Caulerpa, a green siphonous macroalgae, belongs to the Caulerpaceae family with 97 species. It was first described by J V Lamourox in 1809 and derived from the Greek words caulos (stalk/stem) and erpos (creep) (Mehra et al., 2019). Especially, C. taxifolia and C. cylindracea (previously known as C. racemosa var. cylindracea) have attracted attention for the last 30 years due to their invasive properties (Montefalcone et al., 2015) and, more importantly, the various properties of bioactive (especially secondary metabolites) chemicals for defense, communication, growth and development regulation, reproduction, competition, and infection (Erb and Kliebenstein, 2020; Ramawat and Goyal, 2020). Caulerpa secrete linear or monocyclic terpenoids that have aldehyde and enol-acetate functional groups (Mehra et al., 2019). The structure of caulerpin (C₂₄H₁₈N₂O₄), a secondary metabolite and pigment of the C. cylindracea species, was first described by Aguilar-Santos in 1970 (Aguilar-Santos, 1970). Its molecular weight is 398.418 g/mol and its characterized structure is ‘dimethyl-6,13-dihydrodibenzo [b,i] phenazin-5,12-dicarboxylate methyl ester. Caulersin (C₂₁H₁₄N₂O₃) is another bis-indole alkaloid which is isolated from C. serrulata (Su et al., 1997) and from C. racemosa (Yang et al., 2014). Its isomers are caulersin A, B, and C. The molecular weight of caulersin is 342.1004 g/mol (Su et al., 1997). It is characterized by its “central troponoid bridging” bisindole structure (Su et al., 1997). Caulerpenyne is a sesquiterpenoid-structured secondary metabolite which has some bioactivities such as antiproliferative and apoptotic activities (Cavas et al., 2006) and inhibitors of lipoxygenase (Cengiz et al., 2011) and 5-lipoxygenase (Richter et al., 2014), etc. Secondary metabolites of genus Caulerpa are responsible for complex modulation network induced in AMPK, ER Stress, mitochondrial stress, PTP1B inhibition and cell cycle stop pathways, metabolic reprogramming in cancer cells, apoptosis and cell cycle arrest in cancer metabolism (Mehra et al., 2019).

Colorectal cancer (CRC) is the second and third most common diagnosed cancer type in women and men in the world, respectively (Dekker et al., 2019). According to the World Health Organization data, in 2018, CRC caused 1.80 million new cases and 862,000 deaths all around the world (WHO, 2018). Since CRC is a common and fatal cancer type, using novel and effective chemicals for treatment is essential.

The pentose phosphate pathway (PPP) is the pivotal pathway for ribonucleotide synthesis and is the main source of NADPH (the reduced form of Nicotinamide Adenine Dinucleotide Phosphate) (Stincone et al., 2015), which is of great importance for fatty acid synthesis and reactive oxygen species scavenging. PPP branches from glycolysis, and it plays a key role in cancer cells (Patra and Hay, 2014). The oxidative phase of PPP is initiated with hexokinase, which converts glucose to glucose 6-phosphate (G6P). G6P oxidizes (dehydrogenated) to 6-phosphogluconolactone by the rate-limiting enzyme (glucose 6-phosphate dehydrogenase (G6PDH)) to yield NADPH (by reducing NADP+) (Stincone et al., 2015). The other NADPH source of PPP is the conversion of 6-phosphogluconate into ribose (ribulose) 5-phosphate by 6-phosphogluconate dehydrogenase (6PGD). Eventually, the regeneration of GSH, synthesis of DNA, fatty acids, and sterols is achieved by producing 2 mol of NADPH per mole of G6P entering the oxidative phase of PPP (Patra and Hay, 2014). In many solid tumors, overexpression of PPP (especially the enzyme 6PGD) has already been observed (Patra and Hay, 2014; Jin and Zhou, 2019). Furthermore, targeting oxidative phase of PPP for mutant KRAS colorectal carcinomas prevents the recurrence (Gao et al., 2019). Thus, targeting the PPP is a potentially new target for CRC treatment.

In-silico computer-aided methods are commonly used to predict and elucidate the molecular-level behavior of a compound (Dege et al., 2022). Molecular docking is a convenient in-silico method which can be used to evaluate the binding affinity of the ligand on the receptor and can predict the position of the these molecules (Trott and Olson, 2010; Mert Ozupek and Cavas, 2017). ADME provides information about in-silico ADME behavior which is important for medicinal chemistry (Bocci et al., 2017; Dege et al., 2022; Gokce et al., 2022; Pantaleão et al., 2022). Drug-likeness analysis using in-silico is of great importance for evaluating the pharmacokinetic features of fast and cheap (Gokce et al., 2022; Hasan et al., 2022).

