An Experimental Dynamic Investigation of the Influence of Melatonin, Serotonin and Tryptophan on the Stability of the DNA Structure

Abstract

Background: Small molecules play a crucial role in the exploration of physiological pathways and in drug development by targeting deoxyribonucleic acid (DNA). DNA is a central focus for both endogenous and exogenous ligands, which interact directly or indirectly to regulate transcription and replication processes, thus controlling genetic expression in specific cells. Among these molecules, indole derivatives like tryptophan, serotonin, and melatonin are notable for their widespread presence in nature and significant biological effects. Tryptophan, an essential amino acid, serves as a vital structural element in proteins and a precursor for bioactive compounds like serotonin and melatonin, which impact various physiological functions. Methods: Experimental studies have been conducted to reveal the interaction mechanisms of these endogenous indole derivatives with calf thymus DNA (ct-DNA). These investigations involve viscosity measurements and analysis of double-stranded DNA behavior in the presence of indole molecules, using spectrophotometric UV absorption techniques to assess their impact on DNA stability. Additionally, the influence of calcium and magnesium ions on the resulting complexes of these indole derivatives with ct-DNA has been evaluated. Molecular docking validated our findings, offering additional insights into potential DNA–ligand interactions. Utilizing a crystallographic oligomer with an intercalation gap improved docking accuracy, distinguishing intercalation from groove recognition and enhancing assessment precision. Results: Our study offers detailed insights into the interaction patterns of the indole derivatives with DNA and is highly supported by molecular docking analyses: the indole derivatives were predominantly localized between C and G, interacting via π-π interactions and hydrogen bonds and aligning with known data on conventional intercalators. These findings underscore the importance of small compounds’ planar structure and appropriate size, facilitating tight insertion between adjacent base pairs and disrupting regular DNA stacking. Conclusions: Indoles’ physiological roles and potential as drug candidates targeting specific pathways are highlighted, emphasizing their significance as ubiquitous molecules with the ability to modulate biological effects on DNA structure.

1. Introduction

Small molecules are of particular interest in the research and development of new drugs or in revealing physiologic pathways that exhibit biological effects by targeting deoxyribonucleic acid (DNA) [1,2,3,4,5,6,7,8,9]. Exogenous substances, mainly xenobiotics, and many endogenous molecules are extensively studied for their complex interactions with DNA [10,11,12,13,14,15,16,17,18,19,20,21,22] or different proteins [23,24,25,26,27]. Nucleic acids are the main target for a wide range of chemical reactions and physical alterations, causing structural, functional, and informational inexactitudes that accumulate at the nuclear level and transpose themselves into pathogenesis bursting points [28,29].

DNA is the primary target for both endogenous and exogenous ligands, which interact directly or indirectly with it, mainly modulating transcription and replication processes, regulating genetic expression in specific cells. These interactions govern numerous physiological pathways and can also play a role in restraining the pathological development of tumor cells and repairing damaged DNA structures, significantly impacting the cellular environment and the specific reaction of the individual to a series of undesirable biological events [30,31].

DNA, as an unconventional and passive molecular receptor, possesses numerous binding domains and can interact with small molecules through a variety of mechanisms across multiple levels. These interactions play a critical role in balancing the antagonistic processes of nucleic acid degradation and protection. Understanding the precise nature of these interactions is crucial for advancing drug design strategies. By starting with a core moiety or repetitive structural motif that inherently facilitates binding—primarily due to its affinity or physical properties—and subsequently incorporating additional chemical features, it is possible to steer the drug’s effect towards a specific therapeutic target [32,33,34]. The aim of our research is to deepen the understanding of these DNA–small molecule interactions, particularly in the context of drug design, where such insights can inform the development of more effective therapeutic agents.

Indole derivatives such as tryptophan, tryptamine, serotonin, melatonin, skatole, psilocybin, strychnine, ergotamine, bromocriptine, reserpine, vinblastine, and vincristine, are among the most widely prevalent naturally occurring heterocyclic compounds with tremendous biological significance [35]. Tryptophan is an essential amino acid for human cells, a valuable structural component of proteins, and a precursor for various bioactive derivatives, such as serotonin, a crucial neurotransmitter, and the pineal hormone melatonin, a chrono modulator that affects major physiological functions [36,37,38,39,40]. Recognizing the significance of interactions between small molecules and nucleic acids at the molecular level, and acknowledging the importance of indole derivatives in maintaining the molecular homeostasis, our previous work focused on a series of in vitro molecular studies on tryptophan, serotonin, and melatonin [41,42] and clinical studies examining the physiological correlations between indoles and gestational developmental stages [43].

