) for 24–72 h. Then, the cells were exposed to PI (50 μg/mL) containing RNase (100 μg/mL) and Triton X-100 (0.5%) in a darkened room for 10 min²³. Next, we analyzed the stained cells using flow cytometry (BD Biosciences, San Jose, CA, USA). DNA histograms showed the cell distributions in sub-G1, G0/G1, S, and G2/M phases. We used FlowJo software (Ashland, OR USA) to evaluate how cells were spread across the cell cycle.

Annexin V/propidium iodide staining

Apoptotic and necrotic cells were measured using a dual staining method with FITC-Annexin V and PI, to verify the results of previous analysis. The K562 cells were seeded in 6-well plates and treated with the compound (at IC50 value) for 24–72 h. After these times, the collected cells were centrifuged and washed with PBS. Then, the cells were incubated with 100 μL labeling solution (20 μL Annexin V-FITC, 20 μL propidium iodide (PI) solution, 1 mL incubation buffer) for 15 min at room temperature in a dark place. The prepared samples were then analyzed by flow cytometry (BD Biosciences, USA), and the FlowJo software (Ashland, OR, USA) was used for data analysis^24,25.

Caspase-3 enzyme assay

The activity of caspase-3 was measured according to the manufacturer’s instructions using a caspase-3 activity kit (BD Biosciences, USA). Following a 24–72-h treatment with the IC₅₀ value of the compound, the cells were harvested, and a colorimetric activity assay kit was used to caspase-3 activity. Then, the cells were washed with cold PBS and resuspended in cell lysis buffer. Following repeated freeze–thaw lysing, the cells were allowed to sit on ice for 20 min before being centrifuged for 10 min at 12,000 g. After being gathered, the supernatants were mixed with DEVD-pNA. A microplate reader (Bio-TEK, USA) was used to quantify the concentration of pNA, which results from caspase-3's enzymatic conversion of DEVD-pNA, at 450 nm²⁶.

DNA binding assays

Preparation of stock solution

The Ct-DNA stock solution was prepared in Tris-(hydroxymethyl) amino methane hydrochloride (Tris–HCl) buffer (10 mM, pH 7.4), and its concentration was estimated using a UV–vis assay based on the instructions mentioned in previous study²⁷.

UV‒visible absorption spectral studies

In this study, we checked out the UV–vis spectra of Ct-DNA, 4-TM.P, and 4-TM.P-DNA complex using a UV–visible spectrophotometer (PG Instruments Ltd., Leicestershire UK) with a 1-cm path length cuvette. Tris–HCl buffer solution was the corresponding reference solution. The absorption spectra of a constant DNA solution (5.0 × 10^–5 M) with various concentrations of 4-TM.P, as well as the absorption spectra of 4-TM.P (5.0 × 10^–5 M) with and without Ct-DNA were recorded.

Fluorescence studies

Fluorescence measurements were conducted at the JASCO spectrofluorometer (FP6200, Tokyo, Japan). A constant amount of 4-TM.P (1.0 × 10^–3 M) was titrated via different Ct-DNA concentrations, and the change in fluorescence intensity of 4-TM.P was evaluated. Fluorescence emission spectra of 4-TM.P was recorded at three different temperatures (298 K, 308 K and 318 K) in the range of 370–500 nm after excitation at 290 nm wavelength.

Viscosity measurement

Further, the changes in DNA viscosity in the presence of different concentrations of 4-TM.P were monitored by a Ubbelodhe-type viscometer (Julobo, MD-18 V, Germany). Samples with constant DNA concentration (50 μM) and varying compound concentrations of 4-TM.P were prepared separately to obtain different 4-TM.P/DNA ratios. Firstly, it was measured viscosity for the DNA solution was as η°, and every sample’s viscosity was measured separately as η at 25 °C. Plotting the values of the cube root of relative specific viscosity, or (η/η°)^1/3, against the [4-TM.P]/[DNA] concentration ratio was done.

