Looking at Marine-Derived Bioactive Molecules as Upcoming Anti-Diabetic Agents: A Special Emphasis on PTP1B Inhibitors

Diabetes mellitus (DM) is a chronic metabolic disease with high morbimortality rates. DM has two types: type 1, which is often associated with a total destruction of pancreatic beta cells, and non-insulin-dependent or type 2 diabetes mellitus (T2DM), more closely associated with obesity and old age. The main causes of T2DM are insulin resistance and/or inadequate insulin secretion. Protein-tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling pathways and plays an important role in T2DM, as its overexpression may induce insulin resistance. Thus, since PTP1B may be a therapeutic target for both T2DM and obesity, the search for novel and promising natural inhibitors has gained much attention. Hence, several marine organisms, including macro and microalgae, sponges, marine invertebrates, sea urchins, seaweeds, soft corals, lichens, and sea grasses, have been recently evaluated as potential drug sources. This review provides an overview of the role of PTP1B in T2DM insulin signaling and treatment, and highlights the recent findings of several compounds and extracts derived from marine organisms and their relevance as upcoming PTP1B inhibitors. In this systematic literature review, more than 60 marine-derived metabolites exhibiting PTP1B inhibitory activity are listed. Their chemical classes, structural features, relative PTP1B inhibitory potency (assessed by IC50 values), and structure–activity relationships (SARs) that could be drawn from the available data are discussed. The upcoming challenge in the field of marine research—metabolomics—is also addressed.

cancer [16]. Accordingly, multiple studies have been conducted focusing on the development of new PTP1B inhibitors for the treatment of T2DM, obesity, and cancer, but to the authors' knowledge there are no review articles published on this subject. In this sense, the present review aims to provide an overview of the role of PTP1B in T2DM insulin signaling and treatment, and to highlight the most recent findings on several compounds and extracts discovery from marine organisms and their relevance as upcoming PTP1B inhibitors.
Molecules 2018, 23, x 3 of 34 PTP1B inhibitors for the treatment of T2DM, obesity, and cancer, but to the authors' knowledge there are no review articles published on this subject. In this sense, the present review aims to provide an overview of the role of PTP1B in T2DM insulin signaling and treatment, and to highlight the most recent findings on several compounds and extracts discovery from marine organisms and their relevance as upcoming PTP1B inhibitors.

Marine Sources as Upcoming Therapeutic Agents
The marine environment is considered a wide and relatively unexploited source of bioactive compounds with high biodiversity, including fatty acids (especially polyunsaturated fatty acids), proteins, polyphenols, sterols, sulfated polysaccharides, and pigments [32][33][34][35][36]. Indeed, marine algae has been increasingly exploited as renowned sources of metabolites with promising biological effects, including antioxidant, hypoglicemic, hypotensive, hypolipidemic, antibacterial, and antiviral activities [37,38]. Specifically, macroalgae are considered healthy foods as they are rich in minerals and dietary fibers. Traditionally, the Far East and Hawaiian Islands, Japan, Korea, and China consume marine algae as a common component of their diets. Macroalgae species can reach 9000 species and can be classified according to their pigment composition into three classes, i.e., Phaeophyta, Rhodophyta, and Chlorophyta (also known as brown, red, and green algae, respectively) [39].
Unique metabolites from diverse classes have been isolated from different marine plants, with in vivo remarkable pharmacological effects [40], such as anticancer, anti-hyperlipidemic, anti-diabetic, anti-hypertensive, antioxidant, anti-inflammatory, anticoagulant, anti-estrogenic, antibacterial, antifungal, antiviral, immunomodulatory, neuroprotective, and tissue healing properties [41]. More recently, as a result of the characterization of a large number of bioactive metabolites from marine macroalgae, there has been a growing interest in the search for potential applications of macroalgae and their metabolites as functional constituents for human and animal health benefits [42]. Functional constituents of macroalgae have been increasingly used as food supplements as well as for anti-diabetic purposes [40]. Hereby, the possible applications of marine macroalgae and/or macroalgae-derived bioactive metabolites for PTP1B inhibitory effects have been greatly expanded.

Marine Sources as Upcoming Therapeutic Agents
The marine environment is considered a wide and relatively unexploited source of bioactive compounds with high biodiversity, including fatty acids (especially polyunsaturated fatty acids), proteins, polyphenols, sterols, sulfated polysaccharides, and pigments [32][33][34][35][36]. Indeed, marine algae has been increasingly exploited as renowned sources of metabolites with promising biological effects, including antioxidant, hypoglicemic, hypotensive, hypolipidemic, antibacterial, and antiviral activities [37,38]. Specifically, macroalgae are considered healthy foods as they are rich in minerals and dietary fibers. Traditionally, the Far East and Hawaiian Islands, Japan, Korea, and China consume marine algae as a common component of their diets. Macroalgae species can reach 9000 species and can be classified according to their pigment composition into three classes, i.e., Phaeophyta, Rhodophyta, and Chlorophyta (also known as brown, red, and green algae, respectively) [39].
Unique metabolites from diverse classes have been isolated from different marine plants, with in vivo remarkable pharmacological effects [40], such as anticancer, anti-hyperlipidemic, anti-diabetic, anti-hypertensive, antioxidant, anti-inflammatory, anticoagulant, anti-estrogenic, antibacterial, antifungal, antiviral, immunomodulatory, neuroprotective, and tissue healing properties [41]. More recently, as a result of the characterization of a large number of bioactive metabolites from marine macroalgae, there has been a growing interest in the search for potential applications of macroalgae and their metabolites as functional constituents for human and animal health benefits [42]. Functional constituents of macroalgae have been increasingly used as food supplements as well as for anti-diabetic purposes [40]. Hereby, the possible applications of marine macroalgae and/or macroalgae-derived bioactive metabolites for PTP1B inhibitory effects have been greatly expanded.

Ptp1b Inhibitory Activity: In Vitro Findings
Around 300 natural products with PTP1B inhibitory capacity were isolated and characterized from different natural sources, many of them from marine origin [43]. The isolation and identification of sulfircin, a sesterterpene sulfate, from deep-water sponge Ircinia (unknown species), was the first documented marine metabolite possessing PTP1B inhibitory activity [43]. Since then, marine sponges have been considered valuable sources of PTP1B inhibitors with diverse structures [44], such as polybromodiphenyl ether [45], sesquiterpenoids, and sesquiterpene quinones [46]. Nevertheless, the novelty of marine resource screening models has encouraged the development of new studies targeting these resources as upcoming anti-diabetic agents. Marine algae, seaweeds, soft corals, sponges and lichens are considered to be among these models as they were found to exhibit PTP1B inhibitory effects. Tables 1-8 summarize a large number of isolated compounds from marines that have PTP1B inhibitory effects with varying potencies. In the following sections, the PTP1B inhibitory activity of some of these compounds are discussed.

Bromophenols
As the main component of algae, bromophenols may be responsible for the reported anti-diabetic activity of many marine organisms. These compounds arise from the tendency of the phenol moiety to undergo electrophilic bromination to varying degrees (Table 1).

Brominated Metabolites
In 2010, Qin et al. [60] assessed the in vitro PTP1B inhibitory action of two brominated metabolites (compounds 17 and 18) isolated from the red algae Laurencia similis. Both compounds showed PTP1B inhibition with IC 50 values of 3.0 µM and 2.7 µM, respectively. The authors also studied the effect of highly brominated metabolites (compounds 19-23), but their corresponding IC 50 values were much higher than those of the brominated metabolites, ranging from 65.3 µg/mL to 102 µg/mL. This could question the hypothesis that the bromination degree affects the PTP1B inhibitory effect in a directly proportional manner (Table 2).

Polybromodiphenyl Ether Derivatives
Besides the two abovementioned bromophenols (compounds 12 and 13), the Indonesian marine sponge Lamellodysidea herbacea contains six polybromodiphenyl ether derivatives (compounds 24-29). As stated by Yamazaki et al. [45], all of these compounds exhibited in vitro PTP1B inhibitory action with IC 50 values ranging from 0.6 µM to 1.7 µM. The authors also determined the activity of compounds 24 and 25 in Huh-7, a well-differentiated hepatocyte-derived cellular carcinoma cell line that has been increasingly investigated, given its ability to secrete mitogen hepatoma-derived growth factor, responsible for promoting cell growth without depending on other growth factors present in serum. The IC 50 values obtained for these compounds were, respectively, 32 µM and 48 µM (Table 3).

Phlorotannins
Back in 1977, Glombitza was the first to introduce the term phlorotannins [61]. Briefly, they are a characteristic type of integral tannins found in brown algae, Alariaceae, and are basically classified into six main subclasses (Table 4): eckols, fucols, phlorethols, fucophloretols, fuhalols, and isofuhalos [62,63]. Phlorotannins are thought to modulate cellular signaling, leading to the regulation of different body conditions [49]. Eckol and its derivatives (compounds 30-35) isolated from the edible brown algae Ecklonia stolonifera and Eisenia bicyclis were studied by several authors [64][65][66], who found variable anti-diabetic effects. The in vitro PTP1B inhibitory activity was variable, with IC 50 values ranging from 0.6 µM to 55.5 µM, whereas the in vitro α-glucosidase inhibitory action exhibited more potent IC 50 values ranging from 1.4 µM to 141.2 µM.
It is worth mentioning that phlorofurofucoeckol-A (compound 31) showed the lowest IC 50 values for both enzymes. In contrast, phloroglucinol (compound 34), which is actually the building unit of other polymer phlorotannins, had the highest IC 50 values for both enzymes. From this, it is possible to suppose that the activity is due to the product of polymerization, not to the basic monomeric structure.

Diterpenes
Diterpenes isolated from Sarcophyton trocheliophorum Marenzeller, a Hainan soft coral, received pivotal attention from Liang et al. over two consecutive years [73,74]. The authors isolated three compounds (compounds 46-48), and assessed their in vitro PTP1B inhibitory potential. These diterpenes showed variable effects, with IC 50 values ranging from 6.8 µM to 27.1 µM.

Sesterterpenes
Piao et al. [75] evaluated the activity of two sesterterpenoids (compounds 49 and 50) from the sponge Hippospongia lachne found on Yongxing Island against PTP1B through an in vitro study, and found that they exhibited IC 50 values of 5.2 µM and 8.7 µM, respectively.
Aquastatin A (compound 60) is also a fungal metabolite isolated from Cosmospora species ( Table 7) that has received particular attention from several researchers [81,82]. This attention is attributed not only to its low IC 50 value against PTP1B enzyme (0.2 µM), but also due to its selective inhibitory activity against others PTPs, including TCPTP, SHP-2, LAR, and CD45.       Fucoxanthin, compound 61, is a carotenoid that was isolated from Phaeodactylum tricornutum and edible brown seaweeds, such as Eisenia bicyclis (Arame), Undaria pinnatifida (Wakame), and Hi-jikia fusiformis (Hijiki) ( Table 8). Fucoxanthin was found to decrease blood glucose and insulin levels as well as water intake in a diabetic/obese KKAy mice model. A significant reduction in mRNA expression levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α (TNF-α) was observed, which is believed to be involved in insulin resistance induction. A prominent PTP1B inhibitory action was also stated, with an IC 50 value of 4.8 µM [83][84][85].

PTP1B Inhibitory Activity: In Vivo Findings
With regards to the in vivo anti-diabetic activity of marine plants, Shi et al. [53] assessed the in vivo PTP1B inhibitory activity of highly brominated derivatives isolated from the red algae R. confervoides, which contains one or two 2,3-dibromo-4,5-dihydroxybenzyl units, in diabetic rats. They found that R. confervoides extracts led to a significant reduction in serum glucose levels. These in vivo results may confirm that the anti-hyperglycemic activity of R. confervoides is partially attributable to the PTP1B inhibition activity of its constituents [53]. Similarly, Nuño et al. [90] investigated the anti-diabetic activities of the microalgae haptophyte Isochrysis galbana and the ochrophyte Nannochloropsis oculata in a diabetic rat model. Different biochemical parameters were investigated, such as glucose level, body weight, lipoproteins, and nitrogenous compounds. In addition, gastrointestinal (GI) histopathology was studied. Both microalgae studied led to an increase in low-density lipoprotein (LDL) and a decrease in high-density lipoprotein (HDL) levels in both control and diabetic rats. More specifically, I. galbana decreased body weight, glucose, triacylglycerides, and cholesterol levels and exhibited just slight signs of inflammation in the gut. The observed activity could be attributed to their high content in docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acids. The N. oculata-treated diabetic group did not show any changes in clinical values and had negative effects within the GI tract. Further studies are needed to confirm the effective employment of I. galbana as an anti-diabetic functional food.

In Vitro and In Vivo Concerns
In overall, T2DM is a metabolic disease characterized by hyperglycemia and hyperinsulinemia, in which the most common risk factor is overweight or obesity [91]. The development of diabetes may occur as a result of insulin secretion and/or signaling deregulation by insulin receptors (IR) [92]. The action of PTPs on IR themselves or their substrates is an important mechanism in insulin signaling regulation [92]. The role of PTPs in insulin signaling pathways and diabetes has previously been studied using vanadium compounds, which are able to reduce serum glucose levels in both type 1 and type 2 diabetic animal models [93,94]. Vanadium compounds show fundamental in vitro and in vivo insulinomimetic effects. Thus, the oral administration of such compounds promotes the normalization of serum glucose levels in T2DM rats, increasing glucose uptake [95]. These rats exhibited increased levels of hepatic cytosolic PTP activity, which decreased following insulin and vanadate treatment, leading to serum glucose levels normalization. These findings can be explained through PTPs inhibition with consequent improvement of cellular tyrosine phosphorylation [96].
Besides IR recognition, structural studies on PTP1B enzymes led to the identification of JAK2 and tyrosine kinase 2 (TYK2) as potential PTP1B substrates. Following interferon stimulation, both kinases were found to be hyperphosphorylated in PTP1B null fibroblasts [97]. This finding was further confirmed by the negative regulation of leptin-stimulated JAK2 phosphorylation produced by PTP1B, which reduced leptin signaling in in vivo models. Thus, null PTP1B mutation was introduced into leptin-deficient obese ob/ob mice, and a significant decrease in weight gain with an increase in resting metabolic rates was found in PTP1B-deficient ob/ob mice. Moreover, fat pads analysis proposed that the weight variations could be attributed to a decrease in adipose tissue. So, in the absence of leptin, PTP1B loss can reduce weight gain without modifying food intake [98,99]. Moreover, PTP1B-deficient mice had an increased response to weight loss caused by leptin and feeding suppression. The hypothalami of these mice evidenced a marked improvement in leptin-induced Transcription Factor STAT3 phosphorylation, indicating that the introduction of exogenous leptin in PTP1B deficiency will also led to leptin sensitivity enhancement [98,99]. Actually, substrate trapping trials using catalytically inactive PTP1B D181A confirmed that leptin-activated JAK2 is considered a PTP1B substrate, and that leptin signaling reduction is an obesity resistance mechanism in PTP1B null mice.

Human Concerns
In humans, weight loss and improved insulin sensitivity are closely related to decreased PTP activity, along with LAR and PTP1B expression in adipose tissue [100]. It is noticeable that PTP1B activity is not always related to its level of expression.
In obese and diabetic subjects, PTP1B protein levels show a 3-to 5-fold increase in abdominal adipose tissue, while a notable decrease in PTP1B activity was observed per unit of PTP1B protein [101]. It was also observed that total cellular PTP, not increased PTP1B activity, led to a marked raise in adipose tissue in obese individuals. In addition, it has been reported that an increase in PTP activity, but not in PTP1B activity, is accompanied by reduced insulin-stimulated glucose transport, proposing a tissue-specific role in glucose homeostasis for PTP1B [102].
On the other hand, genetic evidence also links PTP1B to diabetes and obesity in humans. Indeed, PTP1B locus maps to chromosome 20 in the region q13.1-q13.2 [103], which is a region recognized as a quantitative trait locus linked to insulin and obesity. A correlation between the role of PTP1B in insulin resistance and various polymorphisms has also been reported. That is, there is a continuing need to identify new PTP inhibitors for diabetes and obesity control.

Concerns in Culture Conditions
The anti-diabetic activity of several microalgae cultured under stressful conditions was also assessed using PTP1B assay [86,87]. Ingebrigtsen et al. [86] evaluated the non-polar fraction of five diatoms isolated from the North Atlantic (i.e., Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus, Skeletonema marinoi, and Porosira glacialis), grown under variable light/temperature conditions. A. longicornis and C. furcellatus extracts displayed anti-PTP1B activity. On the other hand, C. socialis showed activity only when grown under high temperature-low light conditions, whereas P. glacialis showed activity when cultivated only under high temperature-high light conditions. Still, S. marinoi showed no activity in any of the studied conditions. Thus, these findings highlight the importance of culture conditions in activating bioactive metabolites production.
Meanwhile, Lauritano et al. [87] screened a total of 32 crude extracts from microalgal species (four flagellates, seven dinoflagellates, and 21 diatoms) grown under variable culturing conditions. It was found that temperature/light stresses are more important than nutritional stress in microalgal species containing bioactive metabolites that have PTP1B enzyme inhibition.

Conclusions
In general, the data collected emphasize the importance of chemical constituents from different marine species, given their PTP1B inhibition activity, as key targets in T2DM and obesity management.
Although distinct anti-diabetic therapeutic strategies are currently available, there is still an urgent need to find more effective and less toxic pharmacological agents. PTP1B has been established as a promising molecular target for the treatment of both T2DM and its major risk factor, obesity. However, PTP1B inhibitors still present significant problems over the closely related enzymes belonging to the PTP family, due to their poor selectivity, which is the main point to overcome their adverse effects.
As summarized herein, marine metabolites have recently gained significant attention from the scientific community, as they are considered to represent a repository of diverse unexploited bioactivities and structural features that could broaden the chemical library and may provide potential targets for the discovery of novel PTP1B inhibitory agents. However, another challenge must be kept in mind, arising from the limited yields of marine metabolites, which hinder their assessment through in vivo studies. In addition, it should be noted that current studies mainly focus on marine metabolites isolation and characterization as PTP1B inhibitors. However, the underlying mechanisms of action and structure-activity relationships need more attention. Thus, intensive efforts should be made through high-throughput screening of marine metabolites, along with structural optimization and synthesis of new PTP1B inhibitors, in order to identify selective, safer, and more effective PTP1B inhibitory agents in the near future.