Marine Sponges and Bacteria as Challenging Sources of Enzyme Inhibitors for Pharmacological Applications

Enzymes play key roles in different cellular processes, for example, in signal transduction, cell differentiation and proliferation, metabolic processes, DNA damage repair, apoptosis, and response to stress. A deregulation of enzymes has been considered one of the first causes of several diseases, including cancers. In the last several years, enzyme inhibitors, being good candidates as drugs in the pathogenic processes, have received an increasing amount of attention for their potential application in pharmacology. The marine environment is considered a challenging source of enzyme inhibitors for pharmacological applications. In this review, we report on secondary metabolites with enzyme inhibitory activity, focusing our attention on marine sponges and bacteria as promising sources. In the case of sponges, we only reported the kinase inhibitors, because this class was the most representative isolated so far from these marine organisms.


Marine Sponges
Although marine sponges are considered very simple marine organisms, they represent "chemical factories" because they are able to produce a great number of biologically active compounds [29]. There is still an ongoing debate about whether natural products isolated from sponges originated from sponges or from associated bacteria. Several experiments have evidenced that some compounds isolated from sponges are synthesized by their associated microorganisms [15,30]. In the case of enzyme inhibitors, there are also several evidences on their bacterial origin (see below).
Polyketides, terpenoids, and peptides are the most abundant products isolated from sponges, showing inhibitory activities against many enzymes (Figure 1; see also  for the chemical structures of enzyme inhibitors reported in this paragraph).
Recently, the alkylpyridinium salts have been isolated from sponges, showing potent biological activities mainly as enzyme inhibitors. The marine sponge Reniera sarai produce some polymeric 3-alkylpyridinium salts [29]. Cyclostelletamines, cyclic pyridinium alkaloids, from the sponge Stelletta maxima [31] are able to inhibit the reaction of methyl quinuclidinyl benzylate with muscarinic acetylcholine receptors. The sponge Callyspongia fibrosa is the source of some polymers, which inhibit the epidermal growth factors [32]. Among acetylcholinesterase (AChE) inhibitors, an irreversible inhibitor (the onchidal) has been isolated from the mollusk Onchidella binneyi [33], and a pseudozoanthoxanthin-like compound from the coral Parazoanthus axinellae [34]. The enzyme adenine phosphoribosyl transferase of Leishmania tarentolae (L-APRT) has been inhibited by crude extracts from several marine invertebrates, as for example from the ascidian Polysyncraton sp. and from the bryozoan Bugula sp. [35]. Concerning the sponges, an inhibitor of L-APRT activity has been found in Dragmacidon sp. and Polymastia sp. The haplosclerid Callyspongia sp. SS97-23 was the source of three meroterpenoids, ilhabelanol, ilhabrene, and isoakaterpin, very potent inhibitors of L-APRT [36]. The marine sponge Petromica sp. BA99-103 produced halistanol sulfate, another inhibitor of L-APRT [35].
Marine sponges have been also proven as sources of protein kinase (PK) inhibitors. The enzymes, belonging to the protein kinase family, chemically catalyzed the transfer of a phosphate group to a defined substrate from a high-energy molecule. About 2% of all eukaryotic genes are protein kinases, organized in eight main groups [37]: (1) TK (tyrosine kinase); (2) TKL (tyrosine kinase-like); (3) STE (STE20, STE11, and STE7); (4) CK1 (casein kinase 1); (5) AGC (protein kinase A, protein kinase G, and protein kinase C); (6) CAMK (Ca 2+ /calmodulin-dependent kinases); (7) CMGC (Cdk, MAPK, GSK, Cdk-like); (8) RGC (receptor guanylyl cyclase). Kinases play key roles in different regulatory cellular processes, signal transduction, cell proliferation and differentiation, metabolic processes, apoptosis, and so on [2,38]. Because of the very different roles of the kinases, several diseases have as causes the deregulation of these enzymes. Furthermore, misregulation of various kinases has very often been reported in cancerous cells, so anticancer treatments involve kinases to specifically target cancer cells [39]. Kinase inhibitors represented a good challenge for cancer treatments, considering that to date about 130 kinase inhibitors are in different phases of clinical trials [40]. This is the case, for example, of Imitinib (Gleevec, Novartis), a tyrosine kinase inhibitor used in the prognosis for sufferers of chronic myeloid leukemia, now in the pharmaceutical market [41]. Inhibitors for different PK isolated from marine sponges are reported below (Table 1).
Marine sponges have been also proven as sources of protein kinase (PK) inhibitors. The enzymes, belonging to the protein kinase family, chemically catalyzed the transfer of a phosphate group to a defined substrate from a high-energy molecule. About 2% of all eukaryotic genes are protein kinases, organized in eight main groups [37]: (1) TK (tyrosine kinase); (2) TKL (tyrosine kinase-like); (3) STE (STE20, STE11, and STE7); (4) CK1 (casein kinase 1); (5) AGC (protein kinase A, protein kinase G, and protein kinase C); (6) CAMK (Ca 2+ /calmodulin-dependent kinases); (7) CMGC (Cdk, MAPK, GSK, Cdk-like); (8) RGC (receptor guanylyl cyclase). Kinases play key roles in different regulatory cellular processes, signal transduction, cell proliferation and differentiation, metabolic processes, apoptosis, and so on [2,38]. Because of the very different roles of the kinases, several diseases have as causes the deregulation of these enzymes. Furthermore, misregulation of various kinases has very often been reported in cancerous cells, so anticancer treatments involve kinases to specifically target cancer cells [39]. Kinase inhibitors represented a good challenge for cancer treatments, considering that to date about 130 kinase inhibitors are in different phases of clinical trials [40]. This is the case, for example, of Imitinib (Gleevec, Novartis), a tyrosine kinase inhibitor used in the prognosis for sufferers of chronic myeloid leukemia, now in the pharmaceutical market [41]. Inhibitors for different PK isolated from marine sponges are reported below (Table 1).