Molecular docking analysis

The B-DNA structure with PDB ID 1BNA (structure of a B-DNA dodecamer: conformation and dynamics)²⁸ was retrieved from the Protein Data Bank (PDB) database. The structure of 4-TM.P was generated using Avogadro software²⁹, ensuring appropriate atom connectivity and bond angles. The initial structure of 4-TM.P underwent geometrical optimization using Gaussian software. The optimization was performed using the Hartree–Fock algorithm and the ground zero method to attain a stable and energetically favorable conformation. To prepare the B-DNA structure (1BNA) for docking, water molecules and nonessential ligands were removed using UCSF Chimera 1.17.1 software³⁰, a molecular visualization software. The interaction between B-DNA and 4-TM.P was assessed through molecular docking using AutoDock4.2. Then, in our simulation, we utilized a Lamarckian Genetic Algorithm (LGA) to explore the conformational space and predict the binding affinity. The active site on the B-DNA structure was identified by analyzing the binding site residues and their proximity to the DNA helix. The grid box was placed in such a way that it covers both the minor groove and the major groove of DNA, and 30,000 energy evaluations were performed for each of the 1500 docking runs, followed by Ligplot + V.2.2³¹ to analyze the nonbonded interactions between 4-TM.P and B-DNA.

Molecular dynamics simulation

Molecular dynamics (MD) simulations were carried out using Gromacs 2022.4 software³² for DNA-4.TM. The P complex, topology and parameter files for the small molecule 4-TM.P were generated using the ACPYPE script within the Antechamber package³³, which is compatible with the Amber force field. The DNA topology was prepared using the Amber 99 sb force field³⁴. A cubic simulation box with a side length of 1.1 nm was employed. Solvation was achieved using the TIP3P water model, with 9566 water molecules included to solvate the system. To neutralize the net negative charge of DNA, 22 Na + ions were added. Energy minimization was done using the steepest descent algorithm with a maximum force criterion of 30.0 kJ/mol. Temperature equilibration was conducted for 300 ps (15,000 steps) using a velocity-rescaled thermostat³⁵ to ensure a canonical ensemble at 300 K. Subsequently, pressure equilibration was carried out for 300 ps (15,000 steps) using a pressure coupling algorithm to achieve a target pressure of 4.5 × 10–5 bar. The production MD simulation was then run for 100 ns, corresponding to 500 million steps.

Statistical analysis

The experiments were performed in triplicate and expressed as the mean ± SD. The data were evaluated using one/two-way ANOVA analysis with the GraphPad Prism software, version 6.02 (GraphPad Software, CA, USA). Statistical significance was determined at p-values less than 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****).

Results and discussion

Cell viability and growth inhibition

To assess the impact of the investigated compound (4-TM.P) on K562 cell viability, the cells were exposed to different concentrations of 4-TM.P (10–100 µM) for 24, 48 and 72 h. Interestingly, the results of the MTT assay indicated that the growth of K562 cells was inhibited during incubation with 10–100 μM β-ionone-derived ester in a dose- and time-dependent manner. As shown in Fig. 1B and Table 1, the IC50 values of the compounds after 24, 48 and 72 h of exposure were 65 ± 5.0, 50 ± 4.0 and 25 ± 5.0 µM, respectively. After treatment with 100 µM of the compound for 24, 48 and 72 h, only 30 ± 1.0, 28 ± 2.0 and 10 ± 1.0% of the cells, respectively, were viable compared to the control cells. In addition, Dox (as a positive control) showed an inhibitory effect on K562 cells with an IC50 value of 0. 90 µM, after 72 h of incubation (Fig. 1C).

Table 1.

The IC50 values of 4-TM. P for 24, 48 and 72 h.

Compound	IC50
4-TM. P	24 h	48 h	72 h
	65 ± 5.0	50 ± 4.0	25 ± 5.0

Optical inverted microscope image

Induction of apoptosis

The occurrence of apoptosis is characterized by morphological changes including membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic bodies formation^36,37. We observed K562 cells treated with 25 µM of 4-TM.P under inverted and fluorescence microscopes to study their morphology. After 72 h, inverted microscope images revealed that treated cells had apoptotic bodies formed from condensed cytoplasm and nuclear fragments, while control cells remained round (Fig. 2A). We also examined nuclear morphology using a fluorescence microscope. After 72 h with 25 µM of the compound, treated K562 cells showed condensed and fragmented chromatin, indicating apoptosis. Control cells maintained round nuclei without shrinkage or condensation (Fig. 2A). Dying cells had condensed nuclei and appeared green with bright green spots, while living cells were uniformly green with intact nuclei. We further confirmed these microscopic observations by analyzing the genomic DNA from untreated and 4-TM.P-treated K562 cells. DNA from cells treated with 25 μM of 4-TM.P for 72 h exhibited a ladder pattern, indicating the occurrence of apoptosis (Fig. 2B).