In this study, the anticancer activity of phytochemicals of Caulerpa spp. were tested on potential targets (G6PDH and 6PGD) against CRC by using in-silico pharmacokinetic and pharmacodynamic tools. The aim of this study was to investigate the potential Caulerpa-based phytochemicals against fundamental targets (G6PDH and 6PGD) for colorectal cancer treatment.

In this study, 13 of 36 different secondary metabolites of Caulerpa (caulerpin, caulerpenyne, 10,11-epoxycaulerpenyne, caulersin, flexilin, racemosin C, racemosin B, caulerprenylol B, caulerprenylol A, monomethyl caulerpinate, α-tocospiro A, α-tocospirone and furocaulerpin) against crucial targets (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) for colorectal cancer were carried out by using in-silico pharmacokinetic and pharmacodynamic methods. Caulerpin and monomethyl caulerpinate were found to be the most effective metabolites against G6PDH. Caulersin had the lowest affinity score against 6PGD. The pentose phosphate pathway is fundamental for colorectal cancer, thus caulerpin, monomethyl caulerpinate and caulersin play important roles in colorectal cancer treatment through the pentose phosphate pathway.

ADME is an important medicinal chemistry tool that provides information about in-silico ADME behavior (Bocci et al., 2017; Pantaleão et al., 2022). The detailed pharmacokinetic ADME-Tox and drug-likeness results are given in Table 1 and Table 2. The physicochemical properties (molecular weight (g/mol), the number of heavy atoms, the number of aromatic heavy atoms, the number of rotatable bonds, number of H-bond acceptors, the number of H-bond donors, molar refractivity and TPSA (Å)) were calculated for 36 Caulerpa-based phytochemicals. ADME results reveal that the compounds with low molecular weight (≤500 g/mol) tend to absorb well (Daina et al., 2017). In our study, 4 of 36 phytochemicals (sulfoquinovosyldiacyl glycerol, amBiosome, alpha-tocoxylenoxy and caulerpicin) from Caulerpa spp. have high molecular weight (low absorption capacity). The flexibility of bioactive molecules is determined using the number of rotatable bonds (Daina et al., 2017) which should be between 0 and 9. In this study, all the chemicals except sulfoquinovosyldiacyl glycerol and caulerpicin are in the range of this value. Topological polar surface area (TPSA) is based on the fragmental system of phosphorous atoms and polar sulfur. TPSA value should be between 20 and 130 Å for polarity (Daina et al., 2017). In this study, all the 13 phytochemicals were found in this range. Lipophilicity (logP) is crucial for clarifying the effect of chemicals’ absorption, distribution, transportation on physiological systems. In this study, all the samples (36) out of 5 phytochemicals were in the range of logP (-2≤logP≤5). For water solubility, logS (ESOL) was tested. Daina et al. defined the scale of water solubility as insoluble <−10 < poorly <−6 < moderately <−4 < soluble <−2 < very <0 < highly (Daina et al., 2017). The results reveal that caulerpin, caulerpenyne, caulersin, flexilin, racemosin C, racemosin B, monomethyl caulerpinate, and furocaulerpin were found as moderately soluble; 10,11-epoxycaulerpenyne, caulerprenylol B and caulerprenylol A were found as soluble and α-tocospiro A and α-tocospirone were found as poorly soluble.

Before the clinical trials of drug candidates, in-silico toxicity measurement procedure is quite important for better selecting the lead compound (Han et al., 2019). These computational-based toxicity measurement procedures are accurate, accessible, rapid, and common. Both ProTox-II and T.E.S.T servers (freely accessible) were used to identify the adverse effects and toxicity (acute toxicity, carcinogenicity, cytotoxicity, hepatotoxicity, immunotoxicity and mutagenicity) of selected phytochemicals from ADME results. Our results reveal that the toxicity classes of the selected phytochemicals were found to be more than three. For all selected phytochemicals, hepatotoxicity, carcinogenicity, cytotoxicity androgen receptor results were found as inactive.

Drug design and discovery is a step-by-step process, which is costly for companies (Opo et al., 2021). The bioavailability and drug-likeness analysis using in-silico is of great importance for evaluating the pharmacokinetic features of fast and cheap (Hasan et al., 2022).