Non-covalent interactions between small molecules (ligands) and DNA predominantly occur through intercalation and minor groove-binding mechanisms [2,44,45]. In this study, we aimed to thoroughly investigate the interaction of three endogenous indole derivatives with double-stranded calf thymus DNA, alongside the effects of two abundant bivalent cations. Using spectrofluorimetric and UV spectroscopic techniques, alongside ethidium bromide displacement assays, we previously calculated the binding constants of these indole molecules to DNA to elucidate their interaction mechanism [46]. The intercalation of the indole moiety into the DNA structure is hypothesized to reduce the helical twist and lengthen the DNA molecule without direct intrusive interference, potentially modulating the activity of topoisomerases, enzymes that are critical for DNA replication [47].

Building on prior research, our experimental approach sought to provide a more comprehensive understanding of DNA–indole interactions and their implications for DNA stability in the presence of bivalent cations. These interactions were studied to explore their potential effects on critical biological processes, such as gene regulation and enzymatic activity, with particular focus on the modulation of topoisomerase function. We employed a range of techniques, including UV spectroscopy, fluorescence spectroscopy, and viscosity measurements, to characterize the binding patterns between DNA and small molecules [48,49,50]. High-performance hydrodynamic measurements were conducted to further clarify the physicochemical aspects of these interactions. Specifically, assessing the viscosity of DNA solutions provided insights into the structural alterations of the DNA helix as it accommodates intercalating agents. According to the classical intercalation model, the insertion of intercalating molecules between base pairs leads to elongation of the DNA double helix, resulting in increased solution viscosity. In contrast, non-classical intercalation is associated with overstretching of the DNA strands, compressing the double helix and consequently decreasing the viscosity of the solution [51,52].

This comprehensive study not only sheds light on the interaction mechanisms of indole derivatives with DNA but also correlates these effects with broader biological and chemical questions, such as the role of DNA stability in cellular processes and the potential therapeutic modulation of these interactions in disease contexts.

The DNA “melting” point designates the transition from the highly ordered state of the double helix to two independent strands. During the melting process, the hydrogen bonds between the complementary bases pairs disrupt, unwinding the double-helical stack. This separation occurs in vitro through progressive heating of the DNA solution in a buffer solution within a specific temperature range [53]. Throughout this process, absorbance at 260 nm increases, with the temperature corresponding to the inflection point of the absorption curve termed the DNA melting point. The study of DNA denaturation is a crucial method for evaluating the stability of DNA molecules under various physical and chemical conditions. The melting temperature (Tm) of DNA is particularly influenced by its base composition, with guanine–cytosine (G-C) pairs contributing to greater stability due to their three hydrogen bonds, as opposed to the two hydrogen bonds formed by adenine–thymine (A-T) pairs. Consequently, DNA regions rich in G-C pairs exhibit higher melting temperatures. The melting characteristics of DNA are also affected by factors such as denaturation–renaturation temperatures, buffer concentration, solvent composition, pH, and the specific base sequence and concentration [54,55]. Additionally, certain compounds can modulate DNA stability by altering the denaturation–renaturation profile, thereby impacting the temperature at which the DNA double helix denatures.

The aim of our study was to investigate the effects of indole molecules on the denaturation–renaturation behavior of double-stranded DNA within a biological context. Using spectrophotometric UV absorption methods, we sought to assess how these indole compounds influence DNA stability, providing insights into the molecular mechanisms by which small bioactive molecules can modulate DNA structure and function in biological systems. We have also determined the influence of the calcium and magnesium ions on the studied bio-indoles and double-stranded calf thymus DNA complexes. Bivalent cations can modulate the interactions between DNA and other molecules, such as small ligands, proteins, and drugs. By influencing the charge environment and structural conformation of DNA, these cations can affect the binding affinity and specificity of these interactions. Although cations are critical for the formation and stabilization of the nucleic acids’ structure, only a few studies have investigated their binding to the DNA or their influence on DNA–therapeutic substances complex formation [56,57,58]. Herein, using spectrofluorometric methods, we assessed the influence of calcium (Ca²⁺) and magnesium (Mg²⁺) ions on the bonds between DNA and endogenous indole molecules. Ca²⁺ forms direct and indirect bonds with the nitrogenous bases and the phosphoric skeleton from the DNA structure, while Mg²⁺ indirectly reacts through coordination.

2. Materials and Methods

Reagents: all reagents were commercially available and were used without further purification. Melatonin, serotonin, tryptophan, double-stranded calf thymus DNA, phosphate buffer (pH 7.4), dimethyl sulfoxide (DMSO), MgCl₂, and CaCl₂ were purchased from Sigma-Aldrich, St. Louis, MO, USA.

Equipment: LS50 B Perkin Elmer spectrofluorometer, Cary 100 Bio (Varian Inc., Palo Alto, CA, USA) UV-VIS spectrophotometer equipped with thermostatic cell holder and magnetic stirring, Ubbelohde capillary viscometer.