Fig. 2.

Morphological studies in the untreated and treated K562 cells with 25 μM 4-TM.P for 72 h. (A) Images prepared by invert microscope and fluorescence. After treatment with 4-TM.P (at IC50 value) for 72 h, the cells were harvested and stained with AO/EtBr. 4-TM.P induced condensation and fragmentation of the nuclei. (B) DNA electrophoresis of 1.5% agarose gel in control (Ctrl) and treated after 72 h.

4-TM.P arrested the cell cycle in K562 cells

According to previous studies, β-ionone and its derivatives repress tumor cell growth by inducing cell cycle arrest at the G1 phase in various human cancer cells, including prostate, colon, breast, and leukemia^12,38. In this study, we evaluated the anti-cancer effects of synthetic β-ionone-derived ester by determining its cytotoxicity and apoptotic effects on K562 cells in vitro. Our results revealed that the newly synthesized compound could potently induce apoptosis in a time-dependent manner. After 24–72 h of treatment with 4-TM.P (25 μM), the collected samples were analyzed by flow cytometry to evaluate the cell cycle phases. Most of the treated cells in the sub-G1 phase exhibited strong evidence of apoptosis induction. 4-TM.P caused a time-dependent increase in the sub-G1 peak (apoptotic cells). The percentage of cells in the sub-G1 peak was calculated 1.72% in the control cells and increased to 15.6%, 17.1% and 40.5% after 24, 48 and 72 h of treatment, respectively (Fig. 3A). Assessment of the sub-G1 peak of the sub-G1 peak in the cell cycle of treated K562 cells showed increased apoptosis within 24–72 h. Our results indicated that treated cells exhibited G0/G1 cell cycle arrest compared to the control. Generally, the G0/G1 phase population increased after 24 h of treatment, with longer exposure to 4-TM.P further raising G0/G1 cell proportions. This was accompanied by a significant decrease in S- and G2/M-phase cell populations. Our data showed that untreated control cells had 1.72%, 45.7%, 34.9%, and 17.5% in sub-G1, G0/G1, S, and G2/M phases, respectively. In contrast, cells treated with 4-TM.P showed 15.6%, 49.5%, 22.0%, and 12.0% at 24 h; 17.1%, 51.6%, 20.3%, and 10.2% at 48 h; and 40.5%, 34.7%, 14.1%, and 8.55% at 72 h, respectively (Fig. 3A). These findings suggest that DNA breakdown leading to cell death may result from cell cycle arrest and apoptosis induction^23,39.

Fig. 3.

Evaluation of the cell cycle and apoptosis in cells treated with 4-TM.P. (A) Cell cycle analysis of K562-treated cells by flow cytometry at 24, 48 and 72 h after treatment with 25 μM 4-TM.P. The accumulation of cells in the sub-G1 phase indicated the occurrence of apoptosis. (B) Apoptosis was assessed by Annexin-V/PI double staining. After 24, 48 and 72 h, untreated and 4-TM.P-treated K562 cells were harvested and analyzed. Flow cytometric analysis revealed an increased percentage of apoptotic cells. (C) Graph shows changes in caspase-3 activity in the cells treated with 4-TM.P compared to the untreated cells. Results presented as mean ± SD of three independent experiments. Statistical comparison between treated and untreated groups conducted using ANOVA analysis tool. The results were considered statistically significant at p-values of 0.05 (*), 0.01 (**), 0.001 (***), and 0.0001 (****).

