marine drugs Review Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families Isel Pascual Alonso 1, *, Fabiola Almeida García 1 , Mario Ernesto Valdés Tresanco 1,2 , Yarini Arrebola Sánchez 1 , Daniel Ojeda del Sol 1 , Belinda Sánchez Ramírez 3 , Isabelle Florent 4 , Marjorie Schmitt 5 and Francesc Xavier Avilés 6, * 1 Center for Protein Studies, Faculty of Biology, University of Havana, Havana 10400, Cuba; almeidafabiola0406@gmail.com (F.A.G.); marioe911116@gmail.com (M.E.V.T.); yarini1985@gmail.com (Y.A.S.); dojeda9711@gmail.com (D.O.d.S.) 2 Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada 3 Centro de Inmunología Molecular, Habana 11600, Cuba; belinda@cim.sld.cu 4 Unité Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR7245), Muséum National d’Histoire Naturelle, CNRS, CP52, 57 Rue Cuvier, 75005 Paris, France; isabelle.florent@mnhn.fr 5 Université de Haute-Alsace, Université de Strasbourg, CNRS, LIMA UMR 7042, 68000 Mulhouse, France; marjorie.schmitt@uha.fr 6 Institute for Biotechnology and Biomedicine and Department of Biochemistry, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: iselpascual@yahoo.es (I.P.A.); francescxavier.aviles@uab.cat (F.X.A.) Abstract: Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Pep- tidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many Citation: Pascual Alonso, I.; Almeida biochemical and physiological processes. They are also involved in several pathophysiological pro- García, F.; Valdés Tresanco, M.E.; cesses. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids Arrebola Sánchez, Y.; Ojeda del Sol, of proteins or peptide substrates. They are distributed in many phyla and play critical roles in D.; Sánchez Ramírez, B.; Florent, I.; physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and Schmitt, M.; Avilés, F.X. Marine M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing Invertebrates: A Promissory Still hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development Unexplored Source of Inhibitors of of therapeutic agents for human diseases, including cancer, hypertension, central nervous system Biomedically Relevant Metallo disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as Aminopeptidases Belonging to the malaria. The relevance of aminopeptidases has driven the search and identification of potent and M1 and M17 Families. Mar. Drugs selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, 2023, 21, 279. https://doi.org/ 10.3390/md21050279 and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an impor- tant and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with Academic Editor: Claudiu T. foreseen biomedical applications in human diseases. The results reviewed in the present contribution Supuran support and encourage further studies with inhibitors isolated from marine invertebrates in different Received: 27 March 2023 biomedical models associated with the activity of these families of exopeptidases. Revised: 25 April 2023 Accepted: 26 April 2023 Keywords: aminopeptidase; aminopeptidase N; aminopeptidase A; TRH-degrading ectoenzyme; Published: 28 April 2023 leucyl aminopeptidase; enzyme inhibitors; drug-oriented inhibitors; marine invertebrates Copyright: © 2023 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. This article is an open access article Proteolytic enzymes, known as peptidases or proteases, are critical in all living or- distributed under the terms and ganisms [1]. Proteases can act as exo- and/or endopeptidases. They are segregated in conditions of the Creative Commons classes that strongly depend on the chemical nature of the groups involved in catalysis. Attribution (CC BY) license (https:// The recognized mechanistic classes are aspartic, cysteine, glutamic, metallo, asparagine, creativecommons.org/licenses/by/ mixed, serine, threonine, and a group dedicated to unknown catalytic type [1]. 4.0/). Mar. Drugs 2023, 21, 279. https://doi.org/10.3390/md21050279 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2023, 21, x 2 of 37 Mar. Drugs 2023, 21, 279 The recognized mechanistic classes are aspartic, cysteine, glutamic, metallo, asparagine, 2 of 37 mixed, serine, threonine, and a group dedicated to unknown catalytic type [1]. Peptidases are one of the most abundant groups of enzymes in living organisms. Thus,Peptidases in mammals, aremore one ofthan thesix hundred most genesgroups abundant have been assignedin of enzymes toliving them. organisms. They con- Thus, trol theinactivation, mammals,synthesis, more than andsixturnover hundredofgenes haveand proteins been assigned regulate mostto biochemical them. They control and the activation, physiological synthesis, processes, suchand turnover offertilization, as digestion, proteins and regulate growth, most biochemical differentiation, cell and physiological processes, signaling/migration, such asdefense, immunological digestion, fertilization, wound healing,growth, differentiation, and apoptosis [1–3]. They cell signaling/migration, immunological defense, wound healing, and apoptosis are consequently major regulators of homeostasis, ageing, and different human diseases [1–3]. They are consequently such major regulators as cancer, hypertension, of homeostasis, diabetes, ageing,neurodegeneration, inflammation, and different human diseases and Alz- such as cancer, heimer’s disease,hypertension, among others diabetes, inflammation, [4–6]. Proteases neurodegeneration, are also essential for theand Alzheimer’s propagation of disease, among infectious agents,others being[4–6]. majorProteases contributorsare also essential forinthe of pathogenesis propagation several infectious of infectious diseases, agents, being including major contributors the current coronavirusof pathogenesis emergent in several pandemic SARSinfectious COVID-19diseases, [1,7–14].including the current coronavirus emergent pandemic SARS COVID-19 [1,7–14]. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids Among peptidases, of proteins or peptide aminopeptidases substrates. Theycatalyze the cleavage are distributed of the in many N-terminal phyla and play amino crit- acids of proteins or peptide substrates. They are distributed in many phyla ical roles in physiology and pathophysiology [1,8,9,15]. In mammals, they have a wide- and play critical roles incellular spread physiology and pathophysiology distribution in various organs [1,8,9,15]. and are Infound mammals,withinthey cellshave a widespread in many subcel- cellular lular distribution organelles, in theincytoplasm, various organs and are as integral found within membrane cellsorinare proteins, many subcellular exposed or se- organelles, in the cytoplasm, as integral membrane proteins, or are exposed creted extracellularly [8,16–18]. They are mainly metallopeptidases belonging to differ- or secreted extracellularly ent families such [8,16–18]. as M1 andTheyM17, are mainly metallopeptidases although belonging cysteine and serine to different peptidases families are also in- such as M1 and M17, although cysteine and serine peptidases cluded in this group [1]. Among the best representatives of aminopeptidases thatare also included in this are group [1].inAmong currently the focustheofbest representatives biomedical of aminopeptidases investigation, we can pinpoint that theareM1currently in the family neutral focus of biomedical investigation, we can pinpoint the M1 family aminopeptidase (APN, EC 3.4.11.2), glutamyl aminopeptidase (APA EC 3.4.11.7) and neutral aminopeptidase (APN, EC 3.4.11.2), glutamyl thyrotropin-releasing aminopeptidase hormone-degrading (APA EC 3.4.11.7) ectoenzyme (TRH-DE; and ECthyrotropin-releasing 3.4.19.6), and M17 hormone-degrading ectoenzyme (TRH-DE; EC 3.4.19.6), and M17 neutral aminopeptidase, neutral aminopeptidase, also known as leucyl aminopeptidase (LAP, EC 3.4.11.21) (Fig- also known as leucyl aminopeptidase (LAP, EC 3.4.11.21) (Figure 1). These enzymes are ure 1). These enzymes are involved in multiple physiological processes as well as in can- involved in multiple physiological processes as well as in cancer, hypertension, central cer, hypertension, central nervous system disorders, inflammation, immune system dis- nervous system disorders, inflammation, immune system disorders, skin pathologies, and orders, skin pathologies, and infectious diseases, such as malaria, and are current targets infectious diseases, such as malaria, and are current targets for the development of new for the development of new therapeutic drugs [8,9,19]. therapeutic drugs [8,9,19]. Figure Figure 1. 