Marine Bacteria
In the marine environment, several enzyme inhibitors have been isolated from bacteria and actinomycetes for their industrial applications and their use in medicine and agriculture [11] (see Figure 5).

Marine Bacteria
In the marine environment, several enzyme inhibitors have been isolated from bacteria and actinomycetes for their industrial applications and their use in medicine and agriculture [11] (see Figure 5).

Marine Bacteria
In the marine environment, several enzyme inhibitors have been isolated from bacteria and actinomycetes for their industrial applications and their use in medicine and agriculture [11] (see Figure 5).  One of the first isolated marine microbial source of enzyme inhibitors were represented by the Alteromonas sp., from which serine and cysteine protease inhibitors have been isolated [85]. An example of serine protease inhibitors are represented by the marinostatins C-1 and C-2, used as drugs in pancreatitis pathogenesis thanks to its properties to inhibit α-chymotrypsin (IC 50 = 1.0-3.2 µM) [86,87].
Inhibitors for different PK isolated from marine bacteria are summarized in Table 2. Some examples of the chemical structures of enzyme inhibitors from marine bacteria are reported in Figure 6: endogenous monoamine oxidase (MAO) inhibitors, the 2,3-indolinedione (35), from marine Alteromonas sp., increasing the acetylcholine and dopamine in neurotransmission processes (IC 50 = 9.2 µM) [88]. -monastatin, a glycoprotein from pathogenic fish bacteria such as Aeromonas hydrophila and Vibrio anguillarum [89]. It has been applied in the production of cooked fish meat gel [90].  [93]), applied as drugs in diabetes [94], leukemia [95], and cancer [96]. -pyrizinostatin (40), inhibitor of pyroglutamyl peptidase (PGP) from a Streptomyces strain (IC 50 = 21 µM) [97], able to block thyrotropin-releasing and luteinizing hormone-releasing hormones [98]. , used as a drug in diseases caused by an accumulation of uric acid [103]. Table 2. Enzyme, enzyme inhibitors names (together with number compounds in the case the chemical structure have been reported in Figure 6), references, biotechnological applications, and IC 50 values (in micromolar, µM) from marine bacteria. Recently, the actinobacteria have also been considered as producers of enzyme inhibitors. This is the case for 30 strains of marine actinobacteria reported by Ganesan et al. [104], used in treating diabetes. Marine bacteria associated with the sponge Jaspis sp. have been characterized for their production of protease inhibitors [105]. In particular, the associated bacteria Providencia sp. showed inhibitory activity against the protease subtilisin, and Bacillus sp. had inhibitory effects against the metalloproteinase thermolysin.

General Conclusions
The increased incidence of severe diseases, including cancer, prompted the scientific research to find new drugs. This also pushed the scientific communities to explore the marine environment for new pharmaceuticals. In fact, the marine environment has a great richness and biodiversity of microand macroorganisms, also exhibiting biosynthetic pathways to produce secondary metabolites useful both against predators and active compounds for human health attracting the attention of pharmaceutical industries. A very significant example is represented by several classes of enzyme inhibitors isolated from marine sponges and bacteria, as reported in the review. These enzyme inhibitors show cytotoxic activity against many different cancer cell lines (see Table 1). Marine-derived enzyme inhibitors are considered viable alternatives in pharmaceuticals for replacing synthetic drugs to combat, for example, cancer as well as viral, amtiinflammatory, and Marine bacteria associated with the sponge Jaspis sp. have been characterized for their production of protease inhibitors [105]. In particular, the associated bacteria Providencia sp. showed inhibitory activity against the protease subtilisin, and Bacillus sp. had inhibitory effects against the metalloproteinase thermolysin.

General Conclusions
The increased incidence of severe diseases, including cancer, prompted the scientific research to find new drugs. This also pushed the scientific communities to explore the marine environment for new pharmaceuticals. In fact, the marine environment has a great richness and biodiversity of micro-and macroorganisms, also exhibiting biosynthetic pathways to produce secondary metabolites useful both against predators and active compounds for human health attracting the attention of pharmaceutical industries. A very significant example is represented by several classes of enzyme inhibitors isolated from marine sponges and bacteria, as reported in the review. These enzyme inhibitors show cytotoxic activity against many different cancer cell lines (see Table 1). Marine-derived enzyme inhibitors are considered viable alternatives in pharmaceuticals for replacing synthetic drugs to combat, for example, cancer as well as viral, amtiinflammatory, and neurodegenerative diseases. Nonetheless, further studies will be necessary to investigate the human consumer's well being. The general significance of the topic of this review is well underlined by the number of EU-funded projects, as well as by Horizon 2020, which are aimed at improving the exploitation of marine organisms for drug discovery.