Quantitative analysis of apoptosis

In our study, we used variable assessments, including a morphological study, to confirm the occurrence of apoptosis by a β-ionone-derived ester in K562 cells. The movement of phosphatidyl serine (PS) to the cell surface is thought to be a key indicator of apoptosis⁴⁰. To evaluate this hypothesis, we used the annexin V/PI double-staining method to assess the rearrangement of the plasma membrane of cells to visible phosphatidyl serine. After 24–72 h of treatment with 25 μM 4-TM.P, the cells were assessed for early and late apoptosis, as well as necrosis by flow cytometry. As shown in Fig. 3B, PS movement from the inner to the outer plasma membrane (Annx⁺) increased over 24–72 h. The percentages of early apoptotic cells (Annx⁺/PI⁻) in the treated cells were calculated to be 26.86%, 55.91%, and 64.71%, and the percentages of cells undergoing late apoptosis (Annx⁺/PI⁺) were 3.47%, 5.49%, and 6.87% after 24, 48 and 72 h, respectively (Fig. 3B). These findings indicate that the rate of apoptosis increased over time. To understand how the cell cycle relates to apoptosis, we evaluated K562 cells in the sub-G1 phase of the cell cycle. A time-dependent increase in sub-G1 after 24–72 h confirmed the occurrence of apoptosis. These data correspond with previous studies that showed the growth inhibitory and apoptosis-inducing effects of different types of β-ionone derivatives on various types of cancer cells, including human lung, prostate, liver, and breast cancer cells^12,41–43.

Activation of caspase-3 in K562 cells

It has been reported that the activity of caspase-3 increases in the Hepatocellular Carcinoma Cells treated with β-Ionone⁴⁴. In this study, caspase-3 activity was measured to investigate its involvement in the occurrence of apoptosis in the treated K562 cells. As shown in Fig. 3C, compared with untreated cells, K562 cells treated with IC50 values of 4-TM.P significantly (P value < 0.001) increased caspase-3 activity for 2.4, 4.1 and 4.50-fold, respectively, after 24, 48 and 72 h of exposure. These findings confirmed the results of quantitative and qualitative evaluations of apoptosis and showed a significant increase in apoptosis in K562 cells.

DNA binding studies

DNA is considered a common target for various anti-cancer agents. Investigating the interaction between small molecules and DNA is important for designing, synthesizing, or enhancing drugs with improved selectivity and efficacy to affect key biological processes^18,45. The current work presents a study of the interaction between 4-TM.P and Ct-DNA, which were generated using different approaches.

UV‒visible absorption spectral studies

UV–vis absorption spectroscopy is a practical, simple, and effective approach for investigating the binding mode of small molecules to Ct-DNA⁴⁶ by observing changes in the peak positions (blue-shift or red-shift) and intensities of the absorption spectra (hyperchromic or hypochromic effects), which correlate with the strength of the binding. In general, when molecules interact with DNA through covalent binding, a hyperchromic effect occurs, with a red-shifted absorption maximum. On the other hand, the non-covalent mode, specifically intercalative binding, is defined by a hypochromic effect and bathochromic shift, whereas electrostatic binding shows relatively lower hypochromic without bathochromic, and groove binding demonstrates no or slight alteration in UV–vis absorption measurement⁴⁷. Figure 4A shows the absorption maxima of Ct-DNA and 4-TM. P complex. As displayed in Fig. 4A, when the concentration of 4-TM.P increases, the peak intensity at 260 nm increases in the Ct-DNA spectra, which is a typical hyperchromic effect that likely signifies damage to the DNA helix after 4-TM.P binding and the binding mode appear to be non-intercalative⁴⁸. The absorption spectra of 4-TM.P in the presence of different Ct-DNA concentrations are presented in Fig. 4B. The maximum absorbance of 4-TM.P was observed at 268 nm was affected by the addition of Ct-DNA, resulting in a hyperchromic effect with a slight shift in the absorption peak, suggesting complex formation between 4-TM.P and Ct-DNA are distinct from classical intercalation binding and can be interpreted as indicating groove binding. To elucidate the binding strength of 4-TM.P with Ct-DNA, spectral titration data were used to calculate the binding constant (Kb) from Eq. (1)^49,50.