1. Cartoon Cartoon representation of the representation of the3D 3Dstructure structureofofthe thethree three aminopeptidases aminopeptidases focused focused on on by by the the present contribution: (A) human aminopeptidase N (PDB ID: 4fyq), (B) human aminopepti- present contribution: (A) human aminopeptidase N (PDB ID: 4fyq), (B) human aminopeptidase A dase A (PDB ID: 4kx7), and (C) Plasmodium falciparum M17 aminopeptidase PfA-M17 (PDB ID: (PDB ID: 4kx7), and (C) Plasmodium falciparum M17 aminopeptidase PfA-M17 (PDB ID: 4r76). Colors: 4r76). Colors: alpha-helices (cyan), beta sheets (warm pink), and loops (salmon). The zinc atoms alpha-helices are (cyan), shown as gray beta sheets spheres (warmin highlighted pink), and loops a yellow box. (salmon). The zinc atoms are shown as gray spheres highlighted in a yellow box. Marine habitats are an extraordinary source of structurally complex bioactive me- Marine habitats are an extraordinary source of structurally complex bioactive metabo- tabolites, characterized by unique functions with marked biological activities and pol- lites, characterized by unique functions with marked biological activities and polished ished through evolution. These features can be attributed to varied environmental con- through evolution. 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 Mar. Drugs 2023, 21, x 3 of 37 Mar. Drugs 2023, 21, 279 ditions, such as access to/lack of light, high pressure, aqueous environment, ionic con- 3 of 37 centration, pH and temperature changes, scarcity of nutrients, and restricted living spac- es. Marine organisms are an abundant source of bioactive molecules (including saccha- rides, polysaccharides, organisms peptides, are an abundant proteins, source polyketides, of bioactive molecules polyphenolic (including compounds, sterol- saccharides, polysac- like products, alkaloids, quinones, and quinolones, among others), such as charides, peptides, proteins, polyketides, polyphenolic compounds, sterol-like products, al-toxins [20,21], antimicrobial kaloids, peptides quinones, [22,23], antiviral and quinolones, amongcompounds others), such [24], enzymes as toxins andantimicrobial [20,21], enzyme inhibi- pep- tors [25–28], and particularly peptidases [29,30] and peptidase tides [22,23], antiviral compounds [24], enzymes and enzyme inhibitors [25–28], inhibitors of almost all and partic- mechanistic classes [31–42]. These bioactive molecules have a great diversity ularly peptidases [29,30] and peptidase inhibitors of almost all mechanistic classes [31–42]. of chemical structures, These high molecules bioactive potency, andhavediverse a greatspecificities, diversity ofespecially the inhibitors chemical structures, highof potency, metalloen- and zymes (Figures 2–4) [37,43–54]. These inhibitory biomolecules are frequently diverse specificities, especially the inhibitors of metalloenzymes (Figures 2–4) [37,43–54]. involved in nutrition, homeostasis, reproduction, and communication of marine These inhibitory biomolecules are frequently involved in nutrition, homeostasis, reproduc-organisms [27]. Additionally, tion, the high concentration and communication of coexisting of marine organisms organisms inthe [27]. Additionally, a limited area also high concentration makes them very competitive and complex, resulting in the development of coexisting organisms in a limited area also makes them very competitive and complex, of adaptations and behaviors resulting in theaimed at safeguarding development the species. of adaptations andSince most invertebrates behaviors (e.g., sponges, aimed at safeguarding the bryozoans, tunicates, cnidarians, and mollusca snails, among others) lack species. Since most invertebrates (e.g., sponges, bryozoans, tunicates, cnidarians, and morphological defense structures, mollusca peptidase snails, among others)inhibitors are also part lack morphological of mechanisms defense structures,related withinhibitors peptidase protec- are also part of mechanisms related with protection against predators, infection, we tion against predators, infection, and competition [55]. In the present contribution, and review and summarize competition [55]. In thethepresent status of metalloaminopeptidase contribution, we review and inhibitors isolated summarize thefrom ma-of status rine organisms with a focus metalloaminopeptidase on the isolated inhibitors M1 and fromM17 families of enzymeswith marine organisms targeted a focusin on biomed- the M1 ical studies. and M17 families of enzymes targeted in biomedical studies. Figure 2. Figure 2. Structures Structures of of low low molecular molecular weight weight non-peptidic non-peptidic protease protease inhibitors inhibitors of ofdifferent differentmechanis- mecha- nistic classes isolated from marine organisms. Serine peptidase inhibitors: (A) cicloteonamide A tic classes isolated from marine organisms. Serine peptidase inhibitors: (A) cicloteonamide A [56], [56], (B) general structure of cicloteonamide E [56], (C) aeruginosine 298-A, (D) dinosine, (E) oscil- (B) general structure of cicloteonamide E [56], (C) aeruginosine 298-A, (D) dinosine, (E) oscilarina [57]; arina [57]; cystein peptidase inhibitors: (F) tokaramida A [45], (G) discorhabdina P [58], (H) seco- cystein batzellinapeptidase inhibitors: A [59]; aspartic (F) tokaramida peptidases inhibitors:A(I) [45], (G) discorhabdina P [58], N,N-dimetiltiocarbamate, (J) 6 (H) secobatzellina Br-aplisinopsine A [59]; [60]; aspartic peptidases inhibitors: metalloendopeptidase inhibitors:(I) (K)N,N-dimetiltiocarbamate, (J) 6 Br-aplisinopsine jaspisin [43], (L) 1-12-hidroxioctadecanil [60];sul- sodium met- alloendopeptidase inhibitors:sulphate phate [61], (M) calisponginol (K) jaspisin [43],Ageladine [46], (N) (L) 1-12-hidroxioctadecanil A [47], (O) Eckol [62].sodium sulphate [61], (M) calisponginol sulphate [46], (N) Ageladine A [47], (O) Eckol [62]. Mar. Drugs 2023, 21, 279 Mar. Drugs 2023, 21, x 4 of 37 4 of 37 Mar. Drugs 2023, 21, x 4 of 37 Figure Figure 3. Structures of inhibitors isolated from marine invertebrates in complex with target prote- 3. Structures of inhibitors isolated from marine invertebrates in complex with target proteases. ases. (A) ShPI-1 isolated from the sea anemone Stichodactyla helianthus in complex with bovine (A) ShPI-1 isolated trypsin (PDB from Figure the(B) ID: 3m7q), seaShPI-1 anemone 3. Structures Stichodactyla ofininhibitors complex with bovine isolated helianthus from in complex chymotrypsin with (PDB ID: 3t62), marine invertebrates bovine (C) ShPI- in complex trypsin (PDB with target prote- 1 in complex ID: 3m7q), (B) ShPI-1with pancreatic in complex elastase, with and (PDB bovine ID: 3m7Q), chymotrypsin (D) SmCI (PDB isolated ID: from 3t62), ases. (A) ShPI-1 isolated from the sea anemone Stichodactyla helianthus in complex marine (C) an- ShPI-1 inwith complex bovine nelide Sabellastarte trypsin magnifica (PDB ID: in complex 3m7q), (B) with human ShPI-1 in carboxypeptidase complex with bovine A4 (hCPA4) [51]. chymotrypsin Resi- (PDB ID: 3t62), (C) ShPI- with pancreatic elastase, and (PDB ID: 3m7Q), (D) SmCI isolated from marine annelide dues interacting with the S1 subsite have been highlighted as sticks. Proteases and inhibitors are Sabellastarte 1 in complex with pancreatic elastase, and (PDB ID: 3m7Q), (D) SmCI isolated from marine an- magnifica in complex colored with human cyan and orange, carboxypeptidase respectively. A4 (hCPA4) The zinc atom in hCPA4 is shown[51]. Residues as a gray sphere. interacting with the nelide Sabellastarte magnifica in complex with human carboxypeptidase A4 (hCPA4) [51]. Resi- S1 subsite have duesbeeninteracting highlighted as sticks. Proteases and inhibitors with the S1 subsite have been highlighted as sticks. are colored cyan Proteases andand orange, inhibitors are respectively. Thecolored cyan and zinc atom orange, is in hCPA4 respectively. shown asThe zinc sphere. a gray atom in hCPA4 is shown as a gray sphere. Figure 4. Structure of CmPI-II (a serine protease inhibitor isolated from the marine snail Cenchritis muricatus). (A) Family of the 15 lowest energy structures of CmPI-II obtained by NMR. (B) Rib- Figure Figure 4. Structure 4. Structure of CmPI-II (aof CmPI-II serine (a serineinhibitor protease protease inhibitor isolatedisolated from from the the marine marine snailsnail Cenchritis Cenchritis muricatus). (A) Family of the 15 lowest energy structures of CmPI-II obtained by NMR. (B) Rib- muricatus). (A) Family of the 15 lowest energy structures of CmPI-II obtained by NMR. (B) Ribbons- like representation of the lowest energy structure. The β strands (β1–β3) and the α helix are repre- sented in red and blue, respectively. Orange parts represent the hydrophobic nucleus of the protein and green shows disulfide bridges. (C) Prediction of the 3D structure of the CmPI-II/subtilisin A complex. CmPI-II is shown in red and subtilisin A in blue [53]. Courtesy of Prof. Aymara Cabrera Muñoz. Mar. Drugs 2023, 21, 279 5 of 