l-Tryptophan (TRP) is one of the largest essential amino acids and an essential component in the biosynthesis of proteins, muscle, and enzymes, an essential substrate for the production of neurotransmitters and hormones. Acute TRP deficiency results in increased pain sensitivity, auditory startle, increased motor activity, and increased aggression, while chronic TRP deficiency causes ataxia, cognitive impairment and dysphoria with skin hyperpigmentation [1]. Kynurenine (KYN) and serotonin (5-HT) are derived from the TRP metabolic pathway. The kynurenine (KYN) pathway is involved in 95% of TRP degradation, resulting in de novo synthesis of nicotinamide adenine dinucleotide (NAD${}^{+}$) [2]. The pathway begins with the conversion of TRP to KYN catalyzed by two enzymes, tryptophan 2,3-dioxygenase and indolamine 2,3-dioxygenase. Furthermore, depending upon the cell type, KYN is further metabolized through three distinct pathways. (1) Synthesis of 3-hydroxykynurenine (3-HK) in microglia by kynurenine 3-monooxygenase (KMO) and its metabolites 3-hydroxyanthranilic acid (3-HAA) and quinolinic acid (QUIN); (2) Synthesis of anthranilic acid in microglia by kynureninase; and (3) Synthesis of kynurenic acid (KYNA) in astrocytes by kynurenine aminotransferase (KAT) II [3]. QUIN is an endogenous glutamate N-methyl-d-aspartate (NMDA) receptor agonist and KYNA is a noncompetitive antagonist of alpha-7 nicotinic acetylcholine receptor ($\alpha$7nAchR). Hence, abnormalities in the KYN pathway have been implicated in the pathophysiology of depression.

Depression is a heterogeneous disorder, and the exact neuronal mechanisms driving it have not yet been discovered. Evidence also suggests that cholecystokinin (CCK) and its receptors play an important role in the pathogenesis of anxiety-related behaviors and depression [4]. Furthermore, the causal relationship between increased CCKergic neurotransmission in the socially defeated cortex and depressive-like symptoms strongly suggests the relevance of the CCKergic system as a novel target for antidepressant therapy [5, 6]. Anxiety disorders are characterized by excessive fear, and Pavlovian threat conditioning is an adaptive mechanism by which organisms learn to avoid potential threats, thereby increasing their chances of survival [7]. The prefrontal cortex (PFC) is a central brain region in the pathogenesis of depression and it has been suggested that the ventromedial prefrontal cortex (vmPFC) plays an important role in the acquisition of Pavlovian threat conditioning [7]. Amygdaloid CCK${}_2$ receptors appear to be involved in the expression of fear-enhanced startle, as fear-enhanced startle is blocked by systemic injections of CCK${}_2$ antagonists [8]. Furthermore, injections of CCK-8 into the central nucleus of the amygdala increases arousal and fear in rats [9]. Therefore, CCK and its receptors could be one of the prime targets for antianxiety and antidepressant drugs.

Cholecystokinin (CCK) is a 33-amino-acid-long peptide hormone, first identified in the gastrointestinal (GI) tract, that regulates gut motility [10], pancreatic and gastric-acid secretion [11, 12], and gall bladder contractions [13]. It is a secretagogue of insulin and pancreatic polypeptides in non-ruminants [14, 15]. Also, it is one of the most abundant peptides in the central nervous system (CNS) that function as a neurotransmitter and has an inverse correlation of its level with depression and anxiety [16, 17, 18, 19, 20]. CCK levels are elevated in depression [21], and patients with high cerebrospinal fluid (CSF) CCK levels have more suicide attempts than those with low CSF CCK levels [16]. The receptor exists as two subtypes, CCK${}_1$ and CCK${}_2$, previously classified as CCK${}_{\text{A}}$ and CCK${}_{\text{B}}$. CCK${}_1$ is predominant in the GI system and has roles in gallbladder contraction and stimulation of pancreatic secretion [22, 23]. CCK${}_2$ is predominant in the CNS and has roles in satiety, memory, anxiety, osmotic stress, and neuropsychiatric disorders [24, 25]. Several studies have shown that CCK peptides have anxiolytic effects in various experimental models of anxiety and can induce panic attacks in humans [26, 27, 28]. In addition, activation of CCK2 receptors in the brain can cause anxiety. Therefore, selective CCK2 receptor antagonists represent a unique class of anxiolytics [29]. Recently, much effort has been made in developing potent and specific CCK${}_2$ receptor antagonists, which were classified as:

(a) benzodiazepine group: L-365,260, L-36 718 (devazepide), CI-988, YM022, Z-360, YF476 (netazepide), and YM022 [30, 31, 32, 33];

(b) tryptophan dipeptide derivatives: PD-134,308 [28, 34];

(c) ureidoacetamides: RP-73,870 [35];

(d) pyrazolidinones - LY-288,513 [36].