1

Fig. 4.

(A) Absorption spectra of Ct-DNA (5.0 × 10^–5 M) in the absence and presence of increasing amounts of 4-TM.P. (ri = [4-TM.P]/[Ct-DNA] = 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.8) for curves a–i, respectively. (B) 4-TM.P (5.0 × 10^–5 M) in the absence and presence of increasing amounts of ctDNA. (ri = [ctDNA]/ [ 4-TM.P] = 0.0, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4) for curves a–g, respectively. (C) Plot of A_o/A − A_o versus 1/[Ct-DNA] for the titration of 4-TM.P with Ct-DNA.

Here, the absorbance of 4-TM.P and its complex with DNA are A₀ and A, respectively. [Ct-DNA] is the Ct-DNA concentration. The absorption coefficients of 4-TM.P and 4-TM.P -DNA complex are εG and εH − G, respectively. The Kb as the binding constant value was derived from the intercept-to-slope ratio of A₀/(A − A₀) plotted against 1/[Ct-DNA] (Fig. 4C). Based on this calculation, the Kb value is determined to be 7.6 × 10³ M^-1, indicating that 4-TM. P exhibits moderate affinity for DNA binding. Moreover, the Kb value appears to be within the same range as groove binders^51–55. This value is notably lower than the kb values observed for classical intercalators, which typically range from 10⁶ to 10⁷ M⁻¹⁵⁶. This analysis strongly implies the groove binding of 4-TM. P to Ct-DNA instead of intercalation binding. Since the comprehensive mode of interaction cannot be investigated by UV–Vis measurement, additional experiments were conducted.

Fluorescence spectroscopy studies

Steady-state fluorescence

Fluorescence spectroscopy is a prominent method for studying the binding mechanism, binding constant, and type of forces involved in the interactions between biomacromolecules and small molecules. When intercalating agents are inserted into the base pairs of the DNA helix, their rotational motion is restricted; hence, a significant increase in the fluorescence emission of the bound chromophore is normally observed. In the context of groove-binding agents, electrostatic, hydrogen bonding, and hydrophobic forces are close to the sugar-phosphate backbone of DNA. These interactions do not stabilize the groove binders as effectively as intercalation, thereby making it possible to observe a decrease in fluorescence intensity in the presence of DNA⁵⁷.

The fluorescence emission spectra of 4-TM.P values for the different Ct-DNA concentrations at 298 K are shown in Fig. 5. 4-TM.P displayed a prominent emission peak at 418 nm. The fluorescence intensity of 4-TM.P after successive titration with Ct-DNA gradually decreased without any significant shift in the emission peak. This trend suggests that 4-TM. P binds to Ct-DNA via non-intercalative binding. These spectral characteristics were also observed in other groove binders and were similarly interpreted by other investigators^52,58,59.

Fig. 5.

(A) Steady-state fluorescence spectra of 4-TM.P (1.0 × 10^–3 M) with various concentrations of Ct-DNA (0.0–0.011 mM) at 298 K. (B) Steady-state fluorescence spectra of 4-TM.P (1.0 × 10^–3 M) with different concentrations of Ct-DNA at three different temperatures. (C). Plot of log (F0-F)/F versus log [Ct-DNA] at different temperatures. (D) Van’t Hoff plots of 4-TM.P-Ct-DNA systems.

The Stern–Volmer equation (Eq. (2)) was used to analyze the fluorescence quenching mechanism^60,61.

2

Here, F and F0 refer to the fluorescence intensities of 4-TM.P before and after binding with Ct-DNA, respectively. [Q] represents the Ct-DNA concentration. τ0 stands for the average lifetime of 4-TM.P with no quencher. Ksv and Kq are the quenching constant and quenching rate constant, respectively. K_sv values for the interactions of 4-TM. P with Ct-DNA at the three different temperatures were in orders of 10² M⁻¹ (Table 2). This suggests a significant interaction between Ct-DNA and 4-TM. P, which leads to quenching of the fluorescence intensity. Comparing the values of K_sv obtained in the present study with those of other groove binders confirms with groove-binding mode of interaction between Ct-DNA and 4-TM. P^52,62. Intrinsic fluorescence quenching can be divided into two main processes: dynamic and static quenching, which require molecular interactions between the quencher and the fluorophore. During dynamic quenching, the fluorophore and quencher collide during the lifetime of the excited state. Conversely, the quencher and fluorophore collided in the ground state during static quenching. Dynamic and static quenching can be distinguished by their various behaviors under changes in the viscosity and temperature. Dynamic quenching increases because of faster diffusion at higher temperatures, whereas static quenching decreases because the weakly bound complexes dissociate with increasing temperature⁶³.