37 2. M1 and M17 Metalloexopeptidase Inhibitors Isolated from Marine Invertebrates 2.1. Metallopeptidases: General Characteristics and Classification Metallopeptidases constitute the most diverse catalytic type within proteases, since they include both endopeptidases and exopeptidases, cytosolic enzymes, and others that are secreted to the outside of cells, as well as enzymes associated with the plasma membrane and cell organelles. They are widely distributed in all forms of life such as viruses, bacteria, fungi, and plant and animal cells, indicating the important role they play in biological processes. Metalloproteases are included among the hydrolases in which the nucleophilic attack on the peptide bond is mediated by a water molecule. This is a feature they share with aspartic-type peptidases, but in metallopeptidases, a divalent metal cation activates the water molecule [63]. This divalent cation is usually zinc (Zn2+ ) but can sometimes be cobalt (Co2+ ) or manganese (Mn2+ ). The metal ion is held in the protein structure by amino acids that act as ligands. Metallopeptidases can be divided into two large groups based on the number of metal ions required for catalysis. In many metallopeptidases, only one metal ion is required, which frequently is Zn2+ ; however, there is another group of families in which two cocat- alytically acting metal ions are required. Within this group, there are families that have two Zn2+ ions and all the families in which Co2+ or Mn2+ are essential for catalysis. In families where only one metal ion acts, three amino acid residues are required to act as metal ligand coordinators, and in families with cocatalytic ions, only five amino acids are required since one of them acts as a ligand coordinator for both metal ions. All metallopeptidases with cocatalytic ions are exopeptidases, while metallopeptidases with a single metal ion can be both exo- and endopeptidases. Various attempts have been made to classify proteases. The most accepted today is the one initially proposed by Rawlings and Barrett [64,65] that is continuously updated at (https://www.ebi.ac.uk/merops/, accessed on 9 January 2023) [1]. From the general classification of the nine mechanistic classes, they first group the enzymes of each class into families. A family is defined as a group of (homologous) peptidases in which each member shows significant amino acid sequence identity with the “type enzyme” or at least with another member of the family homologous to the type enzyme, mainly in the region of the peptidase that is related to its catalytic activity. The selection criteria used by these authors were very strict, in such a way that they guarantee a common ancestor for the members of a family, which are, therefore, homologous according to the definition of Reeck et al. [66]. Each family is named with a letter denoting the catalytic type (Example: M for metallopeptidases), followed by an arbitrarily assigned number. At a higher level of hierarchy, we find the clan, which is the term used by these authors to describe a group of families, whose members originate from a common ancestor protein but which have diverged to a point where relationships between them cannot be demonstrated by homology in their primary structures. The main evidence for the clan level is the relationship between families in terms of similarities in the three-dimensional structure of their members, in the arrangement of catalytic residues in the peptide structure, as well as similarities in the amino acid sequence around the catalytic residues [1]. Up to now, 16 clans of metallopeptidases have been described: MA, MC, MD, ME, MF, MG, MH, MJ, MM, MN, MO, MP, MQ, MS, MT, and MU, with six of them comprising exo-peptidases. Overall, they form 76 families, with clans MA and MF being two of the most well characterized with enzymes from all living organisms [1]. 2.2. Clan MA: Subclan MA (E) The clan MA is the largest of the metallopeptidases, with a total of 49 families [1], all consisting of enzymes that contain a single Zn2+ in their active sites. This clan is made up of both endopeptidases and exopeptidases, comprising aminopeptidases (families M1, M2, M4, M5, M9, M13, M30, M36, M48, and M61), carboxypeptidases (M2 and M32), peptidyl-dipeptidases (M2), oligopeptidases (M3 and M13), and endopeptidases (families 2.2. Clan MA: Subclan MA (E) The clan MA is the largest of the metallopeptidases, with a total of 49 families [1], all consisting of enzymes that contain a single Zn2+ in their active sites. This clan is made up of both endopeptidases and exopeptidases, comprising aminopeptidases (families Mar. Drugs 2023, 21, 279 6 of 37 M1, M2, M4, M5, M9, M13, M30, M36, M48, and M61), carboxypeptidases (M2 and M32), peptidyl-dipeptidases (M2), oligopeptidases (M3 and M13), and endopeptidases (fami- lies M4, M10, and M12). In the enzymes of the MA clan, the Zn2+ atom is coordinated to M4,protein the M10, and M12). through InHis two theresidues, enzymeswhich of theare MA clan, part Zn2+ atom the HEXXH of the is coordinated motif. In addition to to the protein through two His residues, which are part of the HEXXH motif. the His residues, the catalytic Zn is coordinated by a water molecule and a third resi- 2+ In addition to the 2+ is coordinated by a water molecule and a third due, theHis residues, nature the catalytic of which determines Znthe clan’s subdivision into the MA (E) and MA (M) residue, the subclans. In nature of which the subclan MAdetermines the clan’s (M), the third ligandsubdivision into theofMA can be a residue His(E) or and AspMA (M) within subclans. In the subclan MA (M), the third ligand can be a residue the HEXXHXXGXXH/D signature sequence, while in subclan MA (E) the third ligand is of His or Asp within athe HEXXHXXGXXH/D residue of Glu, located atsignature least 14sequence, whilethe residues after in carboxyl subclan MA (E) theofthird terminus the ligand HEXXH is a residue motif of Glu,5). [1] (Figure located at leastatom The oxygen 14 residues after molecule of the water the carboxyl that terminus of theligand acts as a metal HEXXH is the nucleophilic agent that attacks the carbonyl of the peptide bond to be hydrolyzed. is motif [1] (Figure 5). The oxygen atom of the water molecule that acts as a metal ligand the nucleophilic agent that attacks the carbonyl of the peptide bond to be hydrolyzed. Uniprot Figure 5.5. Uniprot Figure alignment alignment of the of the aminoacidic aminoacidic sequence sequence of theofactive the active site of site of various various M1 family M1 aminopeptidases: AMPE_Human: family aminopeptidases: human aminopeptidase AMPE_Human: E, AMPN_Human: human aminopeptidase human aminopep- E, AMPN_Human: human tidase N, AMPN_Pig: aminopeptidase porcine aminopeptidase N, AMPN_Pig: N, ERAP1_Human: porcine aminopeptidase endoplasmatic N, ERAP1_Human: reticulum ami- endoplasmatic retic- nopeptidase 1, LCAP_Human: human leucyl-cystinyl aminopeptidase, ulum aminopeptidase 1, LCAP_Human: human leucyl-cystinyl aminopeptidase, LKHA4_Human: LKHA4_Human: human leukotriene A4 hydrolase, human leukotriene PSA_Human: A4 hydrolase, PSA_Human: human humanpuromycin sensitive puromycin aminopeptidase, sensitive aminopepti- AMPB_Human: human aminopeptidase B, AMPO_Human: human aminopeptidase O, dase, AMPB_Human: human aminopeptidase B, AMPO_Human: human aminopeptidase O, TRHDE_Human: human thyrotrophin-releasing hormone-degrading enzyme or pyroglutamyl TRHDE_Human: human thyrotrophin-releasing hormone-degrading enzyme or pyroglutamyl aminopeptidase II, AMPQ_Human: human aminopeptidase Q, AMPQ_Mouse: mouse aminopep- aminopeptidase tidase Q. On the II, AMPQ_Human: right human of each sequence, theaminopeptidase access numberQ, AMPQ_Mouse: and identifiers frommouse aminopepti- Uniprot are in- dase Q. The cluded. On the right short nameof each for sequence, each enzyme the access number corresponds toand identifiers Uniprot from Uniprot abbreviations. The are included. rectangle A The shortthe encircles name for eachsequence conserved enzyme corresponds GAMEN relatedto Uniprot with abbreviations. The rectangle the aminopeptidase A of activities encircles these en-the zymes fromsequence conserved M1 family, and the GAMEN rectangle related B encircles with the the consensus aminopeptidase sequences activities of these HEXXH enzymesfrom from the M1 active family,site. andSigns below alignment the rectangle B encirclespoints to othersequences the consensus highly conserved amino HEXXH from acid the residues active inside site. Signs the below M1 family. points alignment * indicates residues to other highlycompletely conserved conserved, amino acid: indicates residuesposition with inside the M1high degree family. of con- * indicates servation of