In this study, the selected metabolites (ligands) have the potential anticancer activity against the selected receptor targets for CRC. According to the results, among the commercial drugs (DHEA and 6ANA), for G6PDH, the best docking energy was exhibited by caulerpin with a vina score of −10.6 kcal/mol, while for G6PDH, the other best docking energy was exhibited by monomethyl caulerpinate with a vina score of −10.5 kcal/mol. For 6PGD, the best docking score among the secondary metabolites of Caulerpa was exhibited by caulersin (−10.7 kcal/mol). (Table 3). In the literature, limited studies are related to the activities of caulerpin on different targets in-silico. In the study by Lorenzo et al. (2015), in-silico molecular docking study of caulerpin and its nine analogs against monoamine oxidase B was carried out. Their results reveal that moldock energy, predicted probability (%) and drug-like score of caulerpin were found as −152%, 58%, and 0.77, respectively. However, the analogs of caulerpin which have non-polar and polar groups showed different moldock energy, predicted probability (%) and drug-like scores. The methods that they used (Volsurf descriptors, structure-based methodology and Random Forest algorithm) are crucial for finding the good drug candidates (caulerpin and its analogs) against monoamine oxidase B (Lorenzo et al., 2015). In the literature, Vitale et al. (2018) carried out an in silico molecular docking evaluation of caulerpin against PPARalpha and PPAR gamma. Their study reveals that the main interaction between ligand (caulerpin) and protein is a hydrophobic interaction. Also, according to their molecular dynamics results, caulerpin makes intermolecular H-bonds with S289 (VI) and S342/R288 (V) (Vitale et al., 2018). Furthermore, antiviral activity of caulerpin against SARS-CoV-2 was tested using in-silico tools (Abdelrheem et al., 2020; Ahmed et al., 2020; Çavaş et al., 2020; El-Mageed et al., 2021).

MM-PBSA analysis is an important and popular method in drug candidate filtration since it is an easy method, and the speed-accuracy balance of the information is high. Estimation of binding free energies with MM-PB(GB) SA for the ligands is automated with the farPPI web server. In our study, the highest and the lowest binding free energies were calculated for monomethyl caulerpinate and caulersin, respectively.

Caulerpin also has some biological activities such as anticancer activity on melanoma cells (Rocha et al., 2007), HIF-1 activation and inhibition of mitochondrial respiration (Liu et al., 2009), antiviral activities against bovine viral diarrhea virus in cattle and herpes simplex virus (Macedo et al., 2012; Pinto et al., 2012; Zhang et al., 2015), pain-sensitizing and spasmolytic effect (Cavalcante-Silva et al., 2014), antituberculosis activity (Canché Chay et al., 2014), antiproliferative activity (Movahhedin et al., 2014), monoamine oxidase inhibitory activity against Alzheimer’s and Parkinson’s disease (Lorenzo et al., 2015), activity on cisplatin-resistant overian cancer and inhibition respiratory complex II activity (Ferramosca et al., 2016), AMPKα1 pathway activation in colorectal cancer cells (Yu et al., 2017) and PPARα and PPARγ agonist activity on hepatocellular cell line (Vitale et al., 2018). Caulersin is a known human protein tyrosine phosphatase-1B inhibitor, which regulates insulin signaling negatively (Yang et al., 2014). The anticancer activity of caulerpin, caulersin, caulerpenyne and 10,11-epoxycaulerpenyne for colorectal cancer data reveal that caulerpin and caulersin are promising anticancer agents against CRC targets and G6PDH and 6PGD could be important targets for CRC.

Inhibition of PPP-enzymes is related with AMPK-activation, HIF-1α degradation, impaired folate metabolism and PP2A-activation (Meskers et al., 2022). G6PDH is the main NADPH production and redox homeostasis contributor (Ghergurovich et al., 2020). The expression level of G6PDH is upregulated and negatively correlated with patients with cancer (Ghergurovich et al., 2020). In different CRC cell lines, the expression levels of G6PDH and 6PGD are quite different. In the study of Polat et al. (2021), the highest and the lowest G6PDH levels were found in HT29 and Caco-2 cell lines, respectively (Polat et al., 2021). Thus, the effectiveness of the selected secondary metabolites could be changed depending on the colorectal cancer cell type.

For the prospects, the not only the in-silico analysis but also the in-vitro experiments of these targets for caulerpin, monomethyl caulerpinate and caulersin should be performed.

Global warming and human activities change the ecosystem structures. Alien members of Genus Caulerpa are widely studied marine algae due to their invasive properties. The present paper proposes an alternative utilization method in medicinal chemistry. The secondary metabolites of Caulerpa spp. attract attention due to their bioactivities. Using the ADME-tox and drug-likeness tests, 13 of 36 secondary metabolites were selected and molecular docking, and molecular dynamics analysis were performed. Caulerpin, monomethyl caulerpinate, and caulersin were found the hit compounds of Caulerpa spp. Against G6PDH and 6PGD, which may play pivotal roles in CRC. Thus, instead of eradication of these algae, the secondary metabolites proposed in this paper (caulerpin, monomethyl caulerpinate) might further be evaluated as promising agents that can be obtained from pharmacy of nature.