All experiments involving the interaction of the indoles with DNA were performed in phosphate buffer, adjusted to pH 7.4. The concentration of the DNA solution was determined by measuring the absorption intensity at 260 nm, using the molar extinction coefficient value of 6600 M⁻¹·cm⁻¹. The absorbances registered at 260 nm and 280 nm gave the ratios of ~1.8–1.9, indicating that the DNA solution was sufficiently free of protein. Serotonin and tryptophan were dissolved in phosphate buffer at the concentration 1.656 × 10⁻⁴ M. Melatonin was dissolved in a solvent mixture of DMSO (minimum quantity necessary for solubilization) and phosphate buffer at a similar concentration. The stock solutions were stored at 4 °C and used within 4 days. We measured the absorbances of mixtures, containing constant concentrations of the tested indoles of 1 µM and increasing concentrations of the nucleic acid. We also measured the absorbances of mixtures, containing constant concentrations of DNA (1 µM) and increasing concentrations of the tested compounds from 1 to 10 µM.

The selected concentrations were carefully chosen to approximate physiological levels, ensuring that the conclusions drawn from the study could be meaningfully extrapolated to in vivo conditions. This approach was intended to enhance the relevance of the findings, allowing for a more accurate prediction of how these interactions might occur within a biological context.

2.1. Viscosity Measurements

The DNA intercalation modes for the three bio-indoles were evaluated using viscosity measurement by assessing the flow-through time of the solutions between the 2 capillaries of the Ubbelohde device. After washing the device with the test solution, five assessments were performed for each solution. The flow-through time was calculated for distilled water, phosphate buffer (pH 7.4), the DNA solution, alone, and in the presence of ten increasing concentrations of the indole derivatives containing constant concentrations of DNA (1 µM) and increasing concentrations of the tested compounds from 1 to 10 µM. The flow-through times of the assessed solutions, measured between the 2 capillaries of the Ubbelohde viscometer, have been accurately recorded. As we used extremely diluted physiological solutions, we calculated the relative viscosity of the studied solutions using the following equation:

η_r = η/η_o = t/t_o, where η_o = viscosity of distilled water; η = viscosity of the solution to be analyzed; t = the flow-through time of the solution to be analyzed; t_o = the flow-through time of distilled water. The results were compared to distilled water, for which η_o = 1.05 ·10³ · kg · m⁻¹ · s⁻¹. Data were presented as (η/η₀)^1/3 versus the ratio of the concentration of the compound to DNA. Viscosity experiments were carried out at 25 ± 0.1 °C.

2.2. DNA Denaturation–Renaturation Spectrometric Assessment

The studies on the influence of bio-indoles on DNA denaturation–renaturation processes monitored the DNA behavior in the presence of indole molecules using UV absorption spectrophotometry. Employed solutions included DNA solution and the assessed intercalation system of indoles–DNA comprising 1000 µL DNA solution, 100 µL indole solution (melatonin, serotonin and tryptophan, respectively), 900 µL phosphate buffer (pH 7.4), mixed together in the reading cell. UV spectra were obtained using the following parameters: spectrum scanning speed 400 nm/min and scanned range 200–400 nm, under continuous mixing. For the DNA solution, UV absorption spectra occurred in the 200–400 nm range, with the solution’s temperature being maintained constant at 30 °C. Then, we increased the temperature of the analyzed solution by 2 °C/1.2 min, from 30 °C to 90 °C, and then decreased it (keeping the temperature gradient) to 30 °C, while recording the spectra. The experiment was repeated for the solutions of the indoles–DNA complexes.

2.3. Spectrofluorometric Studies on Ca²⁺ and Mg²⁺ Influence on the Binding Pattern between DNA and Endogenous Indole Molecules

The influence of Ca²⁺ and Mg²⁺ ions on the interactions between indole molecules and DNA were investigated using a fluorometric technique to measure the fluorescence intensities emitted by the indole molecules. Fluorescence spectra of the DNA–indole complexes were recorded at a 10:1 molar ratio, with varying concentrations of Ca²⁺ and Mg²⁺ ranging from 0.01 mol/L to 0.1 mol/L. The fluorescence spectra were registered using the following wavelengths: melatonin: λ_excitation = 280 nm and λ_emission = 354 nm; serotonin: λ_excitation = 280 nm and λ_emission = 338 nm; tryptophan: λ_excitation = 280 nm and λ_emission = 362 nm. For each concentration, we performed 5 replicates. The results are presented as means ± standard deviation.

2.4. Molecular Docking

We retrieved the structure of 1Z3F (a hexamer d(CGATCG)2 complexed with ellipticine) from the Protein Data Bank [59]. Ellipticine is a typical intercalating agent with antitumor activity [60].

DNA was prepared for docking using YASARA [61]. The DNA–ellipticine complex was minimized by the steepest descent method, with a YASARA force field. Water molecules were deleted. Ellipticine was removed from the complex, and the modified DNA presenting an intercalation gap—d(CGATCG)·(CGATCG)—was used as a target for docking. The docking searching space was set to 96 × 96 × 96 points and a resolution of 0.375 Å to include the entire DNA fragment, as there is no specific binding site [62].