the residues, and . indicates position with mild degree of conservation of residues completely conserved, : indicates position with high degree of conservation of the residues, the residues. and . indicates position with mild degree of conservation of the residues. Thermolysin (EC 3.4.24.27), a secretory endopeptidase, is the model enzyme of the MA clan and its structure, Thermolysin widelya characterized, (EC 3.4.24.27), is a point ofisreference secretory endopeptidase, the model forenzyme the study of of the the enzymes of this clan due to the high structural similarity between them in MA clan and its structure, widely characterized, is a point of reference for the study ofterms of the the organization enzymes of thisof the active clan due center to the [1]. highAmong the most structural studied similarity families between of the them subclan in terms of MA (E) is M1, whose members show a wide distribution in the living world (Table the organization of the active center [1]. Among the most studied families of the subclan 1); furthermore, MA (E) is M1, they are involved whose membersinshow manyafunctions that include wide distribution cell living in the maintenance, growth, world (Table 1); furthermore, they are involved in many functions that include cell maintenance, growth, development, and defense [8]. This family includes enzymes of Gram (+) and Gram (−) bacteria, cyanobacteria, archaea, protozoa, fungi, animals, and plants [1,8]. Mar. Drugs 2023, 21, 279 7 of 37 Table 1. Some members of the M1 family of the MA(E) subclan of metallopeptidases. (Compiled from https://www.ebi.ac.uk/merops/cgi-bin/, accessed on 9 January 2023). IUBMB * Enzyme Merops ID Sources Nomenclature Aminopeptidase N (APN) EC 3.4.11.2 M01.001 Homo sapiens, Sus scrofa Lysyl aminopeptidase - M01.002 Escherichia coli Aminopeptidase A (APA) EC 3.4.11.7 M01.003 Homo sapiens Leukotriene A4 hydrolase (LTA4H) EC 3.3.2.6 M01.004 Homo sapiens Escherichia coli, Arabidopsis Alanyl aminopeptidase (bacterial-type) EC 3.4.11.2 M01.005 thaliana Ape2 aminopeptidase - M01.006 Saccharomyces cerevisiae Aap1’ aminopeptidase - M01.007 Saccharomyces cerevisiae Thyrotropin-releasing hormone-degrading Homo sapiens, Mus musculus, ectoenzyme or Pyroglutamyl-peptidase II EC 3.4.19.6 M01.008 Ratus novergicus (TRH-DE, PPII) Aminopeptidase N (actinomycete-type) - M01.009 Streptomyces lividans Homo sapiens, Arabidopsis Cytosol alanyl aminopeptidase - M01.010 thaliana, Caenorhabditis elegans Insulin-regulated membrane aminopeptidase or EC 3.4.11.3 M01.011 Homo sapiens cystinyl Aminopeptidase (IRAP) Aminopeptidase G - M01.012 Streptomyces coelicolor Aminopeptidase N (insect) - M01.013 Manduca sexta Aminopeptidase B (APB) EC 3.4.11.6 M01.014 Homo sapiens Aminopeptidase H11 (nematode) - M01.015 Aminopeptidase Ey EC 3.4.11.20 M01.016 Gallus gallus domesticus TMA108 protein - M01.017 Saccharomyces cerevisiae Endoplasmic reticulum aminopeptidase 1 - M01.018 Homo sapiens ERAP-1 Tricorn interacting factor F2 - M01.020 Thermoplasma acidophilum Tricorn interacting factor F3 - M01.021 Thermoplasma acidophilum Arginyl aminopeptidase-like 1 - M01.022 Homo sapiens Endoplasmic reticulum aminopeptidase 2 - M01.024 Homo sapiens ERAP-2 Aminopeptidase-1 (Caenorhabditis-type) - M01.025 Caenorhabditis elegans Aminopeptidase Q - M01.026 Homo sapiens Aminopeptidase O (AP-O) EC 3.4.11.- M01.028 Homo sapiens M1 aminopeptidase (Plasmodium spp.) EC 3.4.11.2 M01.029 Plasmodium falciparum Aminopeptidase N2 (insect) - M01.030 Manduca sexta Cold-active aminopeptidase (Colwellia - M01.031 Colwellia psychrerythraea psychrerythraea)-Type peptidase Lysyl aminopeptidase 1 (Streptomyces sp.) - M01.032 Streptomyces albulus Lysyl endopeptidase (Streptomyces albulus) - M01.033 Streptomyces albulus Leukotriene A4 hydrolase (Saccharomyces EC 3.3.2.6 M01.034 Saccharomyces cerevisiae cerevisiae) LePepA g.p. (Legionella pneumophila) - M01.035 Legionella pneumophila * IUBMB: International Union of Biochemistry and Molecular Biology. 2.3. M1 Family of Metalloaminopeptidases The aminopeptidases of the M1 family exist in monomeric or dimeric forms. In eukaryotes, they are generally membrane-associated enzymes such as mammalian APN (i.e., from human or pig), acidic or glutamyl aminopeptidase (APA), adipocyte-derived leucine aminopeptidase, and thyrotropin-releasing hormone-degrading ectoenzyme (TRH- DE), also known as pyroglutamyl peptidase II [16]. Some are cytosolic enzymes, such as leukotriene A4 hydrolase (bifunctional enzyme with aminopeptidase activity) [67] and aminopeptidase B (APB) [68], or associated with the cell wall [9], such as the neutral aminopeptidase (APN, EC 3.4.11.2) of the yeast Candida albicans [69]. The structure of the membrane-bound aminopeptidases of the M1 family, in general, comprises a short intracellular tail attached to the transmembrane domain and a large ectodomain formed, Mar. Drugs 2023, 21, 279 8 of 37 in turn, by 2- or 3-folded and conserved domains. Domain I, N-terminal, has a β-sheet nucleus that, although it is widely exposed to the solvent, contains a hydrophobic region that continues in an anchorage region in the membrane. Catalytic domain II, such as that of thermolysin, contains an active site flanked by a mixed structure of β-sheet and α-helix that is highly conserved throughout the family. Domain III, which is composed of an immunoglobulin-like fold, does not appear in some family members (such as leukotriene A4 hydrolase). Domain IV, C-terminal, is the most variable region within the family. It is completely helical, with such an arrangement that it covers the active site; it is also involved in the dimerization of the mammalian isoforms [8]. Disulfide bridges and abundant glycosylations are generally seen in this extracellular region, and some of these enzymes are surface antigens [1,16]. In the M1 family, a well-conserved motif is the Gly-Ala/X-Met-Glu-Asn (GAMEN/GXMEN) sequence. This sequence, also known as the exopeptidase motif, frequently shows variations in the first two residues, and is very useful for the identification of family members [8,16,70] (Figure 5). Through the technique of crystallography and X-ray diffraction, the three-dimensional structures of several members of this family have been elucidated, such as leukotriene A4 hydrolase in complex with its inhibitor bestatin [71]; tricorn-interacting factor 3 of Thermoplasma acidophilum [72]; Escherichia coli APN (Pep N) in complex with its inhibitor bestatin [73]; Plasmodium falciparum (PfA-M1) alone and in complex with bestatin and low molecular mass analogs [74–76]; human APA [77]; human ERAP-1 [78]; and porcine and human APN in complex with substrates and bestatin [70,79], among others (Table 2) (Figure 6). In all these structures, it can be seen that the catalytic domain of this enzymatic family presents a high structural similarity with thermolysin, despite the fact that in some cases, there is only 7% identity in sequence with the corresponding polypeptide chains [71]. The high availability of M1 aminopeptidase structures, the well-studied active site able to the binding of small molecules, and the well characterized reaction mechanisms, make M1 aminopeptidases ideal candidates for the application of structure-guided inhibitor discovery, including high-throughput screenings in different databases of marine and other natural compounds. These inhibitors have potentialities in different infectious and chronic human diseases [80,81]. 