Enzyme Inhibitors from Gorgonians and Soft Corals

For decades, gorgonians and soft corals have been considered promising sources of bioactive compounds, attracting the interest of scientists from different fields. As the most abundant bioactive compounds within these organisms, terpenoids, steroids, and alkaloids have received the highest coverage in the scientific literature. However, enzyme inhibitors, a functional class of bioactive compounds with high potential for industry and biomedicine, have received much less notoriety. Thus, we revised scientific literature (1974–2022) on the field of marine natural products searching for enzyme inhibitors isolated from these taxonomic groups. In this review, we present representative enzyme inhibitors from an enzymological perspective, highlighting, when available, data on specific targets, structures, potencies, mechanisms of inhibition, and physiological roles for these molecules. As most of the characterization studies for the new inhibitors remain incomplete, we also included a methodological section presenting a general strategy to face this goal by accomplishing STRENDA (Standards for Reporting Enzymology Data) project guidelines.


Introduction
Enzyme inhibitors are ubiquitous in nature and, as key regulators of enzyme activity, play pivotal roles in the physiology of all living forms. At a molecular level, endogenous inhibitors regulate enzymatic processes such as metabolism and nutrition, transport, signal transduction, DNA damage repair, apoptosis, cell differentiation, and cell cycle progression, among others. Therefore, many enzyme inhibitors are directly involved in critical events in the context of health and disease [1][2][3][4][5][6][7][8]. Natural enzyme inhibitors also target exogenous enzymes, such as those from invading or competing organisms. Pathogenic viruses, bacteria, fungi, and parasites usually depend on enzymes as virulence factors to guarantee successful colonization and survival. Additionally, digestive enzymes can be used as part of the offensive armamentarium of living organisms in highly crowded environments to avoid competition. Thus, endogenous enzyme inhibitors result in effective defensive immune or anti-predatory mechanisms against such molecular weapons [9,10]. Consequently, natural enzyme inhibitors have received notorious attention as a research subject and have found numerous biomedical [11,12], industrial [13], and environmental applications [14,15].
Marine environments house many different organisms which produce a huge diversity of bioactive molecules. In particular, they have proven to be a prolific source of novel enzyme inhibitors with unique functional features. Inhibitors for all major enzyme classes Figure 1 graphically resumes relevant statistics on the collected information describing enzyme inhibitors from soft corals and gorgonians. We found and processed over 50 scientific reports, published between 1974 and 2022, which contained 127 inhibitor molecules (Supplementary Tables S1 to S3). Concerning their origin, soft corals were the preferred source for the prospection of new inhibitors in this period ( Figure 1A), accounting for about 64% of the reviewed papers. This tendency was confirmed in the last 5 years when the proportion increased to 81% ( Figure 1B). Considering that 206 original papers from this field (i.e., search terms: "natural product" AND "soft coral" OR "gorgonian" NOT "Review") were deposited in PUBMED in the period 1974-2022, the class of enzyme inhibitors seemed to be a predominant one, representing nearly 25% of the total scientific production.
For classification purposes, the target enzymes for the reported inhibitors were grouped into four major classes (i.e., hydrolases, oxidoreductases, transferases, and translocases) according to the reaction type they catalyze. Hydrolases and transferases were, by far, the most abundant among them ( Figure 1C and Table 1).  For classification purposes, the target enzymes for the reported inhibitors were grouped into four major classes (i.e., hydrolases, oxidoreductases, transferases, and translocases) according to the reaction type they catalyze. Hydrolases and transferases were, by far, the most abundant among them ( Figure 1C and Table 1).   Lastly, we grouped inhibitors by targets and tabulated their structures, potencies (predominantly as half-maximal inhibitory concentrations or IC 50 values), and source organism. Inhibitors were classified into six major classes (i.e., terpenes and terpenoids, steroids, peptides, eicosanoids, alkaloids, and hydroquinones), and showed relatively low structural diversity. As expected, more than half of the inhibitory molecules identified in this period from soft corals and gorgonians were terpenes and terpenoids ( Figure 1D), two classes that have historically dominated the literature on coral-derived natural products. Surprisingly, the functional characterization of novel enzyme inhibitors was biased toward cellular toxicity studies and not toward the in-depth understanding of their inhibitory activity. In the few cases where further enzymological information was available (e.g., inhibition mechanism, the value for the thermodynamic constant K i , or target specificity), we included it within the text.
Elastases are serine proteases belonging to the chymotrypsin-like family. Human neutrophil elastase (HNE) is one of the best-characterized elastases, as it plays important roles in inflammation, the elimination and degradation of extracellular pathogens, and the activation of other proteases [65]. As a key inflammation mediator, the discovery and development of new HNE inhibitors have received significant interest [66].
Kunitz-like peptides 1-3 (PcKuz 1-3), isolated from Palythoa caribaeorum, are a few of the peptide-nature molecules described as enzyme inhibitors from soft corals [59]. However, the weak inhibitory activity against the serine proteases trypsin and elastase, and the results of toxicity tests with zebrafish larvae, lead the authors of this study to suggest that these peptides should be considered Kunitz-type neurotoxins rather than protease inhibitors.
Three prostaglandins (41)(42)(43) with inhibitory activity against elastase were isolated from the gorgonian Plexaura homomalla (Table 3 and Figure 3) [60]. These compounds showed similar potency, in terms of inhibition, in the kinetic assays performed in this study, but no IC 50 or K i values were reported. Based on the results of the kinetics assays, it seems that the presence of the carboxyl group is essential for elastase inhibition, since the molecules with an acetyl ester in this position showed no inhibition on the enzyme [60].