Table 2.

Values of the Stern–Volmer extinction coefficient (Ksv) and Kq for the interaction of 4-TM. P at different temperatures (in Kelvin).

T (K)	Kq × (1010/mol s)	Ksv × (L/mol) × 10–2	R2
298	8.2	8.2	0.9664
308	7.6	7.6	0.9810
318	6.7	6.7	0.9489

The Stern–Volmer plots displayed a linear fit at three different temperatures (Fig. 5B), which indicate that there was only one type of quenching mechanism in the 4-TM.P-Ct-DNA interaction either static or dynamic. The Ksv values decreased with increasing temperature (Table 2), indicating that a static quenching procedure may be a potential mechanism of binding. The Kq values (Table 2) were much greater than the maximum bimolecular quenching rate constant (2.0 × 10¹⁰ L mol⁻¹ s⁻¹). Thus, the fluorescence enhancement indicates a static process characterized by complex formation between 4-TM.P and Ct-DNA.

Equilibrium binding titration

The binding constant (Kb) and the number of binding sites (n) between 4-TM.P and Ct-DNA were ascertained through the following Eq. (3)⁶⁴.

3

Here, F0 and F represent the fluorescence intensity of the 4-TM.P and 4-TM.P-Ct-DNA complex, respectively. [Q] indicates the Ct-DNA concentration. Kb refers to the binding constant, and n is the binding number. By linear regression of F0/ (F0 − F) versus 1/[Q], the Kb values and n were calculated at different temperatures (Table 3 and Fig. 5C). As presented in Table 3, the obtained Kb values were on the order of 10⁴ M⁻¹, which were in the same range as those of DNA groove binders but lower than those of classic intercalators such as the DNA-acridine orange complex (4.0 × 10⁵ L mol⁻¹), confirming that the interaction mode between 4-TM.P and Ct-DNA may undergo groove binding. As mentioned in Table 3, the binding constants decreased with increasing temperature, indicating the stability of 4-TM.P-Ct-DNA complex decreased with increasing temperature, which is consistent with the trend of Ksv. The values of n at different temperatures indicated that 4-TM.P and Ct-DNA interacted at a molar ratio of 1:1.

Table 3.

The binding constant and number of binding sites of 4-TM. P with DNA at different temperatures (298, 308 and 318 °C).

T (K)	Kb (104)	n	R2
298	10.34	1.28	0.9252
308	1.17	1.18	0.9701
318	0.89	0.99	0.9467

Thermodynamic parameters of DNA binding

To identify the binding interaction between 4-TM.P and Ct-DNA, thermodynamic parameters such as the free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) were calculated using Eq. (4)^65,66.

4

here T represents the experimental temperature (298, 308, and 318 K). K and R represent the binding constant and the gas constant (8.314 J mol⁻¹ K⁻¹), respectively. ΔS and ΔH values were estimated from the intercept and slope of the Van’t Hoff plot, respectively (Fig. 5D). ΔG was determined according to Eq. (5)⁶⁶.

5

As mentioned in Table 4, the negative amount of ΔS and ΔH revealed that van der Waals interactions and hydrogen bonding were the main forces involved in the interaction between 4-TM.P and Ct-DNA. The negative ΔG values showed that the interaction process was spontaneous.

Table 4.

The thermodynamic parameters of the interaction between 4-TM. P and DNA at different temperatures (Kelvin).