The chemical structures of melatonin, tryptophan, and serotonin were retrieved from the PubChem database and optimized with YASARA. They were protonated at pH 7.4 and their energy was minimized. They were docked using AutoDock Vina v1.1.2 algorithm within YASARA. A total of 25 docking runs were performed for each ligand. Docking results were returned as the binding energy (ΔG, kcal/mol) and ligand efficiency (ΔG\no. of heavy atoms) of the best binding pose for each ligand. The conformations of the predicted DNA–ligand complexes and molecular interactions were analyzed using BIOVIA Discovery Studio Visualizer (BIOVIA, Discovery Studio Visualizer, Version 17.2.0, Dassault Systèmes, 2016, San Diego, CA, USA).

In our docking experiments, the structural template included both G-C and A-T nucleotide pairs to comprehensively assess ligand–DNA interactions. The docking results indicated that ligands preferentially bound between G-C pairs, likely due to the stronger hydrogen bonding and base stacking interactions that are characteristic of these nucleotide pairs. Given these findings, we focused our analysis on the interactions with G-C pairs, as they were the most relevant to the binding behavior observed in our study.

2.5. Statistical Analysis

Statistical significance of the data was determined using one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls t-test and was determined using the SPSS v26 software. Values were considered as being statistically significant if p-values ≤ 0.05.

3. Results

3.1. Viscometry Assessment

The experimental results obtained following the assessment of the relative viscosity of complexes formed by DNA and increasing concentrations of melatonin are presented in Figure 1a. A decrease in the relative viscosity of the formed complexes was registered, along with the increase in the melatonin–DNA molar ratio. This phenomenon is the effect of the “non-classical” model of intercalation of melatonin in the DNA structure, which supposes the twisting of the double-stranded DNA structure, compressing the double helix and leading to a decrease in the DNA solution’s viscosity.

Figure 1b presents the results of the determination of the relative viscosity of complexes formed by DNA and increasing concentrations of serotonin. The increase in the relative viscosity of the formed complexes was consequent to the increase in the serotonin–DNA molar ratio. This phenomenon is the effect of the “classical” model of serotonin intercalation between the DNA nitrogenous base pairs, which implies that the DNA double helix structure undergoes an elongation process as the nitrogenous base pairs move apart in order to accommodate the serotonin molecules.

In Figure 1c, we present the data regarding the relative viscosity of DNA solution in the presence of increasing concentrations of tryptophan. A non-linear decrease in the relative viscosity of the complexes is depicted, along with the increase in the tryptophan–DNA molar ratio. This phenomenon is the effect of the “non-classical” model of tryptophan intercalation in the DNA structure, compressing the double helix and leading to a viscosity decline. The non-linear binding behavior of tryptophan to DNA could arise from its ability to induce structural changes in the DNA that are not uniform across all binding sites. As tryptophan intercalates into the DNA, it may cause localized unwinding or stretching of the helix, which can alter the overall DNA conformation in a manner that is not simply additive. This effect can lead to a complex relationship between the concentration of tryptophan and the resulting changes in DNA viscosity.

At lower concentrations, tryptophan may induce slight elongation of the DNA, leading to increased viscosity. However, as the concentration increases, the cumulative structural perturbations might lead to DNA compaction or other conformational changes that reduce the viscosity, deviating from a linear relationship.

The dynamics of these biochemical processes have been statistically evaluated using the SPSS v26 software to calculate the Pearson coefficient:

• DNA/melatonin complex viscosity – Pearson coefficient r = −0.98267;
• DNA/serotonin complex viscosity – Pearson coefficient r = 0.9784;
• DNA/tryptophan complex viscosity – Pearson coefficient r = −0.80904.

The most interesting dynamics of the intercalation processes in the DNA structure belong to melatonin and serotonin, compounds that intercalate in the double-stranded DNA structure through different mechanisms. As such, the viscosity of the DNA–melatonin complex showed a statistically significant decrease compared to the viscosity of the DNA–serotonin and DNA–tryptophan complexes (p < 0.001). The viscosity of the DNA–serotonin complex showed a statistically significant increase compared to the DNA–tryptophan complex (p < 0.02). The interaction of indoles with DNA has the potential to modulate gene expression through a variety of mechanisms, including changes in DNA structure, interference with transcriptional machinery, modulation of topoisomerase activity, and induction of epigenetic changes. These effects could have significant biological implications, influencing cellular function, development, and disease processes.

3.2. Denaturation–Renaturation Studies

In view of the spectroscopic studies on the influence of the considered bio-indoles on the denaturation–renaturation processes of double-stranded calf thymus DNA, the denaturation–renaturation DNA behavior was monitored in the absence and in the presence of indole molecules by registering the spectra of the solutions in a cyclic experiment at temperatures ranging from 30 to 90 °C, in a gradient of 2 °C/1.2 min, in ascending and descending order. We obtained, based on the absorptions registered at 260 nm, the hysteresis curves of the double-stranded ct-DNA, under the influence of the three studied indole compounds presented in Figure 2.