2.4. Inhibitors of M1 Family Isolated from Marine Invertebrates 2.4.1. A Specific Inhibitor of Thyrotropin-Releasing Hormone-Degrading Ectoenzyme/Pyroglutamyl Aminopeptidase II Isolated from a Marine Organism Thyrotropin-releasing hormone (TRH), an N-terminal blocked tripeptide (pGlu-His- ProNH2), is mainly produced by brain neurons. Expressed by neurons of the paraven- tricular nucleus of the hypothalamus, TRH is a hypophysiotropic factor that increases the synthesis and release of thyroid stimulating hormone (TSH) and prolactin (PRL) from the adenohypophysis. In other central nervous system (CNS) circuits, it functions as a neurotransmitter and/or neuromodulator [82]. This peptide has therapeutic properties in the treatment of brain and spinal damage and various neurodegenerative disorders [83]. However, TRH effects are of short duration, in part because the peptide is hydrolyzed in blood and extracellular space by TRH-DE, the thyrotropin-releasing hormone-degrading ectoenzyme, a M1 family metallopeptidase. TRH-DE is enriched in various brain regions but is also expressed in peripheral tissues including the anterior pituitary and the liver, which secretes a soluble form into blood. Among the M1 metallopeptidases, TRH-DE is the only member with a very narrow specificity, hydrolyzing preferentially the pGlu-His bond of TRH, its best characterized biological substrate, making it a target for the specific manipulation of TRH activity. TRH-DE presents an anatomical location that correlates partially with TRH receptors in various regions and is very strictly regulated by different hormones and hypothalamic factors, as well as by various pharmacological and pathophys- iological conditions that alter the transmission of TRH-mediated signals. The regulation of TRH-DE activity may be very important for the adjustment of communication mediated Mar. Drugs 2023, 21, 279 9 of 37 by this peptide [84]. Therefore, TRH-DE inhibitors are important tools for studying the physiological functions of this enzyme and TRH in the CNS, as well as for enhancing the different actions of TRH by protecting the degradation of endogenous TRH or exoge- nously administered analogues [85]. TRH-DE inhibition may be used to enhance TRH activity in different pathologies (Figure 7). Only a few synthetic PPII inhibitors have been described [86–88]. Table 2. Crystallographic structures reported for members of the M1 family of metallopeptidases (Compiled from https://www.ebi.ac.uk/merops/cgi-bin/, accessed on 10 January 2023). Crystallographic Codes of the Structures at Enzyme Source Protein Data Bank (PDB) 3EBG, 3EBH, 3EBI, 3Q43, 3Q44, 3T8V, 4J3B, 4K5L, 4K5M, 4K5N, 4K5O,4K5P, 4R5T, 4R5V, 4R5X, 4ZQT, 4ZW3, 4ZW5, 4ZW6, 4ZW7, aminopeptidase N Plasmodium falciparum 4ZW8, 4ZX3, 4ZX4, 4ZX5, 4ZX6, 5XM7, 5Y19, 5Y1H, 5Y1K, 5Y1Q, 5Y1R, 5Y1S, 5Y1T, 5Y1V, 5Y1W, 5Y1X, 6EA1, 6EA2, 6EAA, 6EAB, 6EE3, 6EE4, 6EE6, 6EED, 6SBQ, 6SBR 2DQ6, 2DQM, 2HPO, 2HPT, 2ZXG, 3B2P, 3B2X, 3B34, 3B37, 3B3B, 3KED, 3PUU, 3QJX,4Q4E, 4Q4I, 4XMT, 4XMU, 4XMV, 4XMW, 4XMX, aminopeptidase N Escherichia coli 4XM2, 4XN1, 4XN2, 4XN4, 4XN5, 4XN7, 4XN8, 4XN9, 4XNA, 4XNB, 4XND, 4X03, 4X04, 4X05, 5MFR, 5MFS, 5MFT, 5Y01, 5YQ1, 5YQ2, 5YQB, 6G8B aminopeptidase N Homo sapiens 4FYQ, 4FYR, 4FYS, 4FYT, 5LHD, 6AKT 4FSC, 4FKE, 4FKH, 4FKK, 4HOM, 4NAQ, aminopeptidase N Sus scrofa 4NZ8, 4OU3 ERAP 1 Homo sapiens 2XDT, 2YD0, 3MDJ, 3QNF, 3RJO, 6Q4R 3SE6, 4E36, 4JBS, 5AB0, 5AB2, 5CU5, ERAP 2 Homo sapiens 5J6S, 5KIV aminopeptidase A Homo sapiens 4KX7, 4KX8, 4KX9, 4KXA, 4KXB, 4KXC, 4KXD 1G6W, 1H19, 1HS6, 1SQM, 2R59, 2VJ8, 3B7R, 3B7S, 3B7T, 3B7U, 3CHO, 3CHP, 3CHQ, 3CHR, 3CHS, 3FH5, 3FH7, 3FH8, 3FHE, 3FTS, 3FTU, 3FTV, 3FTW, 3FTX, 3FTY, 3FTZ, 3FU0, 3FU3, leukotriene A4 hydrolase Homo sapiens 3FU5, 3FU6, 3FUD, 3FUE, 3FUF, 3FUH, 3FUI, 3FUJ, 3FUK, 3FUL, 3FUM, 3FUN, 3U9W, 4DPR, 4L2L, 4MKT, 4Ms6, 4RSY, 4RVB, 5AEN, 5BPP, 5FWQ, 5N3W, 5NI2, 5NI4, 5NI6, 5NIA, 5NID, 5NIE, 6ENB, 6ENC, 6END leukotriene A4 hydrolase Saccharomyces cerevisiae (ATCC 2XPY, 2XPZ, 2XQ0 (Saccharomyces cerevisiae) 204508/S288c) cold-active aminopeptidase (Colwellia Colwellia psychrerythraea 3CIA psychrerythraea)-type peptidase (34H/ATCC BAA-681) Thermoplasma acidophilum (ATCC tricorn interacting factor F3 25905/DSM 1728/JCM 9062/NBRC 1Z1W,1Z5H, 3Q7J 15155/AMRC-C165) LePepA g.p. (Legionella pneumophila) Legionella pneumophila 5ZI5, 5ZI7, 5ZIE TATA-binding protein-associated factor Homo sapiens 5FUR, 6MZC, 6MZL, 6MZM IRAP (cystinil aminopeptidase) Homo sapiens 4P8Q, 4PJ6, 4Z7I, 5C97, 5MJ6 A joint project of the Faculty of Biology, University of Havana, Cuba, with the Insti- tute of Biotechnology of UNAM, Mexico, involving a screening in aqueous extracts from 26 Cuban coastline marine organisms (Table 3) resulted in the first natural inhibitor of TRH-DE identified and isolated from the marine annelid Hermodice carunculata (Figure 8); it was named HcPI. As a result of this screening, we also detected inhibitory activities of porcine kidney cortex dipeptidyl peptidase IV in the species Phallusia nigra, Mycale leukotriene A4 hydrolase Saccharomyces cerevisiae 2XPY, 2XPZ, 2XQ0 (Saccharomyces cerevisiae) (ATCC 204508/S288c) cold-active aminopepti- Colwellia psychrerythraea dase (Colwellia psychrery- 3CIA (34H/ATCC BAA-681) thraea)-type peptidase Mar. Drugs 2023, 21, 279 Thermoplasma acidophilum 10 of 37 tricorn interacting factor (ATCC 25905/DSM 1Z1W,1Z5H, 3Q7J F3 1728/JCM 9062/NBRC 15155/AMRC-C165) LePepA g.p. (Legionella microsigmatosa, Condylactis gigantea, Stichodactyla helianthus, and Palythoa caribbaeroum. HcPI Legionella pneumophila is a 580 5ZI5,(molecular Da compound 5ZI7, 5ZIE mass determined by ESI-TOF mass spectrometry), with pneumophila) TATA-binding protein- a possible polymeric structure and the presence of bromine in its structure, as well as Homo sapiens associated factor amide-type bonds.5FUR, HcPI6MZC, 6MZL, 6MZM potently inhibits TRH-DE with a Ki value of 70.3 nmol/L in a IRAP (cystinil aminopep- slow and reversible way, making it one of the most powerful inhibitors described against Homo sapiens 4P8Q, 4PJ6, 4Z7I, 5C97, 5MJ6 tidase) this enzyme [49] (Figure 9A). Mar. Drugs 2023, 21, x 10 of 37 ventricular nucleus of the hypothalamus, TRH is a hypophysiotropic factor that increas- es the synthesis and release of thyroid stimulating hormone (TSH) and prolactin (PRL) from the adenohypophysis. In other central nervous system (CNS) circuits, it functions as a neurotransmitter and/or neuromodulator [82]. This peptide has therapeutic proper- ties in the treatment of brain and spinal damage and various neurodegenerative disor- ders [83]. However, TRH effects are of short duration, in part because the peptide is hy- drolyzed in blood and extracellular space by TRH-DE, the thyrotropin-releasing hor- mone-degrading ectoenzyme, a M1 family metallopeptidase. TRH-DE is enriched in var- ious brain regions but is also expressed in peripheral tissues including the anterior pitui- tary and the liver, which secretes a soluble form into blood. Among the M1 metallopep- tidases, TRH-DE is the only member with a very narrow specificity, hydrolyzing prefer- entially the pGlu-His bond of TRH, its best characterized biological substrate, making it a target for the specific manipulation of TRH activity. TRH-DE presents an anatomical location that correlates partially with TRH receptors in various regions and is very strict- ly regulated by different hormones and hypothalamic factors, as well as by various pharmacological and pathophysiological conditions that alter the transmission of TRH- Figure 6. Cartoon mediated signals. representation The regulation of different of TRH-DE M1 family aminopeptidases: activity may be very (A) pepN from Esche- Figure 6. Cartoon representation of different M1 family aminopeptidases: (A)important pepN fromfor the ad- Escherichia richia coli (PDB ID: 2dq6), (B) Plasmodium falciparum aminopeptidase N PfA-M1 (PDB ID: 3ebh), justment coli of communication mediated by this peptide [84]. Therefore, TRH-DE inhibitors (C)(PDB human ID:ERAP 2dq6),1 (B) (PDB Plasmodium falciparum ID: 3mdj), (D) human ERAP aminopeptidase N PfA-M1 2 (PDB ID: 3se6), (PDBleukotriene (E) human ID: 3ebh), (C) A4 hu- are man important ERAP 1 (PDBtools ID: for 3mdj),studying (D) humanthe physiological ERAP 2 (PDB ID: functions 3se6), (E) of humanthis enzyme leukotriene hydrolase (PDB ID: 1hs6), (F) Saccharomyces cerevisiae leukotriene A4 hydrolase (PDB ID: 2xq0), and A4 TRH in hydrolase the (G)CNS, (PDB as well ID: 1hs6), Thermoplasma (F) as for enhancing Saccharomyces acidophilum tricornthe cerevisiaedifferent actions leukotriene interacting factor of TRH A4(PDB F3 hydrolase by protecting (PDB ID: 1z1w), the ID:Colwellia (H) 2xq0), degra- (G) Ther- psy- dation moplasma of acidophilum chrerythraeaendogenous TRH interacting cold-activetricorn or exogenously aminopeptidase (PDB administered ID:F3 factor 3cia). (PDBColors: analogues alpha-helices ID: 1z1w), [85]. psychrerythraea TRH-DE (cyan), (H) Colwellia inhi- beta sheets bition (warm may pink),be used and loops to enhance (salmon). TRH The zinc activity atoms in are different shown as pathologies gray spheres cold-active aminopeptidase (PDB ID: 3cia). Colors: alpha-helices (cyan), beta sheets (warm pink), (Figure highlighted 7). in a Only yel- a lowsynthetic few box. PPII inhibitors have been described [86–88]. and loops (salmon). The zinc atoms are shown as gray spheres highlighted in a yellow box. 2.4. Inhibitors of M1 Family Isolated from Marine Invertebrates 2.4.1. A Specific Inhibitor of Thyrotropin-Releasing Hormone-Degrading Ectoenzyme/Pyroglutamyl Aminopeptidase II Isolated from a Marine Organism Thyrotropin-releasing hormone (TRH), an N-terminal blocked tripeptide (pGlu- His-ProNH2), is mainly produced by brain neurons. Expressed by neurons of the para- therapeutic applications Figure 7. Potential therapeutic applicationsof oftargeting targetingcentral centralTRH-degrading TRH-degradingectoenzyme ectoenzymeactivity. activi- ty. A joint project of the Faculty of Biology, University of