Screening of Protease Inhibitory Activity in Aqueous Extracts of Marine Invertebrates from Cuban Coast

Abstract

Protease inhibitors have been isolated from many variable sources; however, the need to identify and characterize new molecules has increased with the discovery of new therapeutic targets and the lack of specificity of already identified compounds with inhibitory activity. The goal of this work was to search for inhibitory activity against four proteolytic enzymes already recognized as therapeutic targets: human neutrophil elastase, dipeptidyl peptidase IV, subtilisin from Bacillus licheniformis and cathepsin K in selected marine invertebrates from the Caribbean Sea. A systematic screening was carried out with selected aqueous extracts belonging to 20 species from seven different phyla: Annelida, Bryozoa, Chordata, Cnidaria, Equinodermata, Mollusca and Porifera, all collected at the coast of Havana (Cuba). All extracts showing initial inhibitory activity were characterized in terms of IC50 values and specific inhibitory activity (SIA). Model enzymes were used in the case of human neutrophil elastase (porcine pancreatic elastase) and cathepsin K (papain) for the screening and all positive results were confirmed by testing toward the therapeutic targets. Ten extracts were identified showing inhibitory activity against human neutrophil elastase, for which the most promising values were obtained for Nerita peloronta. Only one extract, Bunodosoma granulifera, showed inhibitory activity against dipeptidyl peptidase IV with rather poor values of IC50 and SIA. Seven extracts showed inhibitory activity against B. licheniformis subtilisin with very good IC50 and SIA values for Lissodendorix isodyctialis, Cenchritis muricatus, and N. peloronta. Finally, eight extracts were positive for cathepsin K with almost similar parameters values among them. All these results confirmed the richness and potential of the marine invertebrate's fauna and indicated new promising sources for the identification of natural compounds with potential application in therapeutics.

From Naturally-Sourced Protease Inhibitors to New Treatments for Fungal Infections

“…Invertebrates are a heterogeneous group of animals (about 1.3 million species) found ubiquitously within the environment, requiring strong defenses (e.g., production of chemicals) to adapt and survive against predators and pathogens, including protease inhibitors as self-defense systems [78,79]. For instance, many compounds with therapeutic potential detected from invertebrates show inhibition profiles against proteases with biotechnological and biomedical interest; although, many more remain to be studied [80][81][82][83]. Here, we present protease inhibitors derived from invertebrates and explore their described antimicrobial properties (Table 2).…”

“…Mollusks present a wealth of natural compounds displaying antimicrobial activity, including 19 within the global marine pharmaceutical clinical pipeline and four approved by the US Food and Drug Administration to date [94]. Notably, over half of the secondary metabolites produced by mollusks have yet to be evaluated for bioactivity, representing a plethora of new avenues to pursue for in vitro, in vivo, and clinical studies [83,95]. For instance, protease inhibitors have been reported from oysters, such as Crassostrea gigas peptides, which are competitive inhibitors of HIV-1 protease with an inhibitory constant (k i ) between 10 and 13 nM [87].…”

Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families

“…Due to the fact that IC 50 for L. isodyctialis and T. ventricosus were lower on the FcB1 strain of P. falciparum than on the PfA-M17 recombinant enzyme, it is very likely that these extracts may contain not only PfA-M17 inhibitors but also other compounds active on other malarial targets. For example, for L. isodyctialis , inhibition of subtilisin from Bacillus licheniformes with an IC 50 value of 3 µg/mL was described by Gonzalez et al [ 39 ]. Another interesting result is the selectivity for rPfA-M17 regarding hLAP of Nerita versicolor extract showing effects on parasite growth with IC 50 in the same order of enzyme inhibition, indicating that this species is also attractive.…”

“…These features can be attributed to varied environmental conditions, such as access to/lack of light, high pressure, aqueous environment, ionic concentration, pH and temperature changes, scarcity of nutrients, and restricted living spaces. Marine organisms are an abundant source of bioactive molecules (including saccharides, polysaccharides, peptides, proteins, polyketides, polyphenolic compounds, sterol-like products, alkaloids, quinones, and quinolones, among others), such as toxins [ 20 , 21 ], antimicrobial peptides [ 22 , 23 ], antiviral compounds [ 24 ], enzymes and enzyme inhibitors [ 25 , 26 , 27 , 28 ], and particularly peptidases [ 29 , 30 ] and peptidase inhibitors of almost all mechanistic classes [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ]. These bioactive molecules have a great diversity of chemical structures, high potency, and diverse specificities, especially the inhibitors of metalloenzymes ( Figure 2 , Figure 3 and Figure 4 ) [ 37 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ].…”