Throughout the denaturation study, we observed, for the DNA solution, a relative leap of absorption, close to the 76 °C point, due to the transformation (denaturation) of the double helix. This phenomenon is registered around the temperature of 81 °C, in the presence of melatonin molecules, and is not registered at all if serotonin is added to the DNA solution. As such, the process of accentuated denaturation of the double-stranded DNA structure does not occur. When adding the tryptophan solution, the DNA denaturation achieves slow progress, with the initiation point of the denaturation process being registered at 67 °C, but the process advances rather slowly.

Tryptophan, being an aromatic amino acid with a planar indole ring, can interact with DNA through intercalation or groove binding. During denaturation, tryptophan’s presence tends to stabilize the DNA double helix to a certain extent but initiates the denaturation process at a lower temperature (around 67 °C). The progression of denaturation is slow, indicating that tryptophan moderately stabilizes the DNA structure, making the strands less prone to separation. However, this stabilization is not strong enough to prevent denaturation but rather slows it down, reflecting a gradual disruption of the hydrogen bonds between base pairs.

Serotonin has a more pronounced stabilizing effect on DNA. The presence of serotonin in DNA solutions appears to significantly inhibit the denaturation process. Unlike in the control (DNA without indoles), where a clear absorption increase occurs due to denaturation, DNA with serotonin does not exhibit this absorption shift, suggesting that the double-stranded structure remains intact. This indicates that serotonin strongly stabilizes the DNA helix, likely through intercalation between base pairs or by reinforcing hydrogen bonding, preventing the strands from separating even at elevated temperatures.

Melatonin also interacts with DNA, but its effects are distinct. In the presence of melatonin, the DNA denaturation occurs at a higher temperature (around 81 °C) compared to the control. This shift indicates that melatonin stabilizes the DNA double helix more effectively than tryptophan but less so than serotonin. The stabilization effect delays the onset of denaturation, requiring higher thermal energy to disrupt the hydrogen bonds and unwind the helix. However, once denaturation begins, the process may proceed more rapidly compared to the gradual denaturation observed with tryptophan.

Indoles can either stabilize or slightly destabilize the DNA double helix, affecting its thermal stability and response to denaturation. The interaction of these indoles with DNA influences the temperature at which the DNA strands separate and how quickly they do so. Serotonin, with its strong stabilizing effect, may protect DNA from denaturation, potentially making it more resistant to environmental stressors. Melatonin also stabilizes DNA but to a lesser extent, delaying but not preventing denaturation. Tryptophan, while also stabilizing, allows for a slow and gradual denaturation, suggesting a more moderate protective role.

These effects have significant implications for understanding how these bioactive molecules might influence genetic stability and integrity in biological systems, particularly in conditions that involve thermal or chemical stress. In therapeutic contexts, these interactions could be leveraged to modulate DNA stability, potentially influencing gene expression, cellular responses to damage, or the efficacy of treatments that rely on DNA-targeting mechanisms.

For the mathematical description of the DNA denaturation–renaturation process in the presence of indoles, we calculated the absorbance variation (Δ absorbance) of the DNA solutions in the presence of indole derivatives. This variation has been computed for temperatures ranging from 30 to 40 °C, which are appropriate for the living cell (

Table 1. Absorbance variation (Δ _absorbance) of the DNA solutions in the presence of indoles, for temperatures ranging from 30 to 40 °C.

Temperature	DENATURATION			RENATURATION
	Δ abs.Mel	Δ abs.5HT	Δ abs.Trp	Δ abs.Mel	Δ abs.5HT	Δ abs.Trp
30 °C	0.263 ± 0.012	0.289 ± 0.014	0.234 ± 0.013	0.204 ± 0.017	0.195 ± 0.009	0.134 ± 0.005
35 °C	0.283 ± 0.011	0.31 ± 0.021	0.251 ± 0.012	0.21 ± 0.011	0.203 ± 0.012	0.142 ± 0.008
40 °C	0.295 ± 0.015	0.323 ± 0.016	0.253 ± 0.011	0.219 ± 0.018	0.211 ± 0.011	0.15 ± 0.011

).

At 35 °C, during the denaturation process, it was found that Δ _absorbance of the DNA–melatonin complex (0.283 ± 0.011) is higher than that of the DNA–tryptophan complex and smaller than the DNA–serotonin complex (0.310 ± 0.021). At the same temperature, the renaturation process displays much smaller values of Δ _absorbance computed for the DNA–melatonin complex (0.210 ± 0.011), DNA–serotonin complex (0.203 ± 0.012), and DNA–tryptophan complex (0.142 ± 0.008). (Trp = tryptophan, 5HT = serotonin, Mel = melatonin).

