Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Ismail Celil Haskologlu; Emine Erdag; Ahmet Ozer Sehirli; Orhan Uludag; Nurettin Abacioglu

doi:10.33069/cim.2024.0012

Chronobiol Med > Volume 6(2); 2024 > Article

Haskologlu, Erdag, Sehirli, Uludag, and Abacioglu: Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Original Article

Chronobiology in Medicine 2024;6(2):87-99.

Published online: June 28, 2024

DOI: https://doi.org/10.33069/cim.2024.0012

Exploring the Therapeutic Potential of Benzoxazolone Derivatives on the Circadian Clock: An In Silico and Hypothetical Approach

Ismail Celil Haskologlu¹

, Emine Erdag²

, Ahmet Ozer Sehirli³

, Orhan Uludag⁴

, Nurettin Abacioglu¹

¹Pharmacology Department, Faculty of Pharmacy, Near East University, Nicosia, Mersin-10, Türkiye

²Pharmaceutical Chemistry Department, Faculty of Pharmacy, Near East University, Nicosia, Mersin-10, Türkiye

³Pharmacology Department, Faculty of Dentistry, Near East University, Nicosia, Mersin-10, Türkiye

⁴Clinical Pharmacy Department, Faculty of Pharmacy, Near East University, Nicosia, Mersin-10, Türkiye

Corresponding author: Emine Erdag, PhD, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Near East University, Near East Boulevard, Nicosia Mersin-10 99138, Türkiye.
Tel: 905338898921, E-mail: emine.erdag@neu.edu.tr

Received May 16, 2024 Revised June 5, 2024 Accepted June 10, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

Pain conditions exhibit variations linked to circadian rhythms. Circadian rhythms, regulated by clock proteins, impact melatonin levels and immune functions. Melatonin, structurally similar to indomethacin, a non-steroidal anti-inflammatory drug, serves as an alternative in pain-related conditions. Literature suggests a correlation between melatonin-dependent regulation of circadian rhythm and reductions in pain complaints. 2(3H)-Benzoxazolone, known for its anti-inflammatory and analgesic properties, is a promising scaffold for drug design. In this study, pharmacophore analysis focused on benzoxazolone derivatives, evaluating their impact on clock proteins, and providing insights into potential chronotherapeutic implications.

Methods

Molecular docking and dynamics simulations were conducted on CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 clock proteins using benzoxazolone and its 5-substituted derivatives. Molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) calculations were employed to analyze binding free energies. Benzoxazolone derivatives, especially 5-nitro-2-benzoxazolone and 5-fluoro-2-benzoxazolone, exhibited elevated binding affinities on clock proteins compared to melatonin and indomethacin, the reference molecules. The interaction profiles and stability of complexes were maintained throughout the simulations.

Results

The results suggest a regulatory impact on CRY proteins, emphasizing their role in circadian rhythm and pain modulation. In addition, the benzoxazolone ring and its derivatives, structurally resembling the core structure of melatonin and indomethacin, demonstrate promising affinities on clock proteins.

Conclusion

These findings provide preliminary data and hypothetically propose benzoxazolone derivatives as potential candidates for dual functionality in analgesic activity and circadian rhythm regulation, warranting further in vitro and clinical investigations.

Keywords: Clock genes; Benzoxazolone; Melatonin; Circadian rhythm; Molecular docking

INTRODUCTION

The majority of patient presentations to healthcare institutions typically involve complaints related to inflammation and pain [1]. The onset of the inflammatory response entails the conversion of arachidonic acid (AA) into either prostaglandin H2 (PGH2) through cyclooxygenase (COX) or into leukotriene A4 (LTA4) via 5-lipoxygenase (5-LO). In the subsequent stages, specialized synthetase enzymes convert PGH2 and LTA4 into bioactive lipid mediators, such as prostaglandin E2 (PGE2) [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) serve as the cornerstone in CIMpharmacological intervention for diverse inflammatory conditions, owing to their versatile anti-inflammatory, antipyretic, and analgesic effects [3]. By inhibiting COX enzymes, NSAIDs attenuate the production of PGE2, thereby contributing to the amelioration of symptoms associated with fever, inflammation, and pain [4].

Some pains have been reported to exhibit variations in their intensity or frequency during day/night times and in well-lit/dark environments [5]. Recent studies have associated certain inflammatory cytokines with circadian rhythms [6,7]. The circadian rhythm is an internal clock system that regulates an organism’s biological processes in a 24-hour cycle. In mammals, the circadian system consists of a central clock within the suprachiasmatic nucleus (SCN) and peripheral clocks found in all cells. The circadian clock, comprising the circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein-1 (BMAL1), is controlled through a feedback loop involving core clock gene proteins. CLOCK and BMAL1 sustain the expression of two inhibitors, cryptochrome (CRY) and period (PER), forming a molecular feedback loop [8]. During the peak phase of the circadian rhythm, certain hormone levels or immune functions may reach their highest levels [9,10].

Melatonin is a hormone that plays a crucial role in regulating the body’s biological clock and circadian rhythms [11]. Melatonin production is generally low during the day, increasing with exposure to sunlight. In the evening and throughout the night, melatonin production rises, serving as a signal that prepares the body for sleep [11]. Melatonin, due to its lipophilic properties, was reported to passively diffuse through cell membranes very recently, which would include those of the SCN cells [12]. This process does not rely on specific transport mechanisms or membrane-bound receptors. Additionally, melatonin’s passive diffusion is a well-accepted concept in the scientific community, supported by its ability to traverse cellular barriers due to its chemical nature [13]. Moreover, the direct passive diffusion of melatonin through cell membranes is widely recognized, and this capability extends to various cell types [12,13]. This implies that melatonin can pass into SCN independently of transport mechanisms like glucose transporters or peptide transporters, which are also pathways for its intracellular delivery and may affect core clock proteins. Some research indicated that melatonin influences the molecular clock proteins which are a group of proteins that regulate circadian rhythms at the cellular level [14]. This influence occurs through modulation of the transcriptional activities of core clock protein complexes such as CLOCK and BMAL1 dimer, and post-translational modifications of clock proteins [14]. Melatonin can influence the expression of these clock proteins, helping to maintain the body’s internal clock and synchronize it with environmental cues such as light and darkness. Therefore, melatonin is integral to the functioning of both the central biological clock and the molecular clock proteins, playing a crucial role in regulating circadian rhythms [14,15].

Circadian rhythms influence pain perception and sensitivity, with pain often exhibiting diurnal variations [16]. Disruptions in circadian rhythms, such as those seen in sleep disturbances or shift work, can exacerbate pain symptoms [16]. Melatonin’s regulation of core clock proteins helps maintain circadian rhythms, which in turn can modulate pain perception and sensitivity. Furthermore, melatonin has anti-inflammatory properties and can modulate immune responses [17]. Core clock proteins are involved in the regulation of inflammatory pathways. By influencing the activity of these clock proteins, melatonin regulates inflammation and its contribution to pain pathology [17]. In addition, circadian rhythms play a role in central sensitization, a process implicated in chronic pain conditions [18]. Melatonin’s effects on core clock proteins may influence the regulation of neuronal excitability and synaptic plasticity, thereby modulating central sensitization and pain processing [18]. Previous research has demonstrated that the pain threshold varies with circadian rhythm, decreasing with the application of twenty-four hours of light exposure, and subsequently returning to normal with the administration of melatonin [5,19]. The results of these studies indicate a correlation between melatonin’s regulation of circadian rhythm and reductions in pain complaints.

Among NSAIDs, indomethacin exhibits notable efficacy in the treatment of certain types of headaches, especially hemicrania continua [20]. Melatonin, with a chemical structure closely resembling indomethacin, has been shown in a previous study to be effective in hemicrania continua-type headaches as an alternative to indomethacin [21]. Additionally, the study explored the use of melatonin instead of indomethacin, including NSAIDs’ prominent side effects, and gastrointestinal disturbances. Discovering compounds with similar chemical structures to indomethacin and melatonin is crucial for reducing side effects observed in NSAIDs [22,23] and maximizing efficiency in chronotherapeutic terms by investigating their effectiveness on circadian rhythm clock genes. Therefore, identifying pharmacophore groups that may create a bioisosteric effect as an alternative to the 5-methoxyindole core present in both indomethacin and melatonin is essential.

In the literature, 2(3H)-benzoxazolone, a heterocyclic compound renowned for its versatile applications in drug design, has garnered significant attention due to its anti-inflammatory and analgesic properties [24,25]. Benzoxazolone derivatives exert their analgesic and anti-inflammatory effects primarily by inhibiting COX enzymes similar to NSAIDs, thereby reducing the production of prostaglandins, which are inflammatory mediators [26]. While their direct interaction with molecular core clock proteins is not documented, their anti-inflammatory properties may influence circadian rhythms, as inflammation and immune responses are known to impact the circadian system [27]. The analgesic and anti-inflammatory effects of benzoxazolone derivatives and melatonin may involve feedback mechanisms that intersect with molecular clock protein regulation.

Despite the abundance of research focused on the analgesic and anti-inflammatory properties of benzoxazolone derivatives [28], a conspicuous gap exists in the literature concerning investigations into the correlation between the benzoxazolone ring structure and its derivatives with circadian rhythms. This study aims to elucidate the impact of the 2(3H)-benzoxazolone core, analogous to the principal ring structures found in both indomethacin and melatonin, as well as its 5-substituted benzoxazolone derivatives on CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 clock gene proteins. The findings are anticipated to highlight potential intersections between benzoxazolone derivatives, melatonin, and clock gene proteins and their relevance to circadian rhythm regulation. The computational insights obtained from this pilot study will serve as a foundational basis for guiding subsequent experimental studies.

METHODS

Ligands and pharmacophore mapping

The pharmacophore models for this study were determined based on the 5-methoxyindole core structure found in the main structures of melatonin and indomethacin. Specifically, the 2(3H)-benzoxazolone ring capable of exhibiting bioisosteric effects on this core structure [29], and its derivatives differing at the 5th position, namely 5-chloro-2-benzoxazolone, 5-bromo-2-benzoxazolone, 5-fluoro-2-benzoxazolone, and 5-nitro-2-benzoxazolone, were designated as ligands. The chemical structures of all molecules were drawn using ChemDraw Pro 12.0 software (PerkinElmer, Inc., Waltham, MA, USA) and converted to mol2 files using LigandScout 4.4.5 [30]. Subsequently, the pharmacophore mapping process was applied through the PharmMapper tool [31]. During this process, the molecules identified were aligned with the pharmacophore models of each target protein listed in the Protein Data Bank (PDB) database (https://www.rcsb.org/), and the compatibility values between each molecule and the pharmacophores were calculated. A strategy for finding the bestfitting pharmacophore for the tested molecules was implemented using genetic algorithms (GA) [31]. In this stage, triangulation of all tested molecules and target pharmacophore models, pairwise alignment, and optimization processes were sequentially carried out [32].

Molecular docking

In this study, the clock proteins CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 were identified as targets to determine the binding affinities of the benzoxazolone core and its 5-substituted benzoxazolone derivatives through pharmacophore mapping analysis. The three-dimensional (3D) structures of CLOCK:BMAL1, PER1, PER2, CRY1, and CRY2 were downloaded from the PDB database (PDB IDs: 4F3L, 4DJ2, 3GDI, 7DLI, and 7V8Y, respectively). All crystal structures referenced by the provided PDB IDs were derived from the organism Mus musculus. Before molecular docking, ligands were prepared through a refinement process, during which water molecules and heteroatoms were removed [33]. To ensure energetically favorable conformations and positions, the ligands were energy minimized using the Universal Force Field (UFF) and converted to PDBQT format with Open Babel in PyRx 0.8 with Lamarckian genetic algorithm [34]. Active pocket scanning was performed on three targets using the CASTp (Computed Atlas of Surface Topography of proteins) tool for the molecular docking study of selected ligands to identify the key amino acid residues involved in binding interactions [35]. The key amino acid residues observed in the active pockets were as follows: CLOCK—ARG126, PHE141, SER143, GLU146, LYS220; PER1—ARG300, ARG312, TYR313, PRO473; PER2—TYR354, LEU359, ARG399; CRY1—LYS11, PHE257, ARG256, LEU386; and CRY2—LEU39, VAL188, CYS196. AutoDock Vina and Discovery Studio Visualizer 2021 software were employed for the molecular docking process [36]. Using AutoDock Vina software, various binding poses and binding affinity values (negative binding energies, ΔG, kcal/mol) between ligands and target genes were determined (Table 1), and interaction graphs were obtained for each complex. Throughout all molecular docking procedures, consistent grid box dimensions of 30× 30×30 Å were utilized.

Molecular dynamics simulations

In this study, a molecular dynamics (MD) simulation lasting 100 ns was conducted using the GROMACS version 5.1.4 (https://www.gromacs.org/), employing the GROMOS96 54A7 force field parameters [37]. With a previously published procedure, the simulation box was filled with simple point charge (SPC) water molecules, arranged in a cubic formation surrounded by periodic boundary conditions [38]. The simulation box created was maintained at a minimum distance of 1.5 nm. Charge neutrality within the complexes formed between the selected ligands and target proteins was preserved throughout the simulation period through the introduction of Na+ and Cl– ions, resulting in a concentration of 0.15 M NaCl. Long-range electrostatic interactions were computed using the particle mesh Ewald (PME) technique [39]. To minimize the energy of the system, the steepest descent method was utilized, followed by a progressive heating process. The progressive heating process consisted of a series of 100 ps NVT (constant number of particles, volume, and temperature) simulations. During this process, all atoms were restrained to a position of 1,000 kJ/mol, and the system’s temperature was gradually increased to 310 K [40]. The simulation was facilitated using V-scale temperature coupling. The simulations were conducted at a temperature of 300 K. The coupling constant used in the equilibration phase was kept at 0.1 ps and the pressure was maintained at 1 bar, controlled by the Parrinello-Rahman barostat, which has been described in previous research [41]. Furthermore, root-meansquare deviation (RMSD) parameter analysis was performed to generate RMSD graphs for ligand-target protein complexes.

MM/PBSA calculations

In addition to the molecular docking method, the binding free energies of all ligand-gene complexes simulated were analyzed using the previously published molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method [42,43].

The MM/PBSA method calculates the binding free energy (ΔG_bind) through the subtraction of the sum of the protein and ligand-free energies (ΔG_{target protein} + ΔG_ligand) from the free energy of the protein-ligand complex (ΔG_complex), as expressed as follows:

ΔG_bind = ΔG_complex – (ΔG_{target protein} + ΔG_ligand). (1)

The binding affinities of the complexes formed by all ligands utilized in the molecular docking process were determined in the last 15 ns of the simulation (Table 2).

Statistical analysis

The data obtained from molecular docking scores and MM/PBSA binding energy calculations were expressed as mean± standard deviation. For obtaining molecular docking scores, the average binding energy values and standard deviation were calculated considering the probabilities of five different docking poses generated over the binding region of each ligand corresponding to the respective clock protein. GraphPad Prism version 8.4.2 (GraphPad Software, Boston, MA, USA) was employed for data analysis. For each analysis, ligands and ligand-clock protein complexes with binding energies more negative than the control group were selected, and ordinary one-way ANOVA variance analysis with post hoc Dunnett test was applied. A p value <0.05 was considered statistically significant for all analyses.

RESULTS

Pharmacophore mapping analyses unveiled that the 2-benzoxazolone core structure shares analogous functional features and three-dimensional configurations with the 5-methoxyindole ring found in indomethacin and melatonin structures. The activity was noted to be potentially influenced by the 5th position in both rings. We demonstrated how different substituents affected the binding affinities of 2-benzoxazolone and 5-substituted benzoxazolone derivatives on target clock proteins, using 5-methoxyindole as a reference molecule. According to the results, the presence of electron-withdrawing groups such as nitro group (-NO₂) and halogens (-F, -Cl, -Br) at the 5th position enhances the expression activity of the benzoxazolone ring on clock genes. In molecular docking studies involving core clock proteins (CLOCK:BMAL1, PER1, PER2, CRY1, CRY2), the three ligands with the highest affinity values were consistently identified as 5-fluoro-2-benzoxazolone, 5-nitro-2-benzoxazolone, and 5-chloro-2-benzoxazolone across all analyses. These ligands exhibited greater binding tendencies when compared to the control (reference) 5-methoxyindole core structure.

The molecular docking analysis revealed that the binding affinities of all tested ligands on the CLOCK:BMAL1 dimer ranged from -5.0 to -5.8 kcal/mol. The ligands exhibiting the most negative binding energy values towards the CLOCK:BMAL1 dimer binding region were determined to be 5-fluoro-2-benzoxazolone (-5.8 kcal/mol), 5-nitro-2-benzoxazolone (-5.6 kcal/mol), and 5-chloro-2-benzoxazolone (-5.5 kcal/mol). In comparison to the reference molecule, 5-methoxyindole, benzoxazolone derivatives containing 5-fluoro and 5-nitro groups demonstrated superior activity, while 5-chloro-2-benzoxazolone exhibited an equivalent binding affinity value to the reference molecule.

The variant analysis of molecular docking scores, measured in kcal/mol for each ligand, is presented in Figure 1. Within the assessed ligands binding to the CLOCK:BMAL1 region, only 5-fluoro-2-benzoxazolone demonstrated a more negative binding energy value than the control ligand. However, this disparity did not reach statistical significance. In the binding region of PER1, both 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone exhibited significantly higher binding energies (p<0.05 and p<0.0001, respectively) compared to the control ligand. Conversely, in the binding region of PER2, only the binding energy of 5-nitro-2-benzoxazolone was found to be statistically significant (p<0.0001) compared to the control ligand.

Furthermore, concerning the binding energy values of ligands in the active region of CRY1, 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone displayed superior and statistically significant binding affinities (p<0.05 and p<0.0001, respectively) in comparison to the control ligand. However, in the CRY2 active region, although 5-chloro-2-benzoxazolone and 5-nitro-2-benzoxazolone exhibited higher binding energies than the control ligand, only 5-fluoro-2-benzoxazolone demonstrated a statistically significant difference (p<0.001) in binding energy values compared to the control ligand.

In addition, using the MM/PBSA method, the binding energy values of the ligands and the binding poses created with CLOCK: BMAL1 dimer were found to be between -151.25±3.42 kJ/mol and -176.34±2.51 kJ/mol. Consistently, the highest binding energy values were calculated as -176.34±2.51 kJ/mol for 5-fluoro-2-benzoxazolone, -171.15±2.42 kJ/mol for 5-nitro-2-benzoxazolone, and -169.05±2.21 kJ/mol for 5-chloro-2-benzoxazolone. The two-dimensional (2D) interaction graphs obtained through molecular docking analysis with CLOCK:BMAL1 illustrated amino acid residues involved in interactions in detail (Supplementary Materials). Similar to the interactions of 5-methoxyindole, all ligands demonstrated pi-anion and pi-cation bonds with ARG126 and GLU146 amino acid residues. Furthermore, in contrast to the 5-methoxyindole structure, benzoxazolone derivatives indicated effective hydrogen bonding with LYS220 amino acid residue in the CLOCK:BMAL1 binding region. Additionally, it was observed that LYS220, along with some ligands, ARG126, PHE141, and SER143 amino acid residues, formed hydrogen bonds in the binding region.

All tested ligands showed binding affinities ranging from -5.0 to -5.8 kcal/mol for the PER1 clock protein. The ligand with the highest binding energy in the PER1 binding region was 5-nitro-2-benzoxazolone with a value of -5.8 kcal/mol, whereas 5-fluoro-2-benzoxazolone and 5-chloro-2-benzoxazolone were ranked second and third, respectively, with binding energy values of -5.6 kcal/mol and -5.3 kcal/mol. The MM/PBSA calculations showed that the binding energy values of the PER1-ligand complexes ranged between -197.02±3.53 kJ/mol and -242.72±2.24 kJ/mol. It was observed that the affinity of all ligands on the PER1 clock protein increased as compared to the binding energy values of CLOCK:BMAL1. The amino acid interactions of ligands within the PER1 active binding region, chemical bonds such as pi-pi tshaped (TYR313), pi-alkyl (ARG312 and PRO473), hydrogen bond (TYR313 and ARG430), and carbon-hydrogen bond (ARG300) were predominantly observed. Ligands containing the 2-benzoxazolone core structure have a significant role in the PER1 binding region by interacting with specific amino acid residues, similar to 5-methoxyindole. Amongst other ligands and the reference molecule in PER2-ligand interactions, 5-nitro-2-benzoxazole exhibits the highest binding affinity value (-6.1 kcal/mol) and MM/PBSA binding energy value (-249.63±2.25 kJ/mol), as with PER1. It was noticed that the ligands having a benzoxazolone core structure had similarities to the reference molecule based on interaction graphs in PER2 interactions. In the PER2 binding region, all ligand interactions, including the reference molecule, showed pipi t-shaped chemical bonds, particularly with the amino acid residue TYR354.

Furthermore, when evaluating the affinities of ligands for the CRY1 and CRY2 clock proteins, it was observed that the binding energies were higher compared to CLOCK:BMAL1 dimer and PER genes—in complexes formed with CRY1, the derivative 5-nitro-2-benzoxazolone exhibited a stronger affinity with a -7.4 kcal/mol negative binding energy value than other ligands and the reference compound (-7.1 kcal/mol). Additionally, in the analysis of the CRY2 protein, the compound with the highest affinity was 5-fluoro-2-benzoxazolone (-6.7 kcal/mol) compared to the binding energy of the reference molecule (-6.4 kcal/mol). Upon comparison of all CRY proteins, it was observed that the ligand affinities for CRY1 were superior to those for CRY2. Furthermore, MM/PBSA calculations for CRY1-ligand complexes showed binding energy values ranging from -253.12±2.41 kJ/mol to -352.36±2.34 kJ/mol. The binding energy value for the complex formed with the reference molecule and CRY1 was calculated as -318.41±3.52 kJ/mol. Compared to this value, ligands with 5-nitro and 5-fluoro benzoxazolone compounds demonstrated stronger binding energies, as observed in other clock proteins.

The variant analysis of MM/PBSA binding energies for each ligand complex is illustrated in a bar chart (Figure 2). Within the complexes formed with CLOCK:BMAL1, it was observed that the ligands 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone displayed more negative binding energies in comparison to the control group. Specifically, the binding energy value of the complex formed between the CLOCK:BMAL1 dimer and 5-fluoro-2-benzoxazolone exhibited statistical significance compared to the control group (p<0.0001). However, the binding energy value of the complex formed between the CLOCK:BMAL1 dimer and 5-nitro-2-benzoxazolone did not attain statistical significance.

Similar to the CLOCK:BMAL1 analysis, among the complexes formed with PER1, it was observed that the ligands 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone manifested more negative binding energies compared to the control group. The binding energy values of the PER1 complexes formed with 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone were both statistically significant compared to the control group (p<0.0001). Conversely, among the complexes formed with PER2, the ligands 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone demonstrated superior and statistically significant binding energy values compared to the control group (p<0.0001). In the case of cryptochrome ligand interactions, the complexes formed with 5-fluoro-2-benzoxazolone and 5-nitro-2-benzoxazolone exhibited statistically significant binding energy values for both CRY1 (p<0.0001) and CRY2 (p<0.0001), in comparison to the control group.

Additionally, when comparing the molecular interactions of the reference molecule with the interactions of other ligands carrying the benzoxazolone core, it has been demonstrated that they interact with common amino acid residues such as PHE257, LYS11, LEU55, ARG256, and LEU386 through pi-pi t-shaped, alkyl/pialkyl, and carbon-hydrogen bond interactions (Table 3). Furthermore, in benzoxazolone derivatives, it has been found that there are interactions with different amino acid residues such as ARG10, ARG51, and ALA388 through hydrogen bonds and halogen (fluorine) bonding. These interactions significantly affected the affinities of ligands for the CRY1 clock protein (Figures 3-5). On the other hand, in CRY2-ligand interactions, amino acid residues such as VAL188, MET193, and CYS196 were found in common in the interaction graphs of benzoxazolone derivatives and the reference molecule. However, in the interactions of benzoxazolone derivatives, leucine amino acid residues were abundant such as LEU39, LEU84, and LEU87.

All ligands, including the reference molecule utilized in the study, involve amino acids such as arginine, glutamate, serine, proline, tyrosine, leucine, and lysine in the interactions formed with CLOCK: BMAL1 and PER proteins. In the binding regions of CRY proteins, amino acids such as alanine, phenylalanine, leucine, methionine, arginine, and cysteine were predominantly observed. Molecular docking and simulation studies disclosed that the 2(3H)-benzoxazolone core exhibited binding affinities on the targeted clock proteins with amino acid residues and binding energies comparable to those of the reference molecule. Nevertheless, the incorporation of electron-withdrawing groups at the 5th position of the 2-benzoxazolone core structure led to distinct interaction patterns and resulted in enhanced binding energies with clock proteins. All examined ligands exhibited acceptable fluctuations within the initial 50–60 ns of the 100 ns simulation period and maintained the stability achieved during the last 30 ns of the simulation.

DISCUSSION

The circadian clock is an intrinsic timekeeping mechanism in biological organisms that follows the daily rhythm. The circadian clock assists organisms in adapting to daily changes in their internal time by regulating various biological and behavioral processes [44]. Functioning through the expression of a set of genes and the production of proteins, the circadian clock’s fundamental component is a protein with basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domains encoded by the CLOCK and BMAL1 (ARNTL) genes [45]. CLOCK and BMAL1 interact with each other, forming a heterodimeric complex. This CLOCK:BMAL1 complex engages with regulatory regions called E-boxes in the PER1, PER2, CRY1, and CRY2 genes throughout the day, activating their transcription and initiating RNA synthesis [45]. Proteins PER and CRY, produced during the day, accumulate and translocate into the nucleus during the night [46]. In the nucleus, these proteins directly interact with the CLOCK:BMAL1 complex, suppressing their transcription through a negative feedback loop. This feedback loop contributes to the regulation of the circadian clock mechanism, allowing the initiation of the next day’s cycle [46]. The complex interaction network among these genes and proteins enables the circadian clock mechanism to maintain its internal time and regulate various biological processes [46].

Drugs targeting circadian clock mechanisms have the potential to be effective in the treatment of a range of circadian rhythm-related disorders including pain [47]. A previous study stated that pain sensitivity is not constant throughout the day but peaks during the nighttime [48]. This pattern is primarily driven by the circadian system, responsible for about 80% of the fluctuation, while sleep-related processes account for the remaining 20%. This highlights the significant role of the circadian system in modulating pain [48]. In another study, it was established that core clock genes collaboratively interact with genes associated with pain modulation [49]. The study stated the exploration of the transcriptional profiles of circadian clock genes, pain-related genes (ADAM11, CACNA1B, CALCA), and the effects of melatonin and opioidrelated genes within the trigeminal ganglion (TG) of humans and mice [49]. The study also identified specific expression patterns of core circadian clock genes, including BMAL1 (ARNTL), CLOCK, PER1/2, and CRY1/2. In addition, the study reported the expression of genes related to melatonin and opioid responses [49]. Opioid-related genes like OGFR and OPRM1 also showed significant expression, which varies between human and mouse TG. It highlighted that melatonin can influence pain perception through its interaction with specific target genes in the TG. In addition to this, Chen et al. [50] stated melatonin’s potential as a multifaceted agent in pain management, linking its natural circadian rhythm-regulating capabilities with its analgesic and anxiolytic effects, thus offering a promising alternative to conventional pain medications with fewer adverse effects. Besides, melatonin, which possesses a chemical structure similar to that of the NSAID indomethacin, has been demonstrated to exhibit anti-inflammatory and antinociceptive properties [51,52]. Indomethacin has been employed in the management of headache disorders, shortly following its introduction as a therapeutic option for pain. In previous research, melatonin has demonstrated efficacy in headaches responsive to indomethacin, encompassing paroxysmal hemicrania and hemicrania continua [21,37,52].

The discovery of pharmacophores similar to the core structure of melatonin and indomethacin, which is the 5-methoxyindole nucleus, enables the development of innovative treatment strategies targeting the circadian rhythm [53]. These strategies may offer alternatives to traditional treatment methods, leading to more specific and effective therapies for pain modulation. An example of an alternative method is pharmacophore mapping analysis which is aimed at identifying the molecular features necessary for a drug to interact with a biological target [54,55]. This analysis provides a summary of the combination of the smallest structural features of a pharmacophore, which can influence specific biological activities including analgesic and anti-inflammatory. Similarly, in our study, pharmacophore mapping analysis has been utilized to select benzoxazolones, as potential drug candidates for the target clock genes from the PDB database. Prior investigations have illustrated the employment of the benzoxazolone ring in assessing analgesic and anti-inflammatory properties [56,57]. It was noted that benzoxazolone scaffold forms the fundamental structure of diverse compounds in pharmacological studies exploring a range of biological effects [58]. In light of previous research, our investigation utilized molecular docking and simulation analyses to illustrate that 2-benzoxazolone and its diverse 5-substituted derivatives may possess pharmacophores similar to the 5-methoxyindole from a chronopharmacological point of view. Significantly, the 5-nitro-2-benzoxazolone and 5-fluoro-2-benzoxazolone emphasized in our study exhibited notably elevated binding energies on clock genes acknowledged for their pivotal role in circadian rhythm regulation, surpassing those of the 5-methoxyindole—a foundational structure within the wellestablished melatonin molecule.

The X-ray structure of the CLOCK:BMAL1 transcriptional activator complex has been presented, allowing for the atomic-level analysis of the multiprotein complexes involved in mammalian circadian clock mechanisms [59]. In addition, X-ray crystallographic structures of the period and cryptochrome genes have been utilized in previous research [60,61]. For instance, studies with mPer gene knockout mice have demonstrated the greater significance of mPer1 and mPer2 compared to mPer3 [62-64]. In our study, using similar crystallographic structures of main clock proteins, we emphasized the significant role of amino acid residues such as leucine, arginine, phenylalanine, serine, proline, cysteine, tryptophan, and methionine which were present in the binding region analysis of all clock proteins. We demonstrated that among the tested ligands, the presence of the electron-withdrawing nitro group at the 5th position, similar to the 5-methoxyindole structure, functions as an essential functional group capable of enhancing affinity by forming hydrogen bonds in the binding regions of clock proteins. Moreover, upon analysis of complexes involving all clock proteins, it can be asserted that the fluorine substituent located at the 5th position of the 5-fluoro-2-benzoxazolone molecule establishes alkyl/pi-alkyl, hydrogen bonding, pianion, pi-cation, and pi-pi T-shaped interactions. Additionally, it contributes to the formation of halogen (fluorine) bonding within the binding regions of target proteins. This feature is identified as one of the crucial factors contributing to increased affinity. The stability analysis throughout the 100 ns simulation period revealed that all complexes created in the study reached stability within this timeframe and maintained their stability. Considering the MM/PBSA calculations and binding affinity values presented in our study, the benzoxazolone core and its 5-substituted benzoxazolone derivatives, as identified in our pharmacophore mapping analysis, exhibit similar affinities and interaction profiles to the 5-methoxyindole structure, position them as effective pharmacophore candidates for both analgesic activity and clock gene expression, especially for cryptochromes.

Cryptochromes, CRY1 and CRY2, engage in interactions with the CLOCK:BMAL1 complex, exerting inhibitory effects on the activity of these transcription factors. Over the diurnal cycle, the CLOCK:BMAL1 complex intricately binds to the promoter regions of PER1, PER2, CRY1, and CRY2, thereby initiating their transcriptional processes [65]. Subsequently, the protein products synthesized by CRY1 and CRY2 translocate to the nucleus, where they suppress the activity of the CLOCK:BMAL1 complex, consequently orchestrating a negative feedback loop that intricately regulates their transcription. This cyclic negative feedback mechanism endures over 24 hours [66]. Melatonin molecule is known to regulate the rhythmic expression of clock genes [14,67]. Hence, ligands carrying a benzoxazolone structure, which is structurally similar to melatonin and indomethacin, have been identified in this study to potentially exert a regulatory effect on clock proteins, particularly CRY. The expression of CRY affects the feedback loop by influencing the inhibition of CLOCK:BMAL1 activity, which in turn regulates the transcription of PER and CRY genes, thereby maintaining the oscillation of the circadian rhythm. Our findings suggest that these ligands might have a regulatory impact on clock proteins and, consequently, could modulate the effects on pain perception along with the circadian rhythm. In our study, when comparing the results of molecular docking and variance analysis, it is noteworthy that in all generated clock gene complexes, ligands derived from benzoxazolone with 5-fluoro and 5-nitro substituents exhibited an increase in affinity compared to the control ligand, 5-methoxyindole, and displayed statistically significant differences in binding energy. This suggests that the addition of fluoro and nitro groups at the 5th position of the benzoxazolone structure makes these compounds promising modulator candidates for clock proteins in pain management.

In our study, upon comparing all binding affinity results and MM/PBSA calculations obtained from ligand-clock protein complexes, the observed increase in binding and interaction tendencies towards CRY proteins suggests that ligands, particularly by expressing CRY proteins, may play a crucial role in regulating circadian rhythm. This study possesses certain limitations. Firstly, while computational methods such as molecular docking and dynamics simulations provide valuable insights, they cannot fully replicate the complexity of biological systems observed in vivo. Thus, future in vitro investigations would be appropriate to provide more comprehensive data. Our study focused on a specific subset of benzoxazolone derivatives and clock proteins. Exploring a broader range of compounds and target proteins could provide a more comprehensive understanding of the potential therapeutic effects of benzoxazolone derivatives on circadian rhythm regulation and pain modulation. The results of the study will provide some preliminary data for clinical studies. Furthermore, this study underscores the benzoxazolone core as a significant pharmacophoric group, hypothesizing the potential utility of 5-fluoro and 5-nitro substituted benzoxazolone rings in the structures of future drug candidates. This could contribute not only to the generation of analgesic activity but also to the regulation of circadian rhythm, consequently modulating pain perception.

Supplementary Materials

The online-only Data Supplement is available with this article at https://doi.org/10.33069/cim.2024.0012.

SUPPLEMENTARY MATERIAL

2D INTERACTIONS OF ALL LIGANDS AND TARGET CLOCK PROTEINS

cim-2024-0012-Supplementary-Material.pdf

NOTES

Conflicts of Interest

The authors have no potential conflicts of interest to disclose.

Availability of Data and Material

The data generated or analyzed during the study are available from the corresponding author upon reasonable request.

Author Contributions

Conceptualization: Ismail Celil Haskologlu, Emine Erdag. Data curation: Ismail Celil Haskologlu, Emine Erdag. Formal analysis: all authors. Investigation: Ismail Celil Haskologlu, Emine Erdag, Orhan Uludag, Ahmet Ozer Sehirli. Methodology: Ismail Celil Haskologlu, Emine Erdag. Software: Emine Erdag. Supervision: Nurettin Abacioglu. Visualization: all authors. Writing—original draft: all authors. Writing—review & editing: all authors.

Funding Statement

None

Acknowledgments

None

Figure 1.

The variance analysis of binding energies (docking scores) of tested ligands in the binding region of different clock genes. The average binding energy values and standard deviation were summarized (A). The binding energies of ligands in the binding site of CLOCK: BMAL1 (B), PER1 (C), PER2 (D), CRY1 (E), and CRY2 (F) were statistically compared with the control ligand (5-methoxyindole). *indicates ligands with higher negative binding energies.

Figure 2.

The variance analysis of molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) binding energy values of different clock gene-ligand complexes. The binding energy values with standard deviations of each complex were summarized (A). The binding energy values of complexes involving CLOCK:BMAL1 (B), PER1 (C), PER2 (D), CRY1 (E), CRY2 (F), and different ligand groups were statistically compared with the control ligand (5-methoxyindole). *indicates ligands with higher negative binding energies.

Figure 3.

A comparison of 3D interactions of amino acid residues in the binding region of CRY1 with 5-methoxyindole (A), the reference molecule, and 5-nitro-2-benzoxazolone (B), the molecule exhibiting the highest binding affinity to CRY1, shows common interacting residues including LYS11, ARG256, and PHE257. Additional hydrogen bonds are illustrated in dark green, highlighting the interaction of the nitro group with TYR35.

Figure 4.

A comparison of 3D interactions of amino acid residues in the binding region of CRY1 with 5-methoxyindole (A), the reference molecule, and 5-fluoro-2-benzoxazolone (B), the molecule exhibiting the second highest binding affinity to CRY1, reveals common interacting residues including ARG256, PHE257, and LEU386. An additional halogen bond is illustrated in blue, highlighting the interaction of the fluorine group with ARG10.

Figure 5.

2D interactions of ligands in the active binding region of CRY1. Interacting amino acid residues of CRY1 and complexes with 5-methoxyindole (A), 2(3H)-benzoxazolone (B), 5-nitro-2-benzoxazolone (C), 5-fluoro-2-benzoxazolone (D), 5-chloro-2-benzoxazolone (E), and 5-bromo-2-benzoxazolone (F). The types of interactions are illustrated with different colors (dark green: conventional hydrogen bonds; light green: carbon-hydrogen bonds; pink: pi-alkyl; blue: halogen (fluorine); dark pink: pi-pi T-shaped).

Table 1.

The binding energy values of ligands against the target clock proteins

Name of ligands	Binding energy (kcal/mol)
Name of ligands	CLOCK:BMAL1 dimer	PER1	PER2	CRY1	CRY2
2(3H)-benzoxazolone	-5.2	-5.1	-5.4	-6.5	-6.2
5-Chloro-2-benzoxazolone	-5.5	-5.3	-5.7	-6.8	-6.5
5-Bromo-2-benzoxazolone	-5.0	-5.0	-5.2	-6.4	-5.9
5-Fluoro-2-benzoxazolone	-5.8	-5.6	-5.9	-7.2	-6.7
5-Nitro-2-benzoxazolone	-5.6	-5.8	-6.1	-7.4	-6.5
5-Methoxyindole	-5.5	-5.4	-5.8	-7.1	-6.4

Table 2.

MM/PBSA calculations of the binding energy values of ligand-protein complexes

Name of ligands	MM/PBSA binding energy (kJ/mol)
Name of ligands	CLOCK: BMAL1 dimer complex	PER1 complex	PER2 complex	CRY1 complex	CRY2 complex
2(3H)-benzoxazolone	-162.32±2.15	-208.32±2.17	-219.15±2.21	-282.42±3.32	-272.62±2.12
5-Chloro-2-benzoxazolone	-169.05±2.21	-219.48±3.13	-233.28±3.18	-291.18±3.15	-286.41±2.34
5-Bromo-2-benzoxazolone	-151.25±3.42	-197.02±3.53	-208.06±2.41	-253.12±2.41	-246.24±2.25
5-Fluoro-2-benzoxazolone	-176.34±2.51	-239.74±2.51	-241.70±3.10	-324.52±2.26	-319.52±3.46
5-Nitro-2-benzoxazolone	-171.15±2.42	-242.72±2.24	-249.63±2.25	-352.36±2.34	-349.60±3.17
5-Methoxyindole	-167.28±3.34	-226.21±3.25	-231.28±2.12	-318.41±3.52	-308.55±2.41

Mean standard deviations are represented as mean±standard deviation. MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area

Table 3.

The binding energy values of ligands in the binding regions of target clock proteins

Name of ligands	Interacting amino acid residues
Name of ligands	CLOCK:BMAL1 dimer	PER1	PER2	CRY1	CRY2
2(3H)-benzoxazolone	Pi-Anion/Pi-Cation: ARG126, GLU146, Hydrogen bond: LYS220	Pi-Pi T-shaped: TYR313, Pi-Alkyl: ARG312, PRO473, Hydrogen bond: TYR313, ARG430, Carbon Hydrogen bond: ARG300	Pi-Alkyl: PRO363, VAL366, Pi-Sigma: THR362, Pi-Pi T-shaped: TYR244, TYR354, Hydrogen bond: GLN367	Pi-Pi T-shaped: PHE257, Pi-Alkyl: LYS11, Hydrogen bond: ARG51	Pi-Alkyl: LEU39, LEU87, VAL188, Pi-Sigma: MET193, Pi-Sulfur: CYS196, Hydrogen bond: CYS196, Carbon Hydrogen bond: ARG197
5-Chloro-2-benzoxazolone	Pi-Anion/Pi-Cation: ARG126, GLU146, Alkyl/Pi-Alkyl: PHE80, PRO182, TYR184, Hydrogen/Carbon Hydrogen bond: LYS220	Pi-Pi T-shaped: TYR313, Alkyl/Pi-Alkyl: ARG312, PRO473, Hydrogen bond: TYR313, ARG430, Carbon Hydrogen bond: ARG300	Pi-Pi T-shaped: TYR354, Alkyl: LEU359, Hydrogen bond: ARG399, ARG401	Pi-Pi T-shaped: PHE257, Alkyl/Pi-Alkyl: LEU55, Hydrogen bond: ALA388	Alkyl/Pi-Alkyl: LEU33, LEU39, LEU84, LEU87, VAL188, Pi-Sigma: MET193, Pi-Sulfur: CYS196, Hydrogen bond: CYS196
5-Bromo-2-benzoxazolone	Pi-Anion/Pi-Cation: ARG126, GLU146, Alkyl/Pi-Alkyl: PRO182, Hydrogen/Carbon Hydrogen bond: LYS220, SER143	Pi-Pi T-shaped: TYR313, Pi-Sigma: TYR313, Pi-Alkyl: ARG312, TYR313, PRO473, Hydrogen bond: ARG430, Carbon Hydrogen bond: ARG300	Pi-Pi T-shaped: TYR354, Hydrogen bond: ARG399	Pi-Pi T-shaped: PHE257, Alkyl/Pi-Alkyl: LEU55, ARG256, Carbon Hydrogen bond: LEU386	Alkyl/Pi-Alkyl: LEU39, LEU84, LEU87, VAL188, Pi-Sigma: MET193, Pi-Sulfur: CYS196, Hydrogen bond: CYS196
5-Fluoro-2-benzoxazolone	Pi-Anion/Pi-Cation: ARG126, GLU146, Hydrogen/Carbon Hydrogen bond: LYS220, Halogen bond: LEU125	Pi-Alkyl: ARG300, ARG312, TYR313, PRO473, Pi-Sigma: TYR313, Pi-Pi T-shaped: TYR313, Hydrogen bond: ARG430, Halogen bond: TYR313	Pi-Pi T-shaped: TYR354, Hydrogen bond: ARG399, Halogen bond: ASP358	Pi-Pi T-shaped: PHE257, Pi-Alkyl: ARG256, Carbon Hydrogen bond: LEU386, Halogen bond: ARG10	Pi-Alkyl: LEU39, VAL49, LEU84, VAL188, Pi-Sigma: MET193, Pi-Sulfur: YS196
5-Nitro-2-benzoxazolone	Pi-Anion/Pi-Cation: ARG126, GLU146, Hydrogen/Carbon Hydrogen bond: ARG126, PHE141, SER143, GLU183, LYS220	Pi-Anion: ASP469, Hydrogen bond: HIS345, SER346, TYR313	Pi-Pi T-shaped: TYR354, Hydrogen bond: ARG399	Pi-Pi T-shaped: PHE257, Pi-Alkyl: LYS11, ARG256, Hydrogen bond: TYR35	Alkyl/Pi-Alkyl: LEU33, VAL188, Pi-Sigma: LEU39, LEU87, MET193, Pi-Sulfur: CYS196, Hydrogen bond: SER89, ARG197
5-Methoxyindole (reference)	Pi-Anion/Pi-Cation: ARG126, GLU146, Hydrogen/Carbon Hydrogen bond: ASP178, THR181, GLU183	Pi-Pi T-shaped: TYR313, Pi-Sigma: TYR313, Pi-Alkyl: ARG300, ARG312, PRO473, Carbon Hydrogen bond: TYR313	Pi-Pi T-shaped: TYR354, Hydrogen bond: ASP358	Pi-Pi T-shaped: PHE257, Alkyl/Pi-Alkyl: LYS11, LEU55, ARG256, Carbon Hydrogen bond: LEU386	Alkyl/Pi-Alkyl: LEU39, VAL49, VAL188, Pi-Sigma: MET193, Pi-Sulfur: CYS196, Hydrogen bond: CYS196, Carbon Hydrogen bond: MET193

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