T (K)	ΔG (kJmol-1)	ΔH (kJmol-1)	ΔS (Jmol-1)
298	− 3.4	− 16.5	− 44
308	− 2.9	− 16.5	− 44
318	− 2.5	− 16.5	− 44

Viscosity studies

The viscosity measurements demonstrated the binding mechanism between 4-TM.P and Ct-DNA because it offers unequivocal evidence of their interaction. The intercalative binding mechanism has been proven to significantly affect the viscosity of DNA solution by requiring substantial space between consecutive base pairs to extend the double helix for small molecules. On the other hand, the effect of groove binders on DNA molecule length was insignificant, resulting in minimal or no impact on DNA solution viscosity⁵². The viscosity of the Ct-DNA solution remained practically constant upon the continuous addition of 4-TM.P (Fig. 6), indicating that 4-TM.P groove binding to Ct-DNA was consistent with the results of the fluorescence and absorption studies.

Fig. 6.

Effect of increasing amounts of 4-TM.P is the viscosity of Ct-DNA (5.0 × 10^–5 M) in Tris–HCl buffer.

Molecular docking study

Molecular docking simulation using AutoDock4.2 revealed that 4-TM.P was placed at the minor groove of the B-DNA, confirming the above experimental results. (Fig. 7A). The binding energy, measured at -8.34 kcal/mol (ΔG), falls within a range indicative of good affinity, particularly for initial lead molecules in drug discovery pipelines. The inhibition constant was 77,267.00 µM for 4-TM.P, which is a weak inhibitor based on conventional thresholds, and the other molecular docking parameters are shown in Table 5. Considering this value in the context of the specific biological target and potential downstream application, further analysis with Ligplot + revealed the formation of a hydrogen bond between O4′ of deoxy-adenosine18 and N2 (at 2-pyrazinoic acid) of 4-TM.P (Fig. 7B), suggesting the presence of specific binding interactions that contribute to the overall affinity. The pyrazine ring in the structure of the synthesized compound appears to be relatively reactive. The presence of nitrogen atoms in the structure of the pyrazine ring is the most important factor in its electrophilicity. Recently, the identification of numerous derivatives of pyrazine, as well as their various pharmacological features, has increased the enthusiasm for research on this nucleus⁶⁷. This specific interaction could be explored further through additional computational studies or mutagenesis experiments to elucidate its role in stabilizing the complex.

Fig. 7.

Molecular docking analysis. (A) Visualizing 3D structure of autodock4.2 best docked model between 4-TM.P (yellow) and DNA with mentioned binding energy of -8.34 kcal/mol, using UCSF chimera1.17.1 software. (B) Interaction analysis of 4-TM.P and DNA utilizing Ligplot + V.2.2.

Table 5.

Docking Results (Lowest Binding Energy Model) for DNA-4-TM.P Interactions.

Binding energy	Inhibition constant (Ki)	Intermolecular energy	Electrostatic energy	Torsional free energy	Total internal energy
− 8.34 kcal/mol	772.67 nM	− 9.83 kcal/mol	− 0.04 kcal/mol	+ 1.49 kcal/mol	− 1.27 kcal/mol