Despite the progress in developing several CCK${}_2$ receptor agonists/antagonists [12, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46], none of them have reached the clinic, because of unfavorable, insufficient, and various biological effects discovered in clinical trials [39]. Only netazepide and Z-360 are currently under clinical development for the management of gastric neuroendocrine tumors and pancreatic cancer, respectively. Therefore, the discovery of selective CCK${}_2$ receptor antagonists with suitable pharmacokinetic profiles is very important.

In drug discovery and development, halogenation plays a vital role in drug optimization by modulating lipophilicity and cell membrane solubility, increasing blood–brain barrier (BBB) permeability and the half-life, and improving membrane binding and CNS delivery of pharmaceutical drugs [47, 48, 49, 50]. In recent years, the halogen bond has received a great deal of attention for hit-to-lead-to-candidate optimization to improve the drug-target binding affinity; as a result, 25% of the marketed drugs are halogenated [49]. Marine sources are predominant in bioactive, structurally diverse, and unique halogenated compounds, of which 45% are Bromo-metabolites [51]. These active metabolites have become structural models in developing synthetic derivatives with superior biological activity [52].

The red alga Symphyocladia latiuscula (Harvey) Yamada is rich in bromophenols with diverse biological activities, including anticancer [53], antibacterial [54], antifungal [55], antiviral [56], free-radical scavenging [57], aldose-reductase inhibitory [58], $\alpha$-glucosidase inhibitory [59], and other properties [60, 61, 62]. Secondary metabolites from this alga contain a 2,3,6-tribromo-4,5-dihydroxybenzyl moiety with various substituents. Recently, we have reported anti-diabetic [63], anti-Alzheimer’s disease [64], anti-tyrosinase [65], and anti-Parkinson’s disease activity [66], of 2,3,6-tribromo-4,5-dihydroxybenzyl derivatives from S. latiuscula. The anti-Parkinson’s disease activity of those derivatives were evaluated via their effect on human monoamine oxidase and dopamine receptors [66]. Monoaminergic neurotransmission is primarily based on G-protein coupled receptors (GPCRs) signaling. The aminergic system is dysregulated in anxiety disorders and major depressive disorder, and antidepressants act directly or indirectly through the GPCRS. Therefore, GPCRs are potential therapeutic targets for intervention in neurodegenerative diseases. However, apart from MAO and dopamine receptors, there are no reports on how natural bromophenols affect other GPCRs receptors.

Therefore, our main objective in this study was to predict novel GPCRs targets via proteocheminformatics modelling (PCM) and characterize the functional effect of natural bromophenols on CCK${}_2$ receptors for the management of CCK-mediated diseases, especially anxiety and depression.

The aim of this study was to predict novel GPCRs targets of three natural bromophenols from Symphyocladia latiuscula via proteochemometric modeling (PCM), characterize the functional effect on CCK${}_2$ receptors and predict the interactions between a compound and the binding site of the target protein for the management of CCK-mediated diseases, especially anxiety and depression. The pathophysiology of major depressive disorder and anxiety is complicated and not fully understood. Selective serotonin reuptake inhibitors (SSRIs), selective norepinephrine reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors are classic antidepressants used to treat depression. However, these drugs require a minimum of 2–4 weeks of continuous treatment to elicit therapeutic relief in depressed patients which leads to prolonged suffering and disability and increases suicide risk [6]. Due to the incomplete effectiveness of current treatment and the burdening neuroscience of fear-related behavior, however, compel the search for novel, more effective therapies [77]. In this regard, we gave a continuous effort to discover novel antidepressants from natural products.

Here, we focused on natural brominated compounds 1‒3 from a red alga S. latiuscula to explore their functional effect on cholecystokinin 2 receptor based on our PCM prediction. We predicted a total of 15 target proteins for compounds 1‒3, and most of the predicted proteins were the same for the three compounds, with different rank orders following the normalization value (Table 1). The CCK${}_2$ receptor was a prime protein target predicted for compound 2; however, the rank order and normalization values were different for 1 and 3. We attribute this difference to their structural differences. So, to verify the PCM prediction and get molecular insight into the test ligands‒CCK${}_2$ receptor interaction, we proceeded with the functional assay followed by molecular docking simulation.

