Alzheimer’s disease (AD) is a neurological disorder that worsens over time and causes significant problems with everyday life and cognitive function. It is one of the most common causes of dementia, with significant social and economic consequences worldwide [1]. Current therapy approaches focus on the cholinergic system, which involves increasing acetylcholine levels by inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Interestingly, the brains of patients with AD exhibit higher levels of BChE activity, even though BChE is generally less selective and breaks down acetylcholine (ACh) more slowly than AChE, particularly in the later stages of the disease [2].

The U.S. Food and Drug Administration (FDA)-approved AChE inhibitors, such as donepezil, rivastigmine, and galantamine, are central to the symptomatic treatment of AD. In addition, memantine, another FDA-approved medication, is employed in the treatment of AD [3]. The primary function of this drug is to act as an antagonist of N-methyl-D-aspartate (NMDA) receptors. In the brain, NMDA receptors are a subtype of glutamate receptors that are essential for the transmission of signals between neurons. The most abundant excitatory neurotransmitter in the brain, glutamate, is essential for memory and learning [3]. Excessive glutamate release in AD can have neurotoxic consequences that cause neuronal overstimulation and ultimately cell death. These FDA-approved medications have a limited ability to slow disease progression. Their systemic effects and lack of targeted distribution in the brain are usually associated with significant adverse effects [4].

Given the limitations of current therapies, there is an urgent need for new strategies that not only improve drug efficacy but also minimize side effects by ensuring more targeted delivery and better timing of drug activation. This is where chronobiology— the study of biological rhythms—can offer a promising solution. Biological rhythms, particularly circadian rhythms, play a crucial role in regulating various physiological processes, including the sleep-wake cycle, mood, and cognitive function [5]. By tailoring the administration and effects of drugs to these natural biological rhythms, therapeutic outcomes can be optimized. Recent advances in photopharmacology, an innovative field of light-driven drug activation, offer a unique opportunity to utilize the principles of chronobiology [5]. Photopharmacology offers a revolutionary approach to integrating photoswitching properties into drug molecules, enabling the control of drug activity with light. This emerging field offers the opportunity to improve the precision of drug delivery and activation, potentially overcoming the limitations of traditional drug treatments [6]. By integrating photoswitching functionality into established AChE inhibitors, the location and timing of drug activation can be controlled, thereby minimizing off-target side effects and improving treatment outcomes [7]. Azobenzenes are highly valued in photopharmacology due to their potent photochemical properties and ease of synthesis [8]. This compound can switch between two isomeric states: the more stable trans (E) form and the less stable cis (Z) form. Light of specific wavelengths can reversibly trigger this switch, allowing precise control over the molecule’s structure and its interactions with biological targets [8]. The E-form of azobenzene is planar, approximately 9 Å in length, and lacks a dipole moment, which may make it better suited for interacting over larger, flat areas. On the other hand, the Z-form is bent, about 6 Å long, and has a dipole moment of 3D, enabling it to engage in more localized interactions or fit into varied binding pockets. Each form includes hydrogen bond acceptors in the azo bond (N=N), which shift in directionality when photoisomerization occurs. This shift influences azobenzene’s ability to form hydrogen bonds: the E-form can interact on opposite sides of the bond, while the Z-form is on the same side (Figure 1). Azobenzene’s ability to switch forms reversibly, along with the structural transformations triggered by light, makes it an effective tool for developing drugs with controllable spatial and temporal activity. This feature may be beneficial for treating diseases where precise drug activity control can significantly improve therapeutic outcomes and minimize side effects [8,9].

In this study, a novel approach for developing photoswitchable forms of FDA-approved drugs using the azobenzene moiety was investigated. This innovation involved the ability to control drug activity using light, specifically targeted at the brain regions affected by AD. Incorporating photoswitchable moieties into these drugs was designed to enable their reversible activation and deactivation, providing dynamic control over their potential pharmacological profiles in alignment with the circadian rhythm of the body. Subsequent molecular docking studies predicted the interactions of these novel photoswitchable derivatives with both AChE and BChE, the latter of which plays a significant role in AD, especially in the later stages [10-12]. By conducting this study, the aim was to demonstrate the enhanced selectivity and binding affinity of these photoswitchable inhibitors, paving the way for a new generation of chronobiologically targeted AD therapies.

The study aimed to assess the binding affinities and stability of different photoswitchable derivatives of well-known neuroactive drugs against cholinesterases and NMDA receptor binding sites using binding energy calculations and MM/PBSA methodologies. The binding energy calculations revealed that the photoswitchable derivatives generally exhibited enhanced affinities compared to their original drug forms (Tables 1 and 2). Photoswitchable derivatives of rivastigmine and donepezil have demonstrated increased binding energy against AChE and BChE receptors compared to their non-photoswitchable forms. In particular, rivastigmine’s planar form showed a notable increase in binding energy registering -9.3 kcal/mol and -7.1 kcal/mol against AChE and BChE receptors, respectively. Similarly, donepezil’s photoswitchable derivatives (both planar and globular) achieved substantial increases in binding energies, with the planar variant achieving -13.4 kcal/mol and -11.8 kcal/mol for AChE and BChE, respectively. These findings suggested that the photoswitchable forms of these drugs may offer stronger and more favorable binding capabilities than their original forms. The most significant enhancements were noted in the photoswitchable galantamine derivatives, where the planar form exhibited exceptionally high binding energies of -13.0 kcal/mol for AChE and -9.2 kcal/mol for BChE. In the NMDA receptor analyses, the photoswitchable memantine derivatives also displayed modest increases in binding energies, with the planar form slightly surpassing the original by exhibiting -6.3 kcal/mol. The MM/PBSA analysis further confirmed these findings, indicating enhanced stability and affinity in the protein-ligand complexes of the photoswitchable derivatives compared to their original counterparts. The MM/PBSA methodology was used to analyze the binding energies of conventional and photoswitchable derivatives of neuroactive drugs, such as rivastigmine, donepezil, galantamine, and memantine, against cholinesterase and NMDA receptor protein complexes. Specifically, the planar form of photoswitchable galantamine achieved the highest MM/PBSA binding energy values, markedly improving upon the non-photoswitchable form’s binding energies. Consistently, both donepezil and rivastigmine derivatives exhibited an increase in MM/PBSA energies when they were in their pho-toswitchable forms, indicating a more stable and favorable binding complex. Among them, the planar configuration of the photoswitchable galantamine showed the most significant increase in MM/PBSA energies, highlighting its improved binding stability (Tables 3 and 4).

For instance, the planar photoswitchable derivative of rivastigmine showed a significant increase in MM/PBSA binding energy, achieving -39.83±0.24 kJ/mol and -31.63±0.24 kJ/mol for AChE and BChE, respectively, markedly higher than the non-photoswitchable form. The results indicated a statistically significant increase in binding energies both for AChE and BChE (p<0.0001) (Figures 2-4). Similarly, donepezil’s planar photoswitchable form displayed an increase to -46.27±0.14 kJ/mol (AChE) and -35.63± 0.35 kJ/mol (BChE), suggesting a statistically significant rise in affinity for both cholinesterases (p<0.0001) and potentially more effective inhibition of enzyme activity. Notably, the photoswitchable derivatives of galantamine, particularly the planar form, presented the most substantial enhancement in binding energies with -71.67±0.15 kJ/mol (AChE) and -50.39±0.43 kJ/mol (BChE), indicating statistically significant (p<0.0001) and favorable interactions. Furthermore, in the context of NMDA receptors, both planar and globular photoswitchable derivatives of memantine exhibited a statistically significant increase in binding energies, reaching -44.26±0.62 kJ/mol (p<0.001) and -42.48±0.38 kJ/mol (p<0.01), respectively, which aligns with the observed trend of improved performance through photoswitchable modifications.

In the interaction diagrams of AChE, BChE, and NMDA receptors, photoswitchable derivatives with the azobenzene moiety exhibit additional electrostatic and hydrogen bonding interactions (Figures 5-8). It has been observed that photoswitchable derivatives with a planar structure tend to interact with a greater number of amino acid residues. Specifically, galantamine designed with azobenzene in the AChE active site, in its planar form, has demonstrated the highest binding energy. This form engages in various interactions, such as alkyl, Pi-alkyl, Pi-sigma, and Pication, through the azobenzene aromatic ring with the amino acid residues LEU48, PRO49, ARG165, and PRO168. These residues may play an additional role in inhibition activity. Similarly, in the BChE active site, the photoswitchable planar form of galantamine exhibited additional electrostatic interactions through amino acid residues ASN228, ASP304, VAL233, and PRO303. A photoswitchable derivative of memantine was observed engaging in hydrogen bonding and amide-Pi-stacked interactions with GLU530, TYR535, and ASN736 in the NMDA receptor active site via the azobenzene aromatic ring.

All of these findings point to the possibility that adding photoswitchable functionality to these medications increases both their binding affinity and the stability of their complexes with the desired receptors. This improvement points to a possible path for the creation of next-generation neurotherapeutics with better clinical outcomes by potentially translating into increased regulated pharmacological profiles and greater therapeutic effectiveness.

Integrating photoswitchable derivatives into the chronobiological framework could revolutionize AD treatment. By aligning drug activation with the body’s natural rhythms, it is possible to optimize drug efficacy and reduce side effects. For instance, using light to activate these derivatives during periods when cognitive function and drug absorption are at their peak can enhance treat-ment benefits. Moreover, this approach aligns with the principle of using light as a zeitgeber to regulate circadian rhythms, further supporting the therapeutic potential of these advanced drug designs [27]. Using this approach, we explored the integration of azobenzene moieties into established neuroactive drugs, introducing a novel approach of photoswitchable control to enhance spatial and temporal precision in AD pharmacotherapy. This method promises to mitigate the challenges associated with non-targeted effects and enhance therapeutic outcomes by allowing reversible activation and deactivation of drug molecules using light, thereby potentially reducing side effects commonly observed with these drugs [28].

Different methods of in vivo light delivery were reported before. Fiber optic cables are integral to the delivery of light to specific areas within the body, particularly in minimally invasive surgical procedures. These cables, composed of flexible glass or plastic fibers, enable the transmission of light over long distances with minimal loss of intensity. In photopharmacology, fiber optics can be used to deliver light to deep tissues, such as the brain or gastrointestinal tract, ensuring that photoswitchable drugs are activated precisely where needed [29]. Another method for achieving localized light activation involves the use of implantable LED devices. These small, biocompatible devices can be surgically placed near target tissues, such as tumors or specific brain regions. Once implanted, the LEDs can be remotely activated, allowing clinicians to control drug activation in a non-invasive manner. The use of such devices is particularly advantageous for therapies requiring repeated or prolonged light exposure, as the devices can be left in place for extended periods [30]. Moreover, several researchers have also pioneered the development of water-soluble photoswitches, which are stable in aqueous environments and effective within the body’s fluids [31,32]. These photoswitches maintain their functionality in vivo, making them ideal candidates for systemic applications. By ensuring that these molecules are both soluble and responsive in biological settings, they offer greater flexibility and potential for clinical use, particularly in systemic therapies where widespread drug distribution is necessary [31,32]. One of the key advantages of these advanced photoswitchable molecules is their potential for systemic application. After administration (either through injection or orally), these drugs can be activated only at the desired sites using targeted light sources. This approach minimizes off-target effects and maximizes therapeutic outcomes, making it particularly useful in conditions such as cancer, where localized treatment is crucial [33]. For instance, in cancer therapy, photoswitchable drugs can be used to selectively target tumor cells by activating the drug only in the tumor area, thus sparing healthy tissues. Similarly, in neurological disorders like epilepsy, implantable LED devices can be used to control drug activation in specific brain regions, offering a new avenue for treatment that is both precise and reversible [34].

In contrast, Siddiqui et al. [35] employed traditional computational methods to identify novel NMDA receptor inhibitors without incorporating photoswitchable functionalities. Their focus on creating a pharmacophore model and conducting extensive molecular docking and dynamics simulations underlined the conventional approach aimed at enhancing our understanding of NMDA receptor interactions and developing new inhibitors against these critical neural components in AD. Although both approaches aimed to improve AD treatment modalities, this study introduces an innovative layer of drug control through photoswitchable mechanisms, which could revolutionize drug delivery and efficacy by aligning drug activity more closely with physiological needs.

Furthermore, the study conducted by Suwanhom et al. [36] investigated novel lawsone-quinoxaline hybrids as dual-binding site acetylcholinesterase inhibitors. This research shares a similar innovative spirit with our study by attempting to enhance inhibitor efficacy by targeting multiple enzyme binding sites. However, in contrast to our method, which emphasizes controllable and reversible drug activation via light-sensitive elements, their strategy centered on structural hybridization to enhance binding and inhibitory efficacy. Both studies shared a commitment to employing sophisticated computational techniques to tackle the intricate pathology of AD. Nevertheless, each approach selected unique methods and targets to improve drug design: developing pharmacophores, synthesizing hybrid molecules, and implementing photoswitchable functionalities [35,36].

The findings from this comprehensive study provided compelling evidence of the significant improvements in binding affinities and stabilities offered by photoswitchable derivatives of well-established neuroactive drugs. The observed improvements in MM/PBSA binding energies for photoswitchable forms of rivastigmine, donepezil, galantamine, and memantine emphasized a promising method for drug enhancement that could lead to improved therapeutic results. Specifically, the notable increases in binding energies for the planar forms of these derivatives indicated not only more effective interactions with drug targets but also suggested the potential for greater drug efficacy in clinical environments.

The binding energies for AChE and BChE targeted with photoswitchable rivastigmine and donepezil indicated a significant increase. This suggests a stronger inhibitory effect on these cholinesterases, which play a crucial role in the development of AD. The remarkable enhancement observed with the photoswitchable planar form of galantamine, exhibiting the highest MM/PBSA energies, may indicate its potential as a superior therapeutic agent against cholinergic deficits observed in dementia. Enhancements observed in photoswitchable memantine derivatives may result in improved regulation of the NMDA receptor, crucial for neural plasticity and memory processes. The modifications may offer greater control of drug activity and may reduce side effects in long-term therapies. Furthermore, the significant results (p< 0.0001 and p<0.01) underscored the reliability of the photoswitchable modifications to consistently produce more favorable interactions with their respective targets.

As expected, this study emphasized the importance of planar forms in photoswitchable derivatives for photopharmacology. These planar forms typically showed improved compatibility with target protein sites’ broad, flat surfaces [37]. This structural alignment allows for more effective and stable interactions with target molecules, particularly enzymes or receptors where a larger contact area can facilitate stronger binding affinity [38]. The planar structure of azobenzene-like photoswitches generally aligns with their trans (E) isomeric state. When exposed to certain wavelengths of light, they can transition to a non-planar, bent cis (Z) state. This reversible change allows for precise control over the shape of the molecule and its interactions with biological targets. This ability to switch is crucial for the timely regulation of biomolecule activity, enabling activation or inhibition when necessary [39,40].

On the contrary, globular forms may not be able to make sufficient interactions in the binding site due to steric hindrance [8]. By utilizing light to control drug activity, the planar forms of photoswitchable derivatives can be activated in specific tissues or cells, minimizing systemic side effects. The planar configuration, when activated by light, ensures that the drug interacts predominantly at the desired site of action. The inherent properties of planar molecules, such as their synthetic accessibility and chemical stability, make them attractive candidates in drug design [41,42]. Interactions involving hydrogen bonding and electrostatic interactions, facilitated by the extended conjugated systems and flat geometry of planar structures, are crucial for the stability and efficacy of molecular interactions in biological systems. Achieving high binding specificity and affinity through these interactions is essential for effective drug action [41,42]. This study corroborated prior research indicating that planar forms, due to their flat shapes, engaged in various robust interactions such as π-π stacking, hydrogen bonding, and electrostatic interactions. Integrating photoswitchable drugs that can be controlled with light makes it possible to harmonize drug activity with circadian rhythm. This approach may enhance the therapeutic impact by ensuring that drugs are most active when the body is naturally more receptive, such as during peak cognitive performance times or when specific biological processes are most active.

The potential revolutionary breakthrough in the treatment of neurodegenerative disorders, particularly AD, may come from the addition of photoswitchable characteristics to neuroactive medicines. The combination of photoswitchable drug compounds with chronobiology might result in a potential treatment approach for AD. It may be possible to provide more focused and efficient treatments by using light to regulate medication action, which would eventually improve patient results and reduce side effects.

To address the potential limitations of photoswitchable drugs in clinical settings, several considerations must be highlighted. One key concern is the unintended activation or deactivation of these drugs due to exposure to environmental light. To mitigate this, careful drug design is essential to ensure that the compounds remain stable and inactive under typical ambient lighting conditions. In clinical applications, technologies such as fiber optics or implantable LEDs can provide controlled, targeted light stimuli, reducing the risk of accidental activation outside of the intended therapeutic environment [33]. Additionally, the long-term effects of using photoswitchable drugs on neurodegeneration require further exploration. While these drugs offer the advantage of reversible and targeted activation [43], it is crucial to investigate whether prolonged use could lead to cumulative toxicity or other adverse effects on neural tissues. Ongoing research will need to focus on assessing the chronic implications of repeated photoactivation to fully understand the therapeutic potential and safety of these innovative compounds.

Despite the limitations of this study, using azobenzene moiety in the planar form to modify the derivatives might be a novel and effective strategy for drug development, particularly in neurological disorders. Although these advancements are promising, further research is necessary to fully explore the pharmacodynamic and pharmacokinetic properties of these compounds.