3.3. Fluorometric Studies Concerning the Influence of Different Concentrations of Mg²⁺ and Ca²⁺

Further experimental results show the relative fluorescence intensities of the indole molecules and double-stranded calf thymus DNA complexes and the influence of increasing concentrations (from 0.01 mol/L to 0.1 mol/L) of Mg²⁺ and Ca²⁺ on the fluorescent signal intensity. Figure 3a,b show representative fluorescence spectra registered during the assessment study of the influence of Mg²⁺ and Ca²⁺ ions on the fluorescent signal intensity of the DNA–melatonin complex.

Figure 4 presents the experimental data obtained from the in vitro evaluation of the influence of Mg²⁺ ions on the fluorescence signals of indole–DNA complexes. Upon the addition of increasing concentrations of Mg²⁺, a decrease in fluorescence was observed across the three analyzed complexes; however, this reduction was statistically insignificant. This outcome likely reflects the strong binding affinity between the indoles and DNA, which remains largely unaffected by the presence of Mg²⁺ ions, indicating that the stability of these complexes is not significantly compromised by Mg²⁺.

Mg²⁺ ions are ubiquitous in biological systems and play a critical role in stabilizing the DNA double helix by neutralizing the negative charges on the phosphate backbone. The finding that Mg²⁺ does not significantly disrupt indole–DNA complexes suggests that these interactions can persist under physiological ionic conditions, making them relevant for in vivo biological processes. This could imply that the biological effects of indole compounds on DNA, such as modulation of gene expression or interaction with DNA-binding proteins, are not easily mitigated by the ionic environment within cells.

Figure 5 presents the experimental data from the in vitro evaluation of the influence of Ca²⁺ ions on the fluorescence signal of the indole–DNA complexes. The introduction of increasing concentrations of Ca²⁺ resulted in a slight reduction in fluorescence. However, this decrease was statistically insignificant, likely due to the robust DNA–indole binding, which remains stable and is not significantly affected by the presence of Ca²⁺ ions.

Ca²⁺ ions play crucial roles in numerous cellular processes, including signal transduction, muscle contraction, and enzyme activity. The fact that Ca²⁺ does not significantly disrupt indole–DNA complexes suggests that these interactions are relevant in physiological conditions, where Ca²⁺ concentrations can fluctuate. This resilience enhances the potential biological significance of indole–DNA interactions, as they would remain intact and functional in vivo, even in the presence of varying levels of Ca²⁺.

3.4. Assessment of the DNA–Ligand Interaction Using Molecular Docking

Using molecular docking, we further explored the intricate interplay between DNA and ligands to elucidate crucial insights into their binding mechanisms. For visualization, we selected the conformations with the optimal binding energy. All ligands bound preferentially between G and C. Intercalation occurred through various mechanisms, including π-π stacking interactions established between the planar polyaromatic system of the ligands and the two flanking bases (Figure 6, Figure 7 and Figure 8), with melatonin having the highest binding energy of the indole derivatives. Other interactions which contributed to the formation of DNA–intercalator complexes were Van der Waals, conventional hydrogen bonding, and carbon hydrogen bonding. For melatonin and tryptophan, interactions occur between the hydrogen atoms bonded to carbon atoms of the ligand and electronegative atoms on the adenine and thymine bases or adenine, respectively. The specific orientation and electronic environment of the adenine and thymine bases facilitate these interactions, making them relevant in the context of the binding affinity observed in our docking study. The same type of interaction was observed between serotonin and guanine. Although carbon hydrogen bonds are generally weaker than conventional hydrogen bonds, which involve nitrogen or oxygen, their presence can still contribute to the stabilization of the ligand–DNA complex. The presence of C-H interactions, alongside π-π stacking and other hydrogen bonds, underscores the multifaceted nature of the binding mechanism between melatonin and DNA, emphasizing the importance of these less conventional interactions in molecular recognition and stabilization of the DNA structure [63].

4. Discussion

The results obtained provide critical experimental evidence elucidating the biochemical mechanisms underlying the direct interaction of DNA with the three bio-indoles studied: melatonin, serotonin, and tryptophan. Viscosity measurements of the melatonin–DNA complex revealed a decrease in relative viscosity with increasing melatonin concentration. This observation supports the “non-classical” model of intercalation, where melatonin induces a twisting of the double-stranded DNA, compressing the helix and resulting in reduced solution viscosity. In contrast, the evaluation of the serotonin–DNA complex showed an increase in relative viscosity as the serotonin–DNA molar ratio increased. This behavior aligns with the “classical” model of intercalation, where serotonin inserts between the nitrogenous base pairs of DNA, causing the helix to elongate as the base pairs separate to accommodate the serotonin molecules. This intercalation process leads to an increase in the viscosity of the DNA solution [5,14,52].

For the tryptophan–DNA complex, we observed a non-linear decrease in relative viscosity as the tryptophan–DNA molar ratio increased. This behavior is indicative of a “non-classical” intercalation mechanism, where tryptophan inserts into the DNA structure in an inconsistent way that compresses the double helix, resulting in a reduction in the viscosity of the DNA solution or elongates its structure, reversing the effect.

Additionally, the viscosity of the DNA–melatonin complex was found to be significantly lower than that of both the DNA–serotonin and DNA–tryptophan complexes (p < 0.001). Furthermore, the viscosity of the DNA–serotonin complex was significantly higher than that of the DNA–tryptophan complex (p < 0.02). These findings confirm that indole molecules interact with DNA through distinct intercalation mechanisms, supporting the conclusions of previous studies [2]. The differences in viscosity reflect the varying degrees of DNA structural alteration induced by each indole, with melatonin leading to the greatest reduction in viscosity, followed by tryptophan, and with serotonin resulting in the highest viscosity due to its classical intercalation mode. Indole-mediated DNA stabilization may have implications for epigenetic regulation. The structural stability of DNA can influence the accessibility of chromatin to enzymes that add or remove epigenetic marks, such as DNA methylation or histone modifications. By stabilizing specific regions of the genome, indoles could affect the pattern of epigenetic modifications, thereby influencing gene expression profiles and potentially altering cellular identity and function.

Throughout the denaturation study, a significant increase in absorption at 76 °C was observed for the DNA solution, indicating the point of double helix denaturation. This increase was noted in the presence of melatonin at approximately 81 °C, and not registered in the presence of serotonin. This suggests that the denaturation process is not as abrupt in the presence of melatonin. Adding tryptophan resulted in a slow evolution of DNA denaturation, with the initial denaturation point registered at 67 °C, and a nearly flat denaturation curve. These findings indicate that the denaturation–renaturation process is affected by the presence of indoles due to their strong interactions with the DNA [55].

Indoles can modulate the thermal stability of the DNA double helix, either enhancing its stability or slightly reducing it, thereby influencing the DNA’s resistance to denaturation. The interaction between indoles and DNA alters the temperature at which the DNA strands separate and the rate at which this separation occurs. Serotonin exhibits a strong stabilizing effect, potentially safeguarding DNA against denaturation and making it more resilient to environmental stressors. Melatonin also contributes to DNA stabilization, though to a lesser degree, delaying the onset of denaturation without fully preventing it. Tryptophan, while providing some stabilization, allows for a slow and gradual denaturation, indicating a more moderate protective influence.

These findings are crucial for understanding the role of bioactive molecules in maintaining genetic stability and integrity, particularly under conditions of thermal or chemical stress. In therapeutic settings, these interactions could be harnessed to modulate DNA stability, thereby impacting gene expression, cellular responses to damage, and the effectiveness of treatments that target DNA.

Experimental data obtained after the in vitro assessment of the influence of Mg²⁺ and Ca²⁺ ions on the fluorescence signal of melatonin–DNA, serotonin–DNA, and tryptophan–DNA complexes revealed that increasing concentrations of cations determined a slight decrease in the fluorescence intensity, suggesting a robust binding pattern between DNA and indole derivatives, minimally influenced by the presence of these ions, at physiological concentrations. The minimal effect of Mg²⁺ and Ca²⁺ on the fluorescence signal suggests that the binding between indoles and DNA may involve interactions that are not heavily reliant on ionic conditions, such as hydrophobic interactions, hydrogen bonds, or π-π stacking. Understanding these binding mechanisms can guide the design of more targeted drugs or biomolecules that leverage these stable interactions with DNA.

Molecular docking further supported our findings, providing supplementary information on the possible DNA–ligand interaction. Small molecules interact with DNA through intercalation or groove recognition. Intercalation induces structural changes in DNA, opening gaps between consecutive base pairs. Using a crystallographic oligomer with an intercalation gap improves docking accuracy, distinguishing intercalation from groove recognition and enhancing assessment precision [64]. We used crystallographic DNA containing a gap to refine our understanding of binding mechanisms and enhance the predictive capabilities of the study.

The assessed indole derivatives were shown to be localized mostly between C and G, interacting with these bases through π-π interactions and hydrogen bonds. These results align with the previously obtained data on conventional intercalators. These do not possess sequence selectivity; their DNA binding is reliant on π-stacking and stabilizing electrostatic interactions. Small aromatic compounds, owing to their planar structure and appropriate size, tightly insert between adjacent base pairs of the DNA double helix, disrupting regular stacking and causing slight unwinding [2]. This alteration modifies the overall structure of DNA, resulting in changes to its physical and chemical properties, including increased rigidity and altered electronic characteristics. Crucially, intercalated ligands can impact DNA replication, transcription, and repair processes [60,61,62,64]. The ability of indoles to stabilize DNA could be harnessed in therapeutic contexts, particularly for diseases characterized by genomic instability, such as cancer. Indole derivatives might be developed as drugs that protect DNA from damage or that modulate DNA dynamics to influence gene expression in beneficial ways. For example, they could be used to enhance the efficacy of existing therapies by preventing the mutation of oncogenes or tumor suppressor genes, or by sensitizing cancer cells to DNA-damaging agents.