Havana, Cuba, with the Insti- tute of Biotechnology of UNAM, Mexico, involving a screening in aqueous extracts from 26 Cuban coastline marine organisms (Table 3) resulted in the first natural inhibitor of TRH-DE identified and isolated from the marine annelid Hermodice carunculata (Figure 8); it was named HcPI. As a result of this screening, we also detected inhibitory activities of porcine kidney cortex dipeptidyl peptidase IV in the species Phallusia nigra, Mycale Mar. Drugs 2023, 21, 279 11 of 37 Table 3. Screening of inhibitory activity of TRH-DE and DPP-IV in aqueous extracts from marine invertebrates collected at the Havana coastline, Cuba (adapted from Pascual et al. [49]). [Protein] Crude Inhibit Activity of Inhibit Activity of Species Phylum Extract (mg/mL) DPP-IV (U/mg) TRH-DE (U/mg) Caulerpa racemosa Chlorophyta 5.86 - - Dictyosphaeria cavernosa Chlorophycota 8.73 - - Halimeda opuntia Chlorophycota 14.72 - - Halimeda incrassata Chlorophycota 10.23 - - Bidens pilosa Magnoliophyta 22.78 - - Ascidia sidneyense Chordata 57.27 - - Molgula occidentalis Chordata 31.41 - - Pyura vittata Chordata 58.53 - - Phallusia nigra Chordata 25.90 93.00 - Microcosmus gamus Chordata 24.45 - - Tectitethya cripta Porifera 0.90 - - Mycale microsigmatosa Porifera 43.50 56.59 - Lima scabra Mollusca 51.55 - - Aplisia dactilomela Mollusca 24.00 - - Zoanthus pullchelus Cnidaria 15.57 - - Plexaura homomalla Cnidaria 15.25 - - Condylactis gigantea Cnidaria 36.60 79.46 - Stichodactyla helianthus Cnidaria 79.50 17.48 - Cassiopea xamachana Cnidaria 3.14 - - Physalia physalis Cnidaria 13.84 - - Palythoa caribaeorum Cnidaria 16.80 133.00 - Bartholomea annulata Cnidaria 56.50 - Hermodice carunculata Annelida 62.41 - 24.00 Sabellastarte magnifica Annelida 67.24 - - Holothuria floridiana Echinodermata 18.84 - - Holothuria mexicana Echinodermata 29.63 - - Inhibitory specificity studies carried out against proteases of all mechanistic classes indicate that HcPI is highly specific for TRH-DE. The specificity of the inhibitory activity was assayed using several enzymes from each mechanistic class of proteinases. In a concentration range from 33 to 660 ng/mL and preincubation times at 37 ◦ C of 5, 10, or 30 min, HcPI was not active against serine (trypsin, chymotrypsin, elastase, and DPP- IV), cysteine (papain, bromelain, and PPI), or aspartic (pepsin and PR-HIV) proteases nor against metalloproteinases (collagenase, gelatinase, ACE, aminopeptidase N, and carboxypeptidase A). It was further confirmed that HcPI inhibits, in vitro, thyroliberinase, a soluble version of TRH-DE that inactivates TRH in the bloodstream. The inhibition is dose-dependent, with a Ki value of 51 nmol/L, similar to that previously described for TRH- DE. The specificity results support the idea that HcPI should be useful to study the role of TRH-DE in different experimental models. HcPI is not toxic in vivo and its intraperitoneal injection in BalbC mice decreases TRH-DE activity in the pituitary and in different brain regions such as the hypothalamus, cerebellum, and olfactory bulb [49] (Figure 9C). The inhibition of TRH-DE in vivo in this experimental model causes a transient increase in the serum concentrations of prolactin (PRL) and thyrotropin (TSH), which indicates an Mar. Drugs 2023, 21, 279 12 of 37 in vivo enhancement of the actions of endogenous TRH when degradation by TRH-DE is decreased. Additionally, studies on cells were performed. First, in primary cultures of adenohypophyseal cells, 45 min of incubation with HcPI produces a decrease in the activity of membrane-associated TRH-DE, highly dependent on the dose of inhibitor tested with an IC50 of 8.3 µg/mL (Figure 9B). Incubation with 8 µg HcPI/mL decreases enzyme activity by 42% from 5 min, an effect stable for at least one hour. Once enzyme inhibition was demonstrated in cultures, the effect of the enzyme on TRH-mediated communication was evaluated, and it was detected that in the absence of TRH in the system, the presence of HcPI (50 µg/mL) does not change the basal levels of secretion of TSH, or PRL. On the other hand, in the presence of TRH (10 nmol/L), the inhibition of TRH-DE by HcPI caused an increase in the levels of PRL released after 30 min by lactotrophs, specialized cells of the adenohypophysis. These results were confirmed in parallel by inhibition of TRH-DE synthesis with the use of antisense RNA, which demonstrated for the first time in a direct way the effects of the regulation of PPII activity on one of the functions of TRH [89]. Intraperitoneal injection of HcPI (1, 5, 20, or 50 µg/g) in mice did not induce any mortality, obvious motor effect, or weight change for up to 15 days. The effect of different doses of HcPI injected intraperitoneally was tested on mouse PPII specific activity. Compared to saline injected animals, PPII activity was significantly decreased 45 min after injection in most of the tissues analyzed; the effect was dose-dependent. Less than 1 mg of the inhibitor per g of animal weight was sufficient to decrease activity by more than 50% in hypophysis; the maximum dose used (5 µg/g) almost completely abolished the activity. The order of potency was as follows: hypophysis > hypothalamus > cerebellum > olfactory bulb (Figure 9) [49]. Other experiments related to the role of PPII in TRH communication within the hypothalamic–adenohypophysis–thyroid axis were continued with the use of animal models. In these studies, HcPI was injected at the beginning of the experiment at a dose of 50 µg/g of animal weight to Wistar rats, dissolved in physiological saline (doses of 5–10 µg/g of animal weight strongly reduce PPII activity in the adenohypophysis and decrease it in CNS regions). The controls received only saline. Four groups of animals were subsequently treated with 1 ng/g animal weight TRH in saline or saline only and slaughtered by decapitation 15 min after the second treatment. Two additional groups were transferred to a cold room kept at 4 ◦ C for 30 min and similarly sacrificed after the end of the experiment. Since cold stress rapidly activates the hypothalamic–adenohypophysis–thyroid axis by increasing concentrations of TRH in the portal hypothalamus–pituitary vessels, this paradigm was used in addition to exogenous administration of TRH, with the objective of evaluating the effects of inhibition of TRH-DE by HcPI on a naturally occurring surge of circulating TSH concentration. Compared to animals that received a single injection of saline, TRH-DE activity is significantly decreased in the hypothalamus and in the pituitary of animals that receive a single dose of HcPI. Similar changes are observed in the activity of serum thyroliberinase. Inhibition of TRH-DE activity by HcPI has no effect on baseline TSH levels, as observed in primary adenohypophyseal cell cultures. However, in animals injected with exogenous TRH or exposed to ambient cold, inhibition of TRH-DE activity by HcPI is associated with a significant increase in serum TSH concentration when compared to control groups that only received saline (Sánchez-Jaramillo et al. 2009). These results demonstrated for the first time the role that TRH-DE exerts on TRH activity and TSH secretion by adenohypophysis and made HcPI a very useful tool for further studies and potential biomedical applications in diseases such as Non-Thyroidal Illness Syndrome (NTIS) in which the levels of thyroid hormones are reduced [90]. 2.4.2. Inhibitors of Aminopeptidase N Isolated from Marine Organisms Neutral aminopeptidases are enzymes that catalyze the cleavage of neutral amino acids from the N-terminus of protein or peptide substrates. They have been classified in several metallopeptidase families, such M1 and M17 [1,8,9]. These enzymes are present in all living organisms, but the diversity of the functions in which they are involved is far from being entirely deciphered. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, Mar. Drugs 2023, 21, 279 13 of 37 M1 family) is the most extensively studied member of the M1 family of zinc-dependent aminopeptidases; it is noteworthy that it catalyzes the cleavage of not only neutral but also basic N-terminal residues. This enzyme, also known as CD13, is widely expressed on cell surfaces of tissues, such as intestinal epithelia and the nervous system. Mammalian APN is a type II membrane protein generally found as a homodimer in several mammalian species. Full-length human APN consists of 967 amino acids with a short N-terminal cytoplasmic domain, a single transmembrane segment, and a large ectodomain containing two catalytic motifs highly conserved across the M1 family: the zinc-binding motif HEXXHX18E and the exopeptidase signature GAMEN [70]. APN plays pivotal roles in many physiological processes, such as pain sensation, sperm motility, cell–cell adhesion, and blood pressure regulation (Figure 10) [8,18]. This enzyme is also up-regulated in human pathologies, such as coronavirus entry, inflammation, immune cell chemotaxis, tumor angiogenesis, and metastasis in several types of cancer, with a strong correlation between the level of APN expression of a cell and its resultant invasive capacity (Figure 10). Dysregulation of APN expression evolves in almost all types of human malignancies, including breast cancer, cervical cancer, ovarian cancer, prostate cancer, non-small-cell lung cancer (NSCLC), liver cancer, colon cancer, cirrhosis gastric cancer, pancreatic cancer, renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (SCC), melanoma, osteosarcoma, and thyroid cancer [19,91]. This makes human APN an attractive target for the treatment of diseases, including cancers (Figure 11) [8,18,19,91–93]. Accordingly, strategies for its inhibition have been developed primarily for the treatment of pain [94,95]. Only Ubenimex (bestatin), a drug inhibitor, is currently approved by the FDA for its uses Mar. Drugs 2023, 21, x 11 of 37 in human pathologies, mainly in cancer [91]. Figure 8. Some of the marine organisms screened for inhibitory activity of TRH-DE and DPP-IV. Figure Some ofcarunculata, (A)8.Hermodice the marine(B) organisms screened Palythoa caribaeorum, (C)for inhibitory Condylactis activity gigantea, and of (D)TRH-DE and DPP-IV. Stichodactyla (A) Hermodice carunculata, helianthus. Pictures (B)ofPalythoa courtesy Professor caribaeorum, (C) Condylactis José Espinosa, PhD, ICIMAR, CITMA,gigantea, Cuba.and (D) Stichodactyla helianthus. Pictures courtesy of Professor José Espinosa, PhD, ICIMAR, CITMA, Cuba. Table 3. Screening of inhibitory activity of TRH-DE and DPP-IV in aqueous extracts from marine invertebrates collected at the Havana coastline, Cuba (adapted from Pascual et al. [49]). [Protein] Crude Extract Inhibit Activity of DPP-IV Inhibit Activity of TRH-DE Species Phylum (mg/mL) (U/mg) (U/mg) Caulerpa racemosa Chlorophyta 5.86 - - Mar. Drugs 2023, 21, 279 14 of 37 Mar. Drugs 2023, 21, x 14 of 37 A B Rat brain TRH-DE Residual activity 0,9 1.8 TRH-DE Specific activity (U/mg) 0,8 1.6 (fraction of control) 1.4 0,7 1.2 0,6 1.0 0,5 0.8 0.6 0,4 0.4 0,3 y =1-0.5*(((3,49415)/(11,2336))+1+(x/(11,2336))-sqrt((((3,49415)/(11,2336))+1+(x 0.2 /(11,2336)))**2-(4*x/(11,2336)))) 0,2 0.0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 HcPI (μmol/L) HcPI (μg/mL) C Hypophysis (U/mg) . 14 a PPII (U/mg) 100 % específica activity 12 10 Placebo ActividadSpecific 8 HcPI 1 μg/g 23 % 6 b 17 % HcPI 3 μg/g 4 bc TRH-DE 3.5 % 2 c HcPI 5 μg/g 0 1 Hypothalamus Hipotálamo Cerebelum Cerebelo TRH-DE Specific activity (U/mg) (U/mg) 80 . 100 % a PPII (U/mg) 140 a 83 % 100 % b activity 120 63 % 60 63.3 % c 100 b específica Specific 80 40 60 Actividad 40 9.2 % 20 TRH-DE c 20 0 0 Bulbo olfatorio Olfactory bulb Corteza cerebralcortex Cerebral TRH-DE Specific activity (U/mg) (U/mg) 180 . 200 PPII (U/mg) a 180 a 160 a a 100 % 75 % activity 140 160 120 ab 63 % 140 específica 120 100 b Specific 100 80 80 60 Actividad 60 40 40 TRH-DE 20 20 0 0 Placebo HcPI 2 μg/g HcPI 4 μg/g Figure Figure9. 9.TRH-DE TRH-DE inhibition inhibition by byHcPI HcPI in in different different enzyme enzyme models. models. (A) Ki determination (A) Ki determination of HcPI ef-of HcPI fecton effect on TRH-DE TRH-DE activity activityininrat ratbrain brainmembranes, membranes, (B) (B) inhibition effecteffect inhibition of HcPI on TRH-DE of HcPI activityactivity on TRH-DE in primary cultures of rat adepnohypophysis, and (C) effect of intraperitoneal injection of different in primary cultures of rat adepnohypophysis, and (C) effect of intraperitoneal injection of different doses of HcPI on mouse TRH-DE specific activity in brain [49]; Different letters indicate a signifi- doses cantofdifference HcPI onbetween mouse TRH-DE treatmentspecific groups, activity in brain [49]; Different letters indicate a significant with p < 0.001. difference between treatment groups, with p < 0.001. Natural inhibitors of human and mammalian APN in general are scarce and have mainly been described from microorganisms [18,96], plants [97], marine invertebrates [36,48,98,99], and more recently from Cuban toad secretions [100,101] (Figure 12). Several compounds from marine organisms have been described with anticancer activities; how- ever, very few of them have been linked with APN inhibition or interaction [102–106]. In Mar. Drugs 2023, 21, 279 15 of 37 Mar. Drugs 2023, 21, x the next section, we review the information available regarding marine invertebrates 16 of 37 Mar. Drugs 2023, 21, x 16 of as 37 a promissory and still unexplored source of inhibitors of APN with biomedical relevance mainly in cancer. Figure 10. Functions of human APN/CD13 (adapted from Amin et al. [19]). 10.Functions Figure10. Figure Functionsofof human APN/CD13 human APN/CD13(adapted from (adapted Amin from et al. Amin et[19]). al. [19]). Figure 11. Up-regulation of human APN in different cancers. Abbreviations: renal cell cancer Figure 11. Up-regulation of human APN in different cancers. Abbreviations: renal cell cancer (RCC), (RCC), hepatocellular carcinoma (HCC), squamous cell carcinoma (SCC), non-small-cell lung car- hepatocellular carcinoma (HCC), squamous cell carcinoma (SCC), non-small-cell lung carcinoma cinoma Figure (NSCLC). 11. Up-regulation of human APN in different cancers. Abbreviations: renal cell cancer (NSCLC). (RCC), hepatocellular carcinoma (HCC), squamous cell carcinoma (SCC), non-small-cell lung car- cinoma (NSCLC). Mar. Mar.Drugs Drugs2023, 21,21, 2023, 279x 16ofof37 17 37 Figure 12.Natural Figure12. Naturalinhibitors inhibitors of of Aminopeptidase Aminopeptidase N. Psammaplin A Psammaplin A Psammaplin A (PsA) is a natural bromothyrosine compound belonging to the open- Psammaplin A (PsA) is a natural bromothyrosine compound belonging to the open- chain-oximinoamidesis bromo-derivates group (Figure 13), isolated from the association chain-oximinoamidesis bromo-derivates group (Figure 13), isolated from the association between two sponges, Poecillastra sp. and Jaspis sp. [107], which represents the first isolated between two sponges, Poecillastra sp. and Jaspis sp. [107], which represents the first iso- natural product containing oxime and disulfide moieties from marine sponges. Sub- lated natural product containing oxime and disulfide moieties from marine sponges. sequently, other natural derivatives, such as biprasin, psammaplin C, psammaplin E, Subsequently, other natural derivatives, such as biprasin, psammaplin C, psammaplin E, psammaplin F, psammaplin G, and psammaplin K (Figure 13), were also isolated and psammaplin F, psammaplin G, and psammaplin K (Figure 13), were also isolated and described [108–112]. described [108–112]. Several biological activities have been described for PsA; it is an antibacterial mainly Several biological activities have been described for PsA; it is an antibacterial main- against Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) ly against Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus due to DNA gyrase inhibition and bacterial DNA synthesis arrest [113]. PsA also inhibits (MRSA) due to DNA gyrase inhibition and bacterial DNA synthesis arrest [113]. PsA al- chitinases that are common in fungi and are crucial for the control of ecdysis in insects [114]. so inhibits chitinases that are common in fungi and are crucial for the control of ecdysis In mammalian cells, PsA inhibits topoisomerase II, an enzyme catalyzing DNA relaxation, in insects [114]. In mammalian cells, PsA inhibits topoisomerase II, an enzyme catalyzing with a high IC50 of 18.8 mM [114]. In 2004, almost at the same time our group described DNA relaxation, with a high IC50 of 18.8 mM [114]. In 2004, almost at the same time our HcPI as an inhibitor of TRH-DE, Shim et al. [48] found that PsA inhibits mammalian APN group described HcPI as an inhibitor of TRH-DE, Shim et al. [48] found that PsA inhibits with a Ki value of 15 µM in a non-competitive way. Structural analogues of PsA, in which mammalian APN with a Ki value of 15 µM in a non-competitive way. Structural ana- phenolic hydroxyl groups were replaced, did not inhibit human or porcine APN, indicating logues of PsA, in which phenolic hydroxyl groups were replaced, did not inhibit human that these groups are crucial in the recognition and inhibition of this enzyme in mammals. or porcine APN, indicating that these groups are crucial in the recognition and inhibition This finding perfectly agreed with the effectiveness of bulky and hydrophobic groups in of this enzyme in mammals. This finding perfectly agreed with the effectiveness of molecules targeting