Simulating binding interaction

Molecular dynamics simulations were carried out to investigate the structural stability and conformational changes of DNA upon binding with 4-TM.P (Fig. 8). The initially docked 4-TM.P 3.5 Å along the x-axis from its docked position and its movement was monitored throughout the simulation. Interestingly, after approximately 3.2 ns, the displaced 4-TM.P molecule migrated back into the minor groove of the DNA, closely resembling its original docked position. The free DNA exhibited remarkable stability throughout the simulation, as reflected by an average RMSD of 0.2902 nm (Table 6). Interestingly, the DNA-4-TM.P complex displayed a lower average RMSD of 0.2411 nm. The plot of RMSD for the free DNA and 4-TM.P-DNA illustrates the dynamic changes in structural deviation between the free DNA and the DNA-4-TM.P (Fig. 8A). Additionally, the RMSD tolerance in the DNA-4-TM.P complex is reduced compared to the free DNA, revealing that DNA-4-TM.P complex is more stable than free DNA. This observation suggests a potential modest enhancement in the overall structural stability of the DNA molecule upon 4-TM.P binding. Local flexibility within the DNA molecule was assessed using the Root Mean Square Fluctuation (RMSF). The free DNA displayed a relatively low degree of atomic fluctuations, with an average RMSF of 0.189 nm (Fig. 8B). However, the DNA-4-TM.P complex exhibited a slightly higher average RMSF of 0.196 nm. Further insights obtained by analyzing the Radius of gyration (Rg), shedding light on the overall compactness and structural changes induced by 4-TM.P binding. The free DNA maintained a compact structure with an average Rg of 1.368 nm (Fig. 8C). The DNA-4-TM.P complex, however, exhibited a marginally lower average Rg of 1.357 nm. This subtle decrease hints at a potential slight compaction of the DNA, possibly due to localized interactions with 4- TM. P. Solvent accessibility also provided valuable information. A plot of the Solvent Accessible Surface Area (SASA) on a time scale is shown in Fig. 8D. The free DNA displayed an average SASA of 47.71 nm², indicating that a specific surface area was accessible to the surrounding solvent. The DNA-4-TM.P complex revealed a slightly higher average SASA of 47.96 nm². This minimal increase suggested that 4-TM.P binding might cause subtle enhancements in the local flexibility of specific DNA residues. While the observed changes were not substantial, these results collectively suggest that 4-TM.P binding exerts a subtle influence on the structural properties of DNA. Upon binding, a potentially slight increase in stability was observed, as indicated by a lower RMSD. Additionally, there was a potential minor compaction, as suggested by the lower Rg. The minimal increase in SASA and RMSF indicates that 4-TM.P binding might cause limited alterations in solvent exposure and local flexibility of the DNA molecule. Therefore, molecular dynamics simulations confirmed the stability of 4-TM.P and DNA complex over a 100 ns simulation, suggesting that the 4-TM.P binding not only facilitates its long-term interaction with DNA but also enhances the stability and compactness of the DNA structure.

Fig. 8.

Comparing the molecular dynamic behavior of free DNA and DNA-4-TM.P (A) free DNA backbone RMSD (blue) compared to that of DNA-4-TM.P (red). (B) RMSF of free DNA (blue) and DNA-4-TM.P (red) complex. (C) Contrast between the free DNA (blue) radius of gyration (Rg) and DNA-4-TM.P (red) against time. (D) Free DNA (blue) against DNA-4-TM.P (red) SASA plot (x-axis is the surface area of the DNA backbone exposed to solvent in nm²).

Table 6.

Summary of structural properties: average and standard deviation of RMSD, RMSF, Rg, and SASA.

Molecule	RMSD (nm)	RMSF (nm)	Rg (nm)	SASA
Free DNA	0.290 ± 0.31	0.189 ± 0.15	1.368 ± 0.13	47.64 ± 0.92
DNA-TMP	0.241 ± 0.16	0.148 ± 0.19	1.357 ± 0.14	47.59 ± 1.18

Conclusion

Taken together, our study showed that 4-TM.P, a small DNA-binding compound derived from β-ionone, inhibits growth and triggers apoptosis in K562 chronic myeloid leukemia cells. The binding interactions of 4-TM.P with ctDNA were evaluated using spectroscopic measurements and computer simulation techniques. The results of these assays showed that 4-TM.P apparently binds to DNA in the minor groove by a non-intercalative mode. As inducing apoptosis in cancer cells is a key strategy for developing anti-cancer agents, this compound appears promising for further pharmaceutical evaluation. It may offer a novel chemotherapeutic approach to treating various human neoplastic disorders. Our prospects for future research will be structural optimization of 4-TM.P and its further evaluations in vivo, as well as studies of its mechanism of action.

Author contributions

KJ and RA, performed most of the experiments, interpreted the results, and wrote the original draft of manuscript. BM and SG performed molecular docking and molecular simulation analyses using the related software. MZ, HK and GD helped with the cell and molecular experiments and helped to interpret the results. NSS and MFD helped to write the manuscript and approved the study. MM as the principal investigator of study, supervising and checking the manuscript, and submitting the manuscript. All authors read and approved the final manuscript.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability

Data Availability All data generated or analyzed during this study are within the article.

Competing interests

The authors declare no competing interests.