5. Conclusions

In the actual context of increasing prevalence of oncological pathologies, most of which are resistant to conventional therapies, there is a persistent need for novel approaches to protect vital cell components and counteract tumor aggression [65,66,67,68,69,70,71,72,73,74].

The interaction between small molecules and double-stranded DNA has been extensively studied for over five decades. Initially, intercalation gathered limited interest due to the prevailing belief that small molecules primarily bind to protein receptors, regulating biological function. However, recent discoveries of nuclear enzymes like topoisomerases have reshaped this understanding, as they modulate the intercalation of various compounds, including certain antitumor drugs and genotoxins. Two parallel research avenues have emerged: developing technology to utilize intercalation in designing safe and effective chemicals, such as pharmaceuticals, nutraceuticals, and agricultural chemicals and investigating intercalation in the mechanism of action of nuclear receptor proteins. Computational modeling has revealed that the fit of certain small molecules into DNA intercalation sites correlates with their biological activity, rather than their binding strength to receptors. Consequently, computational tools have been developed to predict the desirable and undesirable activities of new drug candidates. Multiple lines of experimental evidence suggest that intercalation in double-stranded DNA is a widespread, natural process integral to gene regulation. If proven to be the ultimate target of genomic drug action, intercalation will play a pivotal role in discovering safe and effective pharmaceuticals [75].

The indole ring, a nitrogen heterocyclic compound prevalent in nature under various forms, among which tryptophan, serotonin, and melatonin, plays a crucial role in physiological and biochemical processes in the human organism. Investigating the interactions between indole derivatives and DNA presents an effective approach to discover and develop novel anticancer agents. The potential derivatives containing one or more pharmacophores with diverse mechanisms of action could enhance desired properties and potentially mitigate drug resistance [76,77,78].

In summary, the broader biological implications of indole-mediated DNA stabilization are extensive and multifaceted, encompassing a wide range of cellular processes and potential therapeutic avenues. By influencing gene expression, indoles can modulate the activation or repression of specific genes, which is fundamental to the regulation of cellular function, differentiation, and response to environmental stimuli. This control over gene expression could be particularly relevant in diseases where gene regulation is disrupted, such as in cancer, where restoring normal expression patterns might inhibit tumor growth or enhance the effectiveness of existing treatments.

Furthermore, the stabilization of DNA by indoles plays a crucial role in maintaining genome integrity. Genomic stability is essential for the prevention of mutations that could lead to cancer, genetic disorders, and other age-related diseases. By protecting DNA from damage and preserving its structural integrity, indoles may contribute to the prevention of these conditions, thereby supporting overall cellular health and longevity.

The impact of indole–DNA interactions also extends to the regulation of cellular aging. As cells age, DNA becomes more susceptible to damage, leading to a decline in cellular function and an increased risk of disease. Indole-mediated stabilization of DNA could potentially slow down the aging process by reducing the accumulation of DNA damage, thereby extending the lifespan and delaying the onset of age-related conditions.

Epigenetic regulation is another critical area influenced by indole–DNA interactions. Epigenetic modifications, such as DNA methylation and histone modification, play a vital role in controlling gene expression without altering the underlying DNA sequence. By stabilizing specific regions of DNA, indoles may influence the patterns of epigenetic modifications, thereby affecting long-term gene expression and cellular memory. This could have significant implications for understanding developmental processes, disease progression, and the potential for reversing aberrant epigenetic changes in therapeutic contexts.

In therapeutic applications, the ability of indoles to stabilize DNA offers promising opportunities for drug development. Indole derivatives could be designed to target specific DNA regions, modulating gene expression or enhancing the efficacy of DNA-targeting therapies. For example, in cancer treatment, indole-based drugs could be developed to protect healthy cells from DNA damage while sensitizing cancer cells to chemotherapy. Additionally, the use of indoles in gene therapy could enhance the stability and delivery of therapeutic genes, improving the effectiveness of these treatments.

Overall, these effects underscore the critical importance of understanding how indoles interact with DNA. By elucidating the mechanisms of indole–DNA interactions, researchers can leverage this knowledge to advance both basic biological research and clinical applications. The potential to manipulate DNA stability and function through indole interactions opens up new avenues for therapeutic intervention, offering hope for more targeted and effective treatments for a wide range of diseases. The intricate and specific interactions delineated in the study highlight the substantial potential of these indole moieties in pharmaceutical applications, indicating a vast scope for these promising compounds due to their diverse molecular targets.

6. Limitations of the Study

A limitation of our study is the exclusion of solvent molecules during the docking simulations. This decision was made to simplify the model and focus on the primary interactions between the ligands and DNA, which was our purpose. While this approach allows for a clearer interpretation of these direct interactions, it may overlook the potential influence of solvent-mediated effects, particularly on electrostatic energy calculations. Additionally, counter ions were not explicitly included in our simulations, which could affect the electrostatic environment around the DNA. Future studies will aim to incorporate both solvent molecules and counter ions to provide a more comprehensive understanding of ligand–DNA interactions under physiological conditions.