APN [115]. However, no other in-depth structure–activity or docking bulky and hydrophobic groups in molecules targeting APN [115]. However, no other in- studies of aminopeptidase N inhibition by PsA have been carried out. depth structure–activity or docking studies of aminopeptidase N inhibition by PsA have Psammaplin A possesses antiproliferative activities against various cancer cell lines, in- been carried out. cluding triple-negative breast (TNBC, MDA-MB-231), doxorubicin-resistant human breast Psammaplin A possesses antiproliferative activities against various cancer cell lines, (MCF-7/adr), colon (HCT15), ovarian (SK-OV-3), lung (A549, LM4175), bone (BoM1833), including triple-negative breast (TNBC, MDA-MB-231), doxorubicin-resistant human skin (SK-MEL-2), central nervous system (BrM-2a, XF498) [116–118], and Ishikawa en- breast (MCF-7/adr), colon (HCT15), ovarian (SK-OV-3), lung (A549, LM4175), bone dometrial cells [119]. Some of the mechanisms described to explain the antiproliferative (BoM1833), skin (SK-MEL-2), central nervous system (BrM-2a, XF498) [116–118], and effects of this compound are the induction of cell cycle arrest and apoptosis associated with Ishikawa endometrial cells [119]. Some of the mechanisms described to explain the anti- different factors [119,120]. Pretreatment with PsA was also shown to increase the sensitivity proliferative effects of this compound are the induction of cell cycle arrest and apoptosis of human lung and glioblastoma cancer cells to radiation in vitro [120]. Moreover, this associated with different factors [119,120]. Pretreatment with PsA was also shown to in- compound showed suppressive effects of the invasion and tube formation of endothelial rine sources as well as its poor physiological stability. For these reasons, homodimeric or heterodimeric analogs of PsA have been obtained by chemical synthesis through a disul- fide exchange strategy (Figure 14) [122]. Some of the new synthetic compounds, particu- larly heterodimeric derivatives, displayed higher antibacterial activity than psammaplin Mar. Drugs 2023, 21, 279 A, comparable to clinically used drugs vancomycin and ciprofloxacin [123,124]. Howev- 17 of 37 er, the APN inhibitory activity of all the synthetic psammaplin derivatives (examples in Figures 13 and 14) has not been evaluated, being a still unexplored source of new ami- nopeptidase N inhibitors with biomedical potentialities taking into account the promis- cells stimulated by the basic fibroblast growth factor [48]. These results demonstrate that sory effects of these compounds on several biomedical models [122]. PsA is a new APN inhibitor that can be developed as a new antiangiogenic agent. Figure 13. The Figure 13. The chemical chemical structure structure of of psammaplin psammaplin compounds, compounds,biprasin, biprasin,and andaplysinellin aplysinellinA. A. PsA has also been described as a histone deacetylase (HDAC) inhibitor [119,121]. The mechanism of action underlying the HDAC inhibitory effect of PsA involves a change in the redox state of the disulfide bond. Replacement of the sulfur atom leads to the formation of a mercaptan, which in turn chelates the Zn+ ion present in the characteristic active site of the HDAC enzyme, modifying its conformational state and thus preventing its accessibility to the natural substrate. This new conformational state determines an increase in acetylation levels of histone H3, a well-known epigenetic marker of chromatin structure and function, suggesting selectivity for HDACs. Moreover, PsA also exhibits potent enzyme inhibitory and antiproliferative activities under reduced conditions in cells, which indicates that PsA could be used as a natural prodrug [121]. Although PsA possesses a broad spectrum of bioactivities, its in-depth study has been hindered due to the limited amount of the compound that can be isolated from ma- rine sources as well as its poor physiological stability. For these reasons, homodimeric or heterodimeric analogs of PsA have been obtained by chemical synthesis through a disulfide exchange strategy (Figure 14) [122]. Some of the new synthetic compounds, partic- ularly heterodimeric derivatives, displayed higher antibacterial activity than psammaplin A, comparable to clinically used drugs vancomycin and ciprofloxacin [123,124]. How- ever, the APN inhibitory activity of all the synthetic psammaplin derivatives (examples in Figures 13 and 14) has not been evaluated, being a still unexplored source of new aminopep- tidase N inhibitors with biomedical potentialities taking into account the promissory effects of these compounds on several biomedical models [122]. Mar. Drugs 2023, 21, 279 18 of 37 Mar. Drugs 2023, 21, x 19 of 37 Figure 14. Examples of synthetic homo- and hetero-derivatives of psammaplin A. The blue dashed Figure Examples of 14. highlights line box syntheticthat derivatives homo- and hetero-derivatives showed of psammaplin significant antibacterial A. The effects against blue dashed methicillin- line box highlights resistant derivatives Staphylococcus aureusthat showed (MRSA) duesignificant antibacterial to DNA gyrase effects inhibition and against bacterialmethicillin-resistant DNA synthesis Staphylococcus arrest. The redaureus dashed line box (MRSA) due highlights derivatives to DNA gyrase that and inhibition showed higher bacterial DNA antibacterial activityThe synthesis arrest. than red psammaplin dashed line boxA. The green highlights dashed line derivatives box that highlights showed highera derivative thatactivity antibacterial showed similar than anti- psammaplin A.bacterial The greenactivity to clinically dashed line boxused drugs vancomycin highlights a derivativeand ciprofloxacin. that The yellow showed similar dashed activity antibacterial line box highlights derivatives that possessed 50-fold higher activities than psammaplin A vs. Staphylo- to clinically used drugs vancomycin and ciprofloxacin. The yellow dashed line box highlights coccus aureus and MRSA, in this case mainly by a nonspecific redox-based mechanism (reviewed in derivatives [122]). that possessed 50-fold higher activities than psammaplin A vs. Staphylococcus aureus and MRSA, in this case mainly by a nonspecific redox-based mechanism (reviewed in [122]). Mar. Drugs 2023, 21, 279 19 of 37 Identification of Inhibitory Activity of Mammalian APN in Marine Invertebrates from Cuban Coastline Considering the identification of a highly specific inhibitor of TRH-DE in the marine annelide Hermodice carunculata in aqueous extracts from Cuban marine invertebrates, our group extended the screening to porcine and human APN as targets. As part of a first study carried out in 2011–2015 in aqueous extracts from marine invertebrates belonging to the phyla (Table 4), an APN inhibitory activity is detected in all the extracts evaluated except for Lebrunia danae and Hermodice carunculata, whose extracts display values of enzymatic activity higher than those of the control test of porcine APN (pAPN) (Table 4, Figure 15). The L-Leu-pNA substrate is also hydrolyzed when the assay is performed only in the presence of the extract and activity buffer, indicating the possible presence of a neutral aminopeptidase type activity in these two species. The extracts of the species Bryozoo sp. 2, Diplosoma listerianum, Lisoclinum verrilli, Eucidaris tribuloides, and Ophiocoma echinata were selected as the most promising in terms of the specific inhibitory activity of porcine APN as well as a dose-dependent inhibition behavior [125]. The extracts of the Phallusia nigra, Ascidia sidneyense, Microcosmus guanus, Steinacidia turbinata, and Poticlenum constellatum species do not show inhibition at increasing concentrations, indicating that the initial result is probably due to a component of the extract that interferes with the correct determination of the enzyme activity. All the selected extracts, except that of Lisoclinum verrilli, show slow inhibition, in the order of minutes. On the other hand, for the latter, equilibrium is reached within 1 min of preincubation time, suggesting a fast interaction of the inhibitory components of the extract with the porcine APN. The IC50 values are in the range of 0.11–2.39 mg/mL (Table 4), with the highest efficiency being for the extracts of the two sea squirts (Diplosoma listerianum and Lisoclinum verrilli) and that of Bryiozoo sp2. The active extracts were submitted to clarification treatments (such as 2.5% trichloroacetic acid and heat treatments) to eliminate contaminants (mainly proteins) and to promote dissociation from endogenous inhibitor–target complexes. Clarification increases the specific inhibitory activity of the extracts, suggesting that the procedure should be useful in future works dