Dopamine docking studies of biologically active metabolites from Curcuma longa L. Estudos de docagem molecular de dopamina de metabólitos biologicamente ativos de Curcuma longa L. Estudios de acoplamiento de dopamina de metabolitos biológicamente activos de Curcuma longa L

The dopaminergic system is involved in a wide range of neuropsychiatric and neurodegenerative disorders. The lack of receptor subtype specificity is related to several pharmacological side effects that are observed during therapy among parkinsonian and schizophrenic patients. It is of paramount importance to search for new compounds that act on dopamine receptors with therapeutic potential, higher clinical effectiveness, and fewer adverse effects. In the present study, we performed a molecular docking study of D2, D3, and D4 receptor interactions with 92 metabolites from Curcuma longa using an in silico approach. We sought to identify compounds for possible drug development. A virtual library of compounds was built using molecules that were identified in the phytochemical characterization of C. longa. Protocols that were validated by redocking were applied to a virtual scan of this library using the AutodockResearch, Society and Development, v. 10, n. 7, e59910716992, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i7.16992 2 v4.2.3, Autodock Vina, and Molegro-v6.0 Virtual Docker programs, with four repetitions each. The three-dimensional structures of D2, D3, and D4 receptors in complex with risperidone, eticlopride, and nemonapride were obtained from the Protein Data Bank. Four compounds—stigmasterol, β-sitosterol, cholest-5-en-3-one, and cholestan-3-ol,2methylene-(3β, 5α)—were the most likely to bind D2, D3, and D4 dopamine receptors, suggesting their potential for possible drug development.

All dopamine receptors are seven-transmembrane domain metabotropic receptors that are divided into two main families, D1like receptors (D1 and D5) and D2-like receptors (D2, D3, and D4), that couple to Gs and Gi proteins, respectively (Dong et al., 2020;Martinez et al., 2020). Substances that can interact with dopaminergic receptors have promising therapeutic potential, either by acting as receptor agonists (e.g., pramipexol) or antagonists (e.g., haloperidol).
Dopamine receptor agonists can be used for the symptomatic treatment of patients with early PD and restless leg syndrome (Grimes et al., 2019;Liu et al., 2016). Parkinson's disease is a neurodegenerative disorder that is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (Fontoura et al., 2017;Li et al., 2020;Zhai et al., 2019).
Dopamine replacement therapy can mainly alleviate motor symptoms of the disease. However, levodopa (L-DOPA) and dopamine receptor agonists can cause diverse side effects, such as dyskinesias, motor fluctuations, sleepiness, and hallucination (Kim et al., 2015;Paus et al., 2003;Yun et al., 2017).
Dopamine receptor antagonists are widely used antipsychotic medications that are used for the treatment of schizophrenia, bipolar disorder, and other psychotic conditions (Moncrieff et al., 2020). However, antipsychotics are associated with various side effects, such as sedation, weight gain, metabolic disturbances, sexual dysfunction, gastrointestinal distress, and extrapyramidal movement disturbances (Hynes et al., 2020).
It is of paramount importance to search for new compounds that act on dopamine receptors with therapeutic potential, higher clinical effectiveness, and fewer adverse effects. The drug discovery process is particularly challenging because it is both time-and resource-consuming. However, the recent use of newer technologies and information management has led to the optimization of drug development and design (Makhouri & Ghasemi, 2018).
One promising natural product for the development of pharmacological agents is Curcuma longa L. (Zingiberaceae).
This herbaceous and perennial species originated in Asia and is distributed throughout the tropics. It is a rhizome-like pseudostem plant, popularly known as turmeric (Bortolucci et al., 2020). C. longa is commonly used in the food industry as a food coloring, seasoning, and condiment . The main classes of compounds that are identified in rhizomes of this species are curcuminoids (curcumin, demethoxycurcumin, and bisdesmethoxycurcumin) and oxygenated sesquiterpenes (Marchi et al., 2020). Its pharmacological properties, identified by preclinical studies, include antiinflammatory, gastroprotective, hepatoprotective, antiparasitic, antibacterial, antiviral, antifungal, anti-cancer, antiarthritic, antioxidant, hypolipidemic, and anti-human immunodeficiency virus effects (Araujo & Leon, 2001;Bastos et al., 2009;Krup et al., 2013;Yu et al., 2002). Furthermore, the species also has actions on the immune system, sedative properties, and neuroprotective activity .
To identify potential dopaminergic ligands, the present study performed a molecular docking investigation of D2, D3, and D4 receptor interactions with active metabolites from C. longa using an in silico approach. We sought to identify possible compounds for drug development, following the good standards of scientific research methodology (Pereira et al., 2018).

Virtual library
A virtual library of compounds was built using molecules that were identified in the phytochemical characterization of C. longa by gas chromatography coupled with mass spectrometry and high-performance liquid chromatography (Marchi et al. 2020). The three-dimensional structures were obtained from the PubChem (Kim et al., 2019) and Zinc15 (Sterling and Irwin, 2015) databases. A total of 92 molecules were obtained. The protocols that were validated by redocking were applied to the virtual screening of this library using three different programs, with four repetitions each.

and Autodock
Vina (Trott & Olson, 2010), both implemented in the PyRx-0.9 graphical interface (Dallakyan & Olson, 2015), and Molegro-v6.0 Virtual Docker (Thomsen & Christensen, 2006). The parameters of each program were selected from the redocking of crystallographic ligands in their respective receptors. The protocols were considered validated when redocking returned poses with a root mean square deviation (RMSD) below 3 Å relative to the original conformation of the receptor-ligand complex in four repetitions. In Autodock, the search box was centered on the crystallographic ligand at x, y, z coordinates of 7, 8, -9 for x, 0, -14, 11 for y, and -18, 15, -16 for z (D2, D3, and D4 receptors, respectively. For all three receptors, grid dimensions of 50, a resolution of 0.375 Å, 25 runs, and 2,500,000 energy evaluations were used. In Vina, the same coordinates and dimensions that are described above were used, and the exhaustiveness parameter was changed to 16, and the modes parameter was changed to 25. In Molegro for the D2 receptor, Plants Score (grid) and Moldock Optmizer were used as ranking and search algorithms, with 10 runs and a 15 Å search radius, respectively. For D3 and D4 receptors, the Iterated Simplex search algorithm was selected, and the other parameters were the same as those described above for the D2 receptor.

Results and Discussion
Molecular docking is an important tool that is useful for the rational design of drugs based on structures that can predict how a receptor interacts with a small-molecule ligand to form a stable complex (Raghu et al., 2018). This method has been useful for developing several drugs without expending excessive effort or investment in research, and these drugs subsequently underwent preclinical studies and clinical trials for validation (Chaurasiya et al., 2016;Shruthy & Shakkela, 2014). In the present study, molecular docking was used to simulate in silico interactions between biologically active metabolites from C. longa with D2, D3, and D4 receptors to identify possible new therapeutic targets, which appears to be a common link between neurodegenerative and neuropsychiatric disorders.
Dopamine has been intensively studied as a key neurotransmitter in the central nervous system. Many studies have sought to understand the physiological processes and define the mechanisms of several pathologies, including PD, schizophrenia, substance dependence, and attention-deficit/hyperactivity disorder (Pan et al., 2019;Yang & Tsai, 2017).
Subtypes of DA receptors have high amino acid sequence homogeneity and differ in their distribution, expression, affinity, and functional properties, thus creating challenges in developing receptor subtype-selective agonists and antagonists (Bueschbell et al., 2019;Missale et al., 1998;Neve et al., 2004;Yang et al., 2020). The lack of subtype specificity is related to several side effects that are commonly observed in the treatment of parkinsonian and schizophrenic patients (Briggs et al., 2008;Peschanski et al., 1994;Reichmann, 2016). For some diseases, such as PD and schizophrenia, the therapeutic arsenal only provides symptomatic relief, without significantly altering the underlying pathophysiology (Cuny, 2012;Hou et al., 2019).
Thus, it is urgent to search for new compounds from many sources, including drug synthesis, existing drugs, and natural products, that act on pathways that are involved in neurodegenerative and neuropsychiatric disorders and are safer and more effective for the treatment of these diseases (Durães et al., 2018).
The present study employed redocking validation with risperidone, eticlopride, and nemonapride and D2, D3, and D4 receptor binding sites, respectively, to determine whether the docking protocol is acceptable ( Table 1). The repetitions returned poses with an RMSD below 3 Å, thereby indicating that the protocols were validated and could be applied to screening the virtual library.  (Mishra et al., 2018;Surmeier et. al., 2007). Two isoforms of D2 receptors, D2 long and D2 short, are expressed mainly postsynaptically and presynaptically, respectively. Postsynaptic D2 receptors mediate behavioral and extrapyramidal activity. Presynaptic D2 receptors decrease dopamine release, resulting in a decrease in locomotor activity (Hisahara & Shimohama, 2011). The hyperactivity of dopamine at D2 receptors in the mesolimbic pathway is related to manifestations of positive symptoms of schizophrenia (Brisch et al., 2014).
D3 receptors are abundantly distributed in mesolimbic areas, including the nucleus accumbens, the olfactory tubercle, the amygdala, islands of Calleja, and the striatum (Gurevich & Joyce, 1999;Jaber et al., 1996), and their activation or inhibition is related to symptoms of neurodegenerative and neuropsychiatric diseases, including emotional, behavioral, motivational, and memory fluctuations .
D4 receptors are primarily expressed on pyramidal neurons and interneurons in the prefrontal cortex, but their localization is also found on medium spiny neurons in the basal ganglia (striatum and nucleus accumbens core), throughout the limbic system, and in the thalamus in rodents (Gan et al., 2004;Mrzljak et al., 1996;Tarazi et al., 2004).
Dopamine receptors have long been considered critical players in PD and schizophrenia. In PD, the loss of dopaminergic neurons in the substantia nigra pars compacta leads to dopamine deficiency in the putamen and caudate nucleus, producing classic motor symptoms of PD, such as tremors, trouble moving, and fatigue (Alexander, 2004). Treatment for PD mainly involves dopaminergic-based strategies, which currently remain the best symptomatic treatment to improve quality of life (Miguelez et al., 2020). However, these therapies are limited by various side effects. New alternative treatment strategies, alone or combined with currently available synthetic drugs, are needed.
Schizophrenia is associated with the hyperactivity of dopaminergic systems (Brunelin et al., 2013). Currently available medications focus on the blockade of overstimulated dopamine receptors. For the therapeutic care of schizophrenia patients, these medications are generally effective for treating positive symptoms, but they have strong side effects (Grace, 2012;Miodownik et al., 2019).
The molecular docking study also supported the binding of β-sitosterol, a plant sterol, with D2, D3, and D4 receptors.
Several studies reported various neuroprotective and antioxidant effects of β-sitosterol (Vivancos & Moreno, 2005;Baskar et al., 2012;Muhammad et al., 2017). Additionally, the antidepressant activity of β-sitosterol was evaluated in the tail suspension test and forced swim test in mice. The authors concluded that β-sitosterol exhibits antidepressant-like effects that are mediated by the serotoninergic, dopaminergic, and -aminobutyric acid-ergic systems (Yin et al., 2018). Cholest-5-en-3-one and cholestan-3-ol,2-methylene-(3β, 5α) were other compounds that were identified in the C. longa extract and had affinity for D2, D3, and D4 receptors. These findings should prompt further in vitro and in vivo studies to prove their biological activity.
Finally, the current pharmaceutical armamentarium against neurodegenerative (Youdim & Buccafusco, 2005) and neuropsychiatric (Miodownik et al., 2019) diseases remains limited in terms of both treatment outcome and disease modification. The present results demonstrate that four C. longa compounds interact with dopamine receptors that are involved in the pathophysiology of several neurodegenerative and neuropsychiatric disorders.
The present study has limitations. We performed only a virtual throughput screening. Further high-throughput screening is required, in addition to studies with animal models and clinical trials to further confirm that C. longa metabolites may be potential drug candidates.