The results of a functional assay conducted in the stable transfectant (CHO-CCK${}_2$) indicated that bromophenols 1‒3 are potent CCK${}_2$ receptor antagonists. Bromophenol 1 and 3 are full CCK${}_2$ antagonist that showed 100% antagonist effect at a 100 $\mu$M concentration, whereas the antagonist effect was approx. 82% for 2 at that concentration. All the tested bromophenols had no agonist effect, as indicated by the negative percentage agonist effect. The activity fashion of 1‒3 was similar to our recent studies, where 1 and 3 showed better antidiabetic property by inhibiting protein tyrosine phosphate 1B and $\alpha$-glucosidase enzyme activity, and by increasing insulin sensitivity and glucose uptake in a human liver-cancer cell line [63], anti-Alzheimer’s disease activity via cholinesterase, self-induced A$\beta$${}_{25-35}$ aggregation inhibition [64], anti-Parkinson’s disease activity via the agonist effect on dopamine D${}_3$/D${}_4$ receptors [66], and anti-browning effect by inhibiting melanin content and intracellular tyrosinase levels in $\alpha$-melanocyte-stimulating hormone-induced B16F10 melanoma cells [65].

All test compounds have a 2,3,6-tribromo-4,5-dihydroxybenzyl moiety in common, with different substituents at C-1. Bromophenol 1 has a hydroxymethyl group at C-1, 2 has a methyl ether, and 3 is a dimeric form, in which another 2,3,6-tribromo-4,5-dihydroxybenzyl moiety is attached at C-1 via a methyl ether linkage. Compared to the CCK${}_2$R antagonist effect of 2, bromophenols 1 and 3 had a better effect. However, the exact structure-activity relationship is uncertain, because we had only a few test bromophenols.

To get molecular insight on the promising antagonist effect of 1 and 3 over 2, we did a molecular docking simulation. The CCK${}_2$-binding score for 3 was the lowest (‒7.38 kcal/mol) among the test bromophenols. Bromophenols 1 and 2 bound to the active site of CCK${}_2$ receptor with a similar binding score (approx. ‒5.5 kcal/mol). Also, the interacting residues were almost common for 1 and 2 (Asn353, Tyr380 Trp209, Val349, Val138, Met134, His376, and Tyr380). The only difference is that the Br atom at C2 of 1 had an additional $\pi$-alkyl interaction with Phe227. Likewise, the most potent of them, 3, had unique H-bond interactions with Arg208, Val194, Arg356, and Pro210 that were absent for the binding of 1 and 2. The -OH group at C5, C4’ and C5’ and bromine atom at C2 of 3 had H-bond interactions with Val194, Arg356, Pro210, and Arg208, respectively. The reference antagonist YM022 and 3 had interacting residues Arg356 and Pro210 in common, whereas His376, Met134, and Trp20 are common for YM022, 1, and 2. Residues Arg356 (TM6) and Asn353 (TM6) are active site residues involved in CCK${}_2$R antagonism for non-peptide antagonists [78], and bromophenols formed H-bond interactions with these residues. Two CCK receptor subtypes—CCK${}_1$ and CCK${}_2$ receptors—are 48% identical to each other and differ in terms of molecular structure, distribution, and affinity for the natural ligand (CCK and gastrin). Both subtypes share the same COOH-terminal pentapeptide amide sequence for receptor binding [79]. A study model conducted to discover 1,4-benzodiazepine allosteric ligands targeting CCK receptors suggested six prime amino-acid residues for the selectivity of subtype CCK${}_1$: Asn98 (TM2), Thr117, Thr118 (TM3), Ile329, Phe330 (TM6), and Leu356 (TM7); those for CCK${}_2$ were Thr111 (TM2), Val130, Ser131 (TM3), Val349, Tyr350 (TM6), and His376 (TM7). Asparagine residue is a conserved amino-acid residue in the TM6 of CCK receptor, and Asn353 is responsible for the H-bond interaction with the N4 of the benzodiazepine in CCK${}_2$R [80]. Residues Thr111 (TM2) and His376 (TM7) were most important for binding CCK${}_2$R selective ligands, and Asn353 (TM6) is an anchor residue with excellent enrichment and selectivity for CCK${}_2$R [80]. Likewise, Val349 is a conserved amino-acid residue responsible for CCK${}_2$R binding affinity for nonpeptide antagonists [81]. Our test ligands showed interactions with Asn353, Val349, and His376 and acted as CCK${}_2$receptor-specific antagonists.

Drugs modulate more than one target (six on average) to exert a therapeutic effect [82]. PCM is one of the quantitative bioactivity prediction techniques that predicts the potency or affinity for compound-target pairs [83], enables simultaneous modeling of chemical and biological information and thus compound’s affinity and selectivity across a panel of targets [84]. Nonetheless, the effects of a compound at the cellular or the organism level are poorly understood because PCM cannot account for the interactions of a compound with other unrelated targets [85]. In the present study, though 2 was predicted with the highest normalization value for the CCK${}_2$ receptor, it was the least active among the tested bromophenols in a functional assay. The reason behind this variation might be the ligand-receptor interaction and the ligand-receptor complex stability. This needs to be confirmed via molecular dynamics.

Since halogenation of drug molecules is a common strategy to improve ADME, membrane binding, permeation, and protein-ligand recognition [47, 86, 87, 88], and addition of bromine and chlorine to peptide drugs improves CNS delivery by increasing BBB permeation [48]; these bromophenols 1‒3 with excellent PPB and BBB values [64] could be developed into novel CNS drugs. Marine red algae are promising natural sources that contain around 90% of halogenated compounds [89]. Because of a wide array of biological activities of halogenated compounds and little halogen content in other natural products, structural modification of natural products by halogenation is emerging [49].

The sulfated octapeptide (CCK-8s) is the most abundant molecular form of CCK in the brain, with 400 pmol/g tissue content [90]. Earlier studies had reported an increase in behavioral arousal and fear in rats after CCK-8 injection into the central nucleus of the amygdala [91, 92]. CCK${}_2$ receptors have been strongly implicated in anxiety [93]. The systemic or intracerebral injection of the CCK-like peptides (CCK${}_2$ or CCK${}_1$/CCK${}_2$ non-selective agonists) exhibited an anxiogenic-like effect in various animal models [94, 95]. However, co-treatment with CCK${}_2$ antagonist LY288513 blocked those effects; when administered alone, it attenuated anxious behavior in animals. Also, other highly selective CCK${}_2$ receptor antagonists PD134308 and PD135158 were as effective as was diazepam to antagonize aversive behavior [96].

Food intake releases gastrin that binds to the CCK${}_2$ receptor on enterochromaffin-like (ECL) cells and aids the synthesis and secretion of histamine, which stimulates parietal cells for acid secretion by proton pump via histamine H2-receptor and muscarinic M3-receptor [97]. Mental health is well-linked with the gut function [98, 99, 100] because of microbiota-gut-brain axis functions in a bidirectional manner in the regulation of depressive-like behaviors [101, 102]. Therefore, these CCK${}_2$ antagonist bromophenols can regulate depression-like behaviors.

Our study highlights the in vitro CCK${}_2$ antagonist effect of only three bromophenols in the red alga Symphyocladia latiuscula and predicts the binding mode. In silico molecular dynamics studies predicting the stability of ligand-receptor complexes are lacking. Penetration into the central nervous system and stability of the ligand-receptor complex remain to be studied in vivo. The small number of test compounds limited the structure-activity relationship. The effects of the tested bromophenols at the cellular or organismal level remain unknown because proteochemometric modeling cannot account for interactions between compounds and other unrelated targets.

In the future, a more detailed understanding of CCK${}_2$ receptor signaling and regulation, especially using in vivo models, will be critical to ensure the activity of these natural bromophenols as anxiolytics and antidepressants.

Natural bromophenols 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (3) from Symphyocladia latiuscula are potent CCK${}_2$ antagonists with good binding scores and interactions with prime residues at TM7 of the receptor in a similar manner to a reference antagonist YM022. This study suggests that bromophenols 1–3 are natural CCK${}_2$ antagonists that could be novel therapeutic agents for CCK${}_2$-related diseases, especially anxiety and depression, and provides a foundation for future studies to elucidate the molecular mechanism in animal models of anxiety and depression.

PP participated in study design, isolation, treatment, and biochemical analysis, and drafted the manuscript. SEP and SHS performed the molecular docking studies. FMF performed PCM modeling. HAJ was involved in spectral analysis. JSC conceived the study, coordinated the study, and interpreted the data. All authors read and approved the final manuscript.

The research reported here was conducted at Eurofins Cerep, France according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and was authorized from the Research Ministry to manipulate GMO, with a certificate number 28099.

The authors thank Prof. Dr. Hye Jin Park (Changshin University, Republic of Korea) for providing the leafy thalli of Symphyocladia latiuscula (Harvey).

This research was supported by the National Research Foundation of Korea (NRF) grant funded by Ministry of Science and ICT (No. 2020R1C1C1008331 [HNIBR202100303]).

The authors declare no conflict of interest. Pradeep Paudel is serving as one of the Guest editors of this journal. We declare that Pradeep Paudel had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Gernot Riedel.