1. Introduction
Marine natural products are a rich source of potential drugs because of their high biodiversity, low toxicity, suitability for oral applications, and a variety of biological actions [
1,
2,
3]. According to the global marine pharmaceutical pipeline website (
www.marinepharmacology.org), 15 marine-derived drugs have been approved by the United States Food and Drug Administration (US FDA), and 32 marine-derived compounds are currently in various phases of clinical trials for drug development. Most have been developed or are under development as anticancer drugs, whereas a few are targeted for diseases other than cancer, such as pain, hypertriglyceridemia, Alzheimer’s disease, and viral infections. Considerable scientific and technological advances in analytical methods and functional assays in the past decade have opened up new opportunities for the exploration of marine natural products as novel potential therapeutic agents for more diverse disease entities [
4,
5,
6].
Membrane transport proteins are important targets for drug discovery and delivery [
7,
8]. Transport proteins are essential for translocating solutes across the plasma or intracellular membranes, thus maintaining homeostasis. Transport proteins include channels and membrane transporters that are divided into three distinct classes: solute carriers (SLC), ATP-binding cassette (ABC) transporters, and ATPase ion pumps [
9,
10]. According to the data from The HUGO Gene Nomenclature Committee (
https://genenames.org/), transport proteins account for approximately 6% of all human protein-coding genes: 427 genes of SLC superfamily, 312 genes of ABC transporters, 125 genes of ATPase, and 291 genes of channels. The loss or alteration of the function of membrane transporters and channels causes a wide variety of human diseases, including channelopathies [
11,
12,
13]. Membrane transporters are rapidly emerging as potential drug targets but have remained understudied until recently [
14,
15]. Moreover, since membrane transporters function as drug transporters, they play key roles in the absorption, distribution, and elimination of drugs and determine the therapeutic efficacy and adverse reactions of drugs in the process of drug discovery and development [
16,
17,
18].
Aquaporins (AQPs) are a family of small integral membrane proteins that primarily transport water across cell membranes along osmotic gradients. To date, 13 AQP subtypes have been found in mammals (AQP0-12), some of which permit transcellular passage of glycerol and urea as well as water (AQP3, 7, 9, and 10), and are thus called aquaglyceroporins [
19]. AQPs are key players in maintaining normal water balance and energy homeostasis in the body [
20,
21]. AQPs have been implicated in multiple disorders including diabetes insipidus, brain edema, cancer, obesity, cataracts, and neuromyelitis optica [
22,
23,
24,
25,
26]. In addition to AQP-based diseases, AQP modulators have potential therapeutic utility in various pathological conditions for correcting abnormal transepithelial fluid and glycerol transport [
20,
21,
27]. Notwithstanding the vigorous efforts to identify aquaporin modulators, limited progress has been made in the development of aquaporin-based therapeutics [
28]. The relatively low hit rate, poor druggability due to toxicity, and lack of specificity makes the exploration of new AQP-targeted drugs challenging [
29,
30]. Because marine natural products not only possess greater structural and chemical diversity but also less toxicity than synthetic chemical libraries, the difficulty in finding promising lead compounds as AQP modulators using synthetic compound libraries encouraged us to conduct the present study to screen marine natural products for novel drugs targeting AQPs.
3. Discussion
In this study, we aimed to identify potential AQP modulators in marine natural products. Although none of the marine natural compounds tested had significant effects on osmotic water permeability, we found several compounds that significantly affected glycerol permeability in mouse erythrocytes. We confirmed the modulatory effects of stichoposide C and rhizochalin on aquaglyceroporins through transepithelial glycerol transport assays using AQP3-expressing human keratinocytes.
Natural products, including plants, animals, and minerals, continue to be the best sources of novel drugs owing to their structural diversity and biodiversity [
5,
6]. In 2023, 10 new natural products or their direct derivatives were drug approved by the US FDA, which corresponds to 18% of total 55 new drugs on the market [
32]. Because the total number of species and biochemical diversity in oceans are higher than those on land, marine natural products are more invaluable sources for potential drug discovery than terrestrial products [
3,
4]. Moreover, natural products released into water are rapidly diluted and, therefore, need to be highly potent to have any effect. These advantages of marine natural products, along with the growing appreciation for functional assays and phenotypic screening, may further contribute to the revival of interest in natural products for drug discovery and development [
33,
34].
Triterpene glycosides are widely distributed not only in plants, but also in marine invertebrates, especially echinoderms, octocorals, and sponges [
35,
36]. Many investigators have attempted to develop marine triterpene glycosides as candidate anticancer agents based on in vitro and in vivo studies [
37,
38,
39]. Stichoposides C and D are triterpene glycosides extracted from sea cucumbers of the family
Stichopodidae [
40,
41]. Stichoposide C is a quinovose-containing hexoside, whereas stichoposide D is a glucose-containing hexoside (
Figure 7). Stichoposide C is an active membrane-acting agent with anticancer activity, and is more potent than stichoposide D [
42,
43]. In the present study, stichoposide C strongly stimulated glycerol permeability in murine erythrocytes and AQP3-expressing human keratinocytes, whereas stichoposide D did not. Based on the notion that a linear tetrasaccharide fragment in triterpene glycosides is essential for the actions leading to the modification of the cellular membrane [
37,
44,
45], having quinovose, rather than glucose, as a second monosaccharide unit in glycosides seems to be critical for interaction with 5(6)-unsaturated sterols of cellular membranes in contact with aquaglyceroporins or direct interaction with channel proteins. Otherwise, STC-specific activation of the sphingomyelinase-ceramide pathway might account for the different effects on aquaglyceroporins [
38]. However, further studies on the structure–activity relationship of these molecules are needed to improve the efficacy and safety of these compounds in activating aquaglyceroporins.
Rhizochalin is a marine two-headed sphingolipid-like natural product isolated from the sponge
Rhizochalina incrustata [
46,
47]. These compounds differ from classical sphingolipids in the presence of polar groups at α,ω-positions, which contain a terminal methyl group instead of hydroxymethyl group unlike sphingoid bases, thus representing a unique class of bipolar lipids [
48,
49]. Rhizochalin and its analogs have been reported to have antibacterial, antifungal, and cytotoxic activities [
50,
51,
52,
53]. In the present study, the peracetyl aglycon of rhizochalin exhibited the most potent inhibitory effect on glycerol permeability in murine erythrocytes and human AQP3-expressing epithelial cells. It might be assumed that the absence of a glucopyranosyl sugar moiety on one polar end, together with the presence of three acetyl moieties on the other polar end of two-headed bipolar lipids (
Figure 7), favors an intimate interaction with aquaglyceroporin or neighboring membrane lipids and regulatory proteins. The molecular mechanism underlying the inhibitory effect of the peracetyl aglycon of rhizochalin on aquaglyceroporins and the structure–activity relationship of rhizochalin analogs require further investigation.
Aquaglyceroporins mediate various physiological and pathophysiological processes [
54,
55,
56]. Water-selective AQPs function primarily in water and salt homeostasis, whereas water/glycerol-transporting aquaglyceroporins are primarily involved in energy metabolism and lubrication. Because aquaglyceroporin dysfunction is implicated in various human diseases and symptoms, including obesity, nonalcoholic fatty liver disease, psoriasis, cancer, polyuria, and glyceroluria, functional modulators of aquaglyceroporins are promising therapeutic targets for a wide range of clinical conditions [
24,
57,
58,
59]. A few dozen aquaglyceroporin modulators have been reported to date, but none have been developed or are under development. Metal compounds, such as HgCl
2 and
p-chloromercuribenzene sulfonate (pCMBS), were first identified as AQP inhibitors, including aquaglyceroporins, and are still the most commonly used inhibitors in functional assays [
60]. The gold compound Auphen ([Au(phen)Cl
2]Cl) and its copper analog Cuphen ([Cu(phen)Cl
2]) inhibited AQP3 and AQP7 [
61,
62]. Recently, several aquaglyceroporin inhibitors were identified by screening a commercially available library of small molecules: DFP00173 for AQP3, Z433927330 for AQP7, and HTS13286 for AQP9 inhibitors [
63,
64]. Natural compounds were also reported to have modulating effects on aquaglyceroporins which are usually from plants, such as glycolic acid, 18β-glycyrrhetinic acid, curcumin, chrysin and phloretin [
59]. However, the application of aquaglyceroporin modulators in clinical trials is still far from being implemented, possibly because of the lack of selective and potent modulators that can be administered in vivo.
In this study, we searched for potential AQP modulators among marine natural products and found that stichoposide C and the peracetyl aglycon of rhizochalin were potential aquaglyceroporin modulators. Since mouse erythrocytes express AQP7 and transepithelial glycerol transport was measured using human AQP3-expressing cells, it can be stated that stichoposide C and the peracetyl aglycon of rhizochalin function as modulators of AQP3 and AQP7. AQP3 activators may exert therapeutic effects on defects in the hydration, lubrication, and proliferation of the skin, bladder, vagina, and respiratory system, whereas AQP7 activators may be applicable as useful adjuvants for obesity treatment. Therefore, AQP3 and AQP7 inhibitors are worthy of development as anticancer drugs. Further studies are needed to verify the therapeutic effects of stichoposide C and the peracetyl aglycon of rhizochalin in animal models of human diseases or in 3D bioengineered disease models.
4. Materials and Methods
4.1. Marine natural products
The marine natural products were provided by Dr. V. A. Stonik at the Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Science, Vladivostok, Russian Federation. Thirty two compounds tested were extracted from marine natural products. The compounds were dissolved in phosphate-buffered saline (PBS) or dimethyl sulfoxide (DMSO) as 1–2 mM stock solutions, and diluted to working concentrations ranging from 100 nM to 1 μM.
4.2. Erythrocyte preparation
Thirty CD1 mice, 6–8 weeks old, were used in this experiment. The mice were anesthetized with avertin (2,2,2-tribromoethanol-tert-amyl alcohol, 250 mg/kg intraperitoneally). Blood was collected from the inferior vena cava in heparinized tubes. Freshly obtained whole blood was centrifuged at 120 × g for 10 min to remove the plasma and buffy coat. The erythrocytes were washed three times and suspended in PBS to adjust the final hematocrit to 2%. All animal protocols were approved by the Dong-A University Medical School Institutional Animal Care and Use Committee (DIACUC-10-6-1).
4.3. Stopped-flow light scattering measurements
Water and glycerol permeabilities were measured via light scattering using a stopped-flow apparatus (SFM-3, Biologic, France). The erythrocyte suspension was subjected to a 300 mM inwardly directed gradient of either sucrose or glycerol. The kinetics of decreasing cell volume were measured from the time course of 90° scattered light intensity at 530 nm. The osmotic water and glycerol permeability coefficients were calculated from the time course of light scattering using the following equations:
dV(t)/dt = Pf x SAV x MVW x (Cin - Cout)
where V(t) is the relative red cell volume as a function of time, SAV is the red cell surface area-to-initial volume ratio, MVW is the molar volume of water (18 cm
3/mole), and C
in and C
out (mole/cm
3) are the initial concentrations of the intracellular and extracellular solutes, respectively. The average erythrocyte surface area and volume were 7.2 × 10
-7 cm
2 and 3.1 × 10
-11 cm
3, respectively [
31].
4.4. Tissues preparation and immunohistochemistry
CD1 mouse kidneys were fixed in 10% neutral buffered formalin for 48 h, paraffin-embedded, and sectioned at 5 μm using a microtome (RM 2125RT, Leica, Wetzlar, Germany). Paraffin sections were deparaffinized with xylene, serially rehydrated in 100%, 95%, and 70% ethanol, and boiled for 15 min in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, and pH 6.0) for antigen retrieval. After blocking with 5% bovine serum albumin for 2 h, the sections were incubated with the following primary antibodies overnight at 4°C at a dilution of 1:300: anti-AQP1, AQP2, AQP3, and AQP4 (Merck Millipore, Billerica, MA, USA); anti-AQP5, AQP6, and AQP7 (Alomone Labs, Jerusalem, Israel); anti-AQP8 (Mybiosource, CA, USA); and anti-AQP9 (Bioss, Woburn, MA, USA). Subsequently, the sections were incubated with secondary anti-rabbit horseradish peroxidase (HRP)-linked IgG (Dako, Glostrup, Denmark) for 40 min at 25°C, and HRP activity was determined by adding DAB+ chromogen (Dako). The sections were mounted using Marinol Mount (Muto Pure Chemicals, Tokyo, Japan) solution and xylene (Muto Pure Chemicals) (1:1), and then visualized using a digital slide scanner (Pannoramic Midi, 3DHistech, Budapest, Hungary) at the Neuroscience Translational Research Solution Center (Busan, South Korea).
4.5. Cell cultures
HaCaT cells, an immortalized human keratinocyte line, were stably transfected with a plasmid encoding yellow fluorescent protein (YFP) and either human AQP1 (YFP-hAQP1) or human AQP3 (YFP-hAQP3) (gifts from Dr. Alan Verkman at the University of California, San Francisco, California, USA) and selected using G418 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were grown in DMEM medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Hyclone™, Cytiva, Logan, UT, USA), penicillin G (100 U/mL), streptomycin (100 μg/mL) and maintained at 37°C in a humidified 5% CO2 incubator.
4.6. Measurement of transepithelial glycerol transport
YFP-hAQP1-HaCaT cells and YFP-hAQP3-HaCaT cells were seeded at a density of 2 × 104 cells/well onto 6.5 mm, 0.4 μm pore size Transwell permeable supports (Corning Life Sciences, Tewksbury, MA, USA), cultured for 3–5 days to a confluent state, and serum-deprived for 2–3 days before the experiments. Once the cells reached a confluent state, the integrity of the cellular barriers was assessed by transepithelial electrical resistance (TEER) using an epithelial voltohmmeter. When the TEER values were above 200 Ω.cm2, transepithelial flux experiments were performed.
Glycerol transport across HaCaT cell monolayers was determined by measuring the glycerol concentrations of the culture medium in the upper and lower compartments, as well as in the Transwell inserts. The cells were pretreated with marine natural products or HgCl2 for 10 min, and 20 mM glycerol was added to the bath solution on the basal side of the cells. Aliquots (10 µL) of samples were collected from the medium of the opposite side from glycerol addition at the indicated time points. Glycerol concentrations were measured using a glycerol assay kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions.
4.7. Statistical analysis
All the data were expressed as mean ± standard deviation (SD). The statistical significance of differences between groups was analyzed using Student’s t-test or one-way ANOVA. P<0.05 was considered statistically significant.
Author Contributions
Conceptualization, J.Y.K. and H.R.B.; methodology, J.W.I., J.H.L. and H.R.B.; software, S.J. and M.S.; validation, J.W.I., S.J. and H.R.B.; formal analysis, J.W.I. and H.R.B.; investigation, J.W.I. and J.H.L.; resources, J.Y.K. and V.A.S.; data curation, J.W.I. and H.R.B.; writing—original draft preparation, J.W.I., J.H.L. and H.R.B.; writing—review and editing, M.S. and H.R.B.; visualization, J.W.I. and J.H.L.; supervision, , J.Y.K. and H.R.B; project administration, H.R.B.; funding acquisition, H.R.B. All authors have read and agreed to the published version of the manuscript.