3CLpro Enzyme (EC 3.4.22.69)
The main protease of severe acute respiratory syndrome coronavirus is critical for the replication cycle of these viruses [67]. The main protease of SARS-CoV-2 is a 3CLpro cysteine protease that performs the cleavage of 12 nonstructural proteins of the virus. Inhibitors of this protease have proved to be effective in the inhibition of viral replication in cell-based assays [68]. An inhibitor of the SARS-CoV-2 3CLpro protease, described as a cyclized merosesquiterpenoid with a new carbon scaffold and composed by a highly substituted chromene core, was found in Duva florida (Table 3 and Figure 3) [61]. This compound, named Tuaimenal A (44), showed no inhibitory activity against other cysteine proteases (Fasciola hepatica cathepsin L1 and L3, and human cathepsin L) or serine proteases (trypsin, chymotrypsin, and thrombin), suggesting a particular specificity towards 3CLpro protease. Cell-based studies have shown that Tuaimenal A (44) has low toxicity in cells that are sensitive to other protease inhibitors, highlighting the potential of this molecule as a hit in the development of new anti-coronavirus drugs.
Punaglandins are highly functional cyclopentadienone and cyclopentenone prostaglandins, chlorinated at the endocyclic R-carbon position. A group of these molecules, isolated from the soft coral Telesto riisei, were used to investigate the inhibition mechanism previously proposed for dienone prostaglandins [76]. In this study, the punaglandins PNG 2 (55), PNG 3 (56), PNG 4 (57), Z-PNG 4 (58), and PNG 6 (59) showed inhibitory activity against the ubiquitin isopeptidase using in vitro and in vivo assays (Table 4 and Figure 4) [73]. The use of these punaglandins demonstrates that the presence of chloride in endocyclic carbon increases the inhibitory activity in comparison to unchlorinated prostaglandins. Additionally, the results obtained suggest that the inhibitory activity of the prostaglandins is related to the olefin-ketone conjugation and the reactivity of the endocyclic carbon [73].

Figure 7. Oxidoreductase inhibitors isolated from soft corals and gorgonians (compounds 83-91).
Structures include inhibitors of 5α-Reductase, Cytochrome P450 1A, and Tyrosinase.  2.2.2. Cytochrome P450 1A (EC 1.14.14.1) Cytochrome P450 enzymes are a superfamily with high versatility in drug metabolism, detoxification of xenobiotics, and biosynthesis of endogenous compounds [99]. Particularly, cytochrome P4501A is one of the most important enzymes involved in the tumorigenesis induced by environmental pollution [100]. Therefore, this enzyme has been considered as an attractive target for the development of anti-cancer drugs [99]. The cembranoids 12(S)hydroperoxylsarcoph-10-ene (88), 8-epi-sarcophinone (89), and ent-sarcophine (90) were isolated from the Red Sea soft coral Sarcophyton glaucum (Table 6 and Figure 7) [97]. The results of the inhibition assays with these compounds revealed some interesting structure-function relationships. For instance, the differences obtained for the inhibitory potency for ent-sarcophine (90) and sarcophine (33) suggest that the configurations of atoms C1 and C6 are essential for the inhibition of the cytochrome P450 1A [ Tyrosinase is a key enzyme which catalyzes a rate-limiting step in melanin synthesis, and the downregulation of its activity constitutes the most prominent approach for the development of melanogenesis inhibitors [101]. Thus, tyrosinase inhibitors are considered as promising candidates for the development of skin whitening agents [101]. The tyrosinase inhibitor 4-(phenylsulfanyl)butan-2-one (91) is isolated from the Formosan soft coral Cladiella australis (Table 6 and Figure 7). The kinetic characterization of this compound showed that it acts as a noncompetitive inhibitor of the mushroom tyrosinase, with a K i value of 3.45 × 10 −5 M. Additionally, in vitro cell-based assays revealed that 4-(phenylsulfanyl)butan-2-one (91) has low cytotoxicity towards several human cell lines [98].

Tyrosine Kinase p56 lck (TK) (EC 2.7.10.2)
The tyrosine kinase p56 lck is a lymphocyte-specific protein tyrosine kinase that is a member of the Src family of non-receptor protein kinases [102]. This kinase is involved in the phosphorylation of several intracellular signaling proteins such as protein kinase C, phosphoinositide 3-kinase, and Zeta-chain-associated protein kinase 70 [103].

IKKbeta Kinase (EC 2.7.11.10)
IKKbeta is one of the two catalytic units that compose the kinase complex IkB [108]. IKKbeta is involved in nuclear factor-KB signaling, and hence in the pathogenesis and progression of inflammatory diseases [109]. The cembranoids 3,4-epoxy,13-oxo,7E,11Z,15cembratriene (94), and 3,4-epoxy,13-oxo,7E,11E,15-cembratriene (95) were isolated from the soft coral Sarcophyton sp. and both showed inhibitory activity against the IKKbeta kinase ( Table 7 and Figure 8) [105]. In the same study, the carotenoid astaxanthin (96) was isolated from the gorgonian Subergorgia sp., and it showed inhibitory activity towards the IKKbeta kinase [105]. However, it is likely that astaxanthin is not produced by Subergorgia sp., but instead acquired from a marine bacteria or algae [110]. EGFR is a member of the epidermal growth factor receptor family. It was the first member of this family with supporting evidence linking its overexpression with cancer [111]. This relationship has been established for several types of cancer, including laryngeal [112], esophageal [113], and non-small cell lung cancer [114], among others. Thus, EGFR inhibitors are considered as attractive candidates for developing anticancer drugs. The pachycladin A (97) is a diterpenoid isolated from the soft coral Cladiella pachyclados and was able to inhibit the kinase enzymatic activity of the EGFR kinase 8 (Table 7 and Figure 8) [106]. The inhibition of pachycladin A (97) seems to be selective towards EGFR kinase since no inhibition was detected against the two other kinases from the EGFR family (HER2 and HER4).

Compound IC 50 (µM) Source Classification Reference
Human tumor-related protein kinases  Casitas B-lineage lymphoma proto-oncogene B (Cbl-b) is an E3 ubiquitin ligase that acts as an important regulator of the immune response [125]. Targeting this enzyme is a promising approach for the treatment of autoimmune diseases and cancer [126,127]. The Sinularamides A−G (107−113), isolated from the soft coral Sinularia sp., are a group of diterpenoids that inhibits Cbl-b enzyme [122]. The most potent of these compounds was the sinularamide C (109), displaying an IC 50 value in the low micromolar range (Table 8 and Figure 9).

Farnesyl Protein Transferase (EC 2.5.1.58)
The farnesyl protein transferase is an enzyme that adds a 15-carbon isoprenoid to a cysteine amino acid of Ras protein [128]. Oncogenic mutations of Ras protein are quite common in cancer, making the protein that is involved in its post-transcriptional modification a promising way to block Ras signal transduction [129,130]. The molecule 1aR,4E,8E,11S,11aR,14aS,14bS)-1a,5,9,14a-tetramethyl-12-methylene-13-oxo-1a,2,3,6,7,10, 11, 11a,12,13,14a,14b-dodecahydrooxireno [2 ,3 :13,14]cyclotetradeca [1,2-b]furan-11-yl acetate (114), with inhibitory activity towards recombinant human farnesyl protein transferase, was isolated from the soft coral Lobophytum cristagalli [123]. Kinetic assays suggest that this inhibitor competes with the Ras protein, substrate of the farnesyl protein transferase. The apparent K i value determined in this kinetic characterization was 0.17 µM. However, the same inhibitor is noncompetitive with respect to the farnesyl pyrophosphate substrate. In the same study, this compound also inhibited the enzyme geranyl protein transferase-1, closely related to the farnesyl protein transferase, but with lower potency (IC 50  Glutathione S-transferases (GST) are a family of enzymes that catalyze the conjugation of glutathione to a wide number of xenobiotics, making them more hydrophilic and facilitating their elimination [131]. Although GST protects the cell from toxic products, it also reduces the effectiveness of certain anticancer drugs [132]. The use of inhibitors of GST could, therefore, increase the sensitivity of tumor cells to anticancer drugs [133].
Crude and aqueous extracts of the gorgonian Plexaura homomalla have shown inhibitory activity towards the GST enzyme from the gastropod Cyphoma gibbosum. The structural analysis of these extracts revealed the presence of several series of prostaglandins (115)(116)(117)(118)(119). The use of commercially available prostaglandins that represents the diversity of classes found in P. homomalla confirmed the capacity of the molecules 15(S)-PGA2 (115) (Table 8 and Figure 9). The results of the enzymatic assays revealed that compounds with the cyclopentenone ring showed the highest inhibitory activity [124].

Translocases
Vacuolar H(+)-pyrophosphatase is an electronic proton pump present in most land plants and in other organisms such as algae, bacteria, protozoa, and archaebacteria [134]. In plants, this enzyme participates in the cytosolic hydrolysis of PPi and in vacuole acidification [135]. Specific inhibitors of the enzyme would be useful for investigating its physiological role and its biochemical characteristics. Acylspermidines A (120), B (121), C (122), D (123), and E (124) (Figure 10) from the soft coral Sinularia sp. inhibit plant vacuolar H(+)-pyrophosphatase from Vigna radiata cv. Wilczek [136]. These acylspermidine derivates showed a noncompetitive inhibition toward the vacuolar H(+)-pyrophosphatase. However, these compounds did not inhibit the vacuolar H+-ATPase, plasma membrane H+-ATPase, mitochondrial ATPase, or cytosolic PPase, suggesting a specific inhibitory activity against the vacuolar H(+)-pyrophosphatase. Glutathione S-transferases (GST) are a family of enzymes that catalyze the conjugation of glutathione to a wide number of xenobiotics, making them more hydrophilic and facilitating their elimination [131]. Although GST protects the cell from toxic products, it also reduces the effectiveness of certain anticancer drugs [132]. The use of inhibitors of GST could, therefore, increase the sensitivity of tumor cells to anticancer drugs [133].
Crude and aqueous extracts of the gorgonian Plexaura homomalla have shown inhibitory activity towards the GST enzyme from the gastropod Cyphoma gibbosum. The structural analysis of these extracts revealed the presence of several series of prostaglandins (115)(116)(117)(118)(119). The use of commercially available prostaglandins that represents the diversity of classes found in P. homomalla confirmed the capacity of the molecules 15(S)-PGA2 (115) (Table 8 and Figure 9). The results of the enzymatic assays revealed that compounds with the cyclopentenone ring showed the highest inhibitory activity [124].

Translocases
Vacuolar H(+)-pyrophosphatase is an electronic proton pump present in most land plants and in other organisms such as algae, bacteria, protozoa, and archaebacteria [134]. In plants, this enzyme participates in the cytosolic hydrolysis of PPi and in vacuole acidification [135]. Specific inhibitors of the enzyme would be useful for investigating its physiological role and its biochemical characteristics. Acylspermidines A (120), B (121), C (122), D (123), and E (124) (Figure 10) from the soft coral Sinularia sp. inhibit plant vacuolar H(+)pyrophosphatase from Vigna radiata cv. Wilczek [136]. These acylspermidine derivates showed a noncompetitive inhibition toward the vacuolar H(+)-pyrophosphatase. However, these compounds did not inhibit the vacuolar H+-ATPase, plasma membrane H+-ATPase, mitochondrial ATPase, or cytosolic PPase, suggesting a specific inhibitory activity against the vacuolar H(+)-pyrophosphatase.

Walking the Whole Path to Describe an Enzyme Inhibitory Activity: What Next, after Screening and Structure Elucidation?
Previous sections reveal that, despite the number of enzyme inhibitors identified in the last five decades from gorgonians and soft corals, none of them, to date, possess a complete enzymological characterization. Remarkably, in many of these works, not even an IC 50 value is calculated, but only an inhibition percentage at a fixed inhibitor concentration. This trend, which is extendable to other natural sources, reflects both (i) the lack of a uniform procedure for the enzymological characterization of inhibitors from marine organisms and (ii) the challenge that this goal represents for users unfamiliar with enzymology.
The STRENDA project provides guides for the minimal information set required for reporting enzymological data [38,39,137]. For inhibitors, report requirements include reversibility and time-dependency, inhibition type, Ki value, and the method used for its estimation [37]. Using these parameters as a reference, many reports of enzyme inhibitors, and particularly those from marine sources, appear noticeably incomplete. Although several factors might be involved, we believe that the scarcity of fully characterized inhibitors might be due to the lack of expertise of many laboratories in studying this bioactivity. Therefore, we believe that a brief description of critical steps might encourage the study of a higher number of novel enzyme inhibitors from this prominent source. Figure 11 presents a general strategy for characterizing new enzyme inhibitors independent of their nature and origin, adapted from the one proposed by Copeland [138]. Although it admits some flexibility in the order of steps, the selected approach is, in our opinion, intuitive and the most convenient to implement in unexperienced laboratories. In brief, the strategy consists of five steps: (1) obtaining a preliminary assessment of inhibitory potency (i.e., the range of molar concentrations in which it effectively inhibits the target enzyme) by determining half-maximal inhibitory concentration (IC 50 ); (2) determining inhibition reversibility and temporal dependency; (3) elucidation of inhibition type; (4) estimation of K i value; and (5) a specificity validation step.
3. Walking the Whole Path to Describe an Enzyme Inhibitory Activity: What Next, after Screening and Structure Elucidation?
Previous sections reveal that, despite the number of enzyme inhibitors identified in the last five decades from gorgonians and soft corals, none of them, to date, possess a complete enzymological characterization. Remarkably, in many of these works, not even an IC50 value is calculated, but only an inhibition percentage at a fixed inhibitor concentration. This trend, which is extendable to other natural sources, reflects both (i) the lack of a uniform procedure for the enzymological characterization of inhibitors from marine organisms and (ii) the challenge that this goal represents for users unfamiliar with enzymology.
The STRENDA project provides guides for the minimal information set required for reporting enzymological data [38,39,137]. For inhibitors, report requirements include reversibility and time-dependency, inhibition type, Ki value, and the method used for its estimation [37]. Using these parameters as a reference, many reports of enzyme inhibitors, and particularly those from marine sources, appear noticeably incomplete. Although several factors might be involved, we believe that the scarcity of fully characterized inhibitors might be due to the lack of expertise of many laboratories in studying this bioactivity. Therefore, we believe that a brief description of critical steps might encourage the study of a higher number of novel enzyme inhibitors from this prominent source. Figure 11 presents a general strategy for characterizing new enzyme inhibitors independent of their nature and origin, adapted from the one proposed by Copeland [138]. Although it admits some flexibility in the order of steps, the selected approach is, in our opinion, intuitive and the most convenient to implement in unexperienced laboratories. In brief, the strategy consists of five steps: (1) obtaining a preliminary assessment of inhibitory potency (i.e., the range of molar concentrations in which it effectively inhibits the target enzyme) by determining half-maximal inhibitory concentration (IC50); (2) determining inhibition reversibility and temporal dependency; (3) elucidation of inhibition type; (4) estimation of Ki value; and (5) a specificity validation step. Figure 11. Proposed strategy for the characterization of reversible enzyme inhibitors from marine organisms according to STRENDA guidelines. The strategy is presented as a flowchart with five sequential steps. As alternative approaches or methodologies are possible in each step, two specific sequences are proposed according to the properties of the inhibitor (e.g., classic, rapid reversible inhibitor vs. tight-binding, slow reversible inhibitor).
Determining a preliminary estimate of inhibitor potency (Step 1) is critical, as this determines the most appropriate approach for further characterization steps ( Figure 11). After estimating the IC50 value by quantifying the inhibition percentage at different inhibitor concentrations, the assessed inhibitors can be classified as classic (i.e., IC50 >> [E]0) or tight binding (i.e., IC50 ≈ [E]0). Importantly, caution is recommended when comparing IC50 Figure 11. Proposed strategy for the characterization of reversible enzyme inhibitors from marine organisms according to STRENDA guidelines. The strategy is presented as a flowchart with five sequential steps. As alternative approaches or methodologies are possible in each step, two specific sequences are proposed according to the properties of the inhibitor (e.g., classic, rapid reversible inhibitor vs. tight-binding, slow reversible inhibitor).
Determining a preliminary estimate of inhibitor potency (Step 1) is critical, as this determines the most appropriate approach for further characterization steps ( Figure 11). After estimating the IC 50 value by quantifying the inhibition percentage at different inhibitor concentrations, the assessed inhibitors can be classified as classic (i.e., IC 50 >> [E] 0 ) or tight binding (i.e., IC 50 ≈ [E] 0 ). Importantly, caution is recommended when comparing IC 50 values, as they reflect not only the potency of the inhibitor but also the experimental conditions used for its estimation [139].
Step 2 consists of assessing the reversibility of the enzyme-inhibitor interaction. This property can be determined by the jump dilution assay [138][139][140] or alternative interactionbased techniques [35,36,141,142]. Although irreversible and pseudo-irreversible inhibitors exist in nature [143][144][145][146], most of the enzyme inhibitors described so far from marine organisms belong to the reversible class. Other relevant aspects of inhibition, such as association (i.e., time-dependent inhibition) and dissociation kinetics, are also investigated at this stage.

Elucidation of inhibition type (
Step 3) and the estimation of Ki value (Step 4) are typically performed simultaneously by measuring enzyme velocity while varying substrate and inhibitor concentrations. The potency of competitive, noncompetitive, and uncompetitive inhibitors variate distinctively as substrate concentration increases [139], a fact exploited to identify their inhibition mode [138,140]. Once the inhibition modality has been elucidated, diverse mathematical approaches are available to estimate the Ki value for classical inhibitors [147][148][149]. On the other hand, determining Ki values for tight-binding inhibitors requires a specific methodology [150,151], as many of the assumptions applied to derive the equations for classic inhibitors are no longer valid in such systems.
The strategy is completed with a specificity validation step (Step 5) to discard unspecific actuators or PAINS (pan-assay interference compounds), which can act over many different proteins or interfere with bioassays through diverse mechanisms [152,153]. As these compounds can occur in nature [154], the specificity of novel inhibitors should be assessed against related and unrelated off-target reporters [155]. Additionally, experimental conditions can be conveniently manipulated (e.g., by adding to the reaction mix appropriate amounts of detergents such as Triton X-100) to minimize the occurrence of aggregators, the most common cause of artificial enzyme inhibition during screenings [155][156][157][158][159].
Due to the limited scope of this issue and for the sake of space, the presented strategy is not an exhaustive guide for the functional characterization of new inhibitors. In contrast, it aims to summarize relevant concepts and set a framework to address this goal, in practice burdensome enough to cause a halt in the study of potentially interesting enzyme inhibitors. The infrastructure required for performing most enzymatic reactions is readily available in many academic institutions. Numerous substrate and enzyme preparations are commercially available at affordable prices and basic enzymology is part of almost every formation program in biological sciences. In addition, many enzymes are considered important biomolecules for industrial, biomedical, and environmental applications, making their activity modulation by specific inhibitors a topic of particular interest. Paradoxically, in-depth enzymological characterization of inhibitors seems to be the prerogative of only a few laboratories, resulting in the accumulation of reports describing bioactivity prospection in natural extracts with no follow-up. This is particularly true for, but not exclusive to, organisms of marine origin, which have constituted an abundant source of enzyme inhibitors with unique properties. Therefore, we hope this effort can contribute to democratizing and popularizing the procedures required in enzymological practice to minimally characterize, according to STRENDA suggestions, new enzyme inhibitors from marine organisms. In addition, this work might also help to propagate the message of the STRENDA project, focusing on the uniformity, quality, and completeness of experimental data, and hence reproducibility, in enzymological practice. Adherence to these standards should significantly facilitate relaunching of high-quality enzymological research in the context of marine organisms.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md21020104/s1, Table S1: Complete dataset of enzyme inhibitors included in the manuscript., Table S2: Dataset of enzyme inhibitors isolated from soft corals, Table S3: Dataset of enzyme inhibitors isolated from gorgonians. For convenience, the three tables were included as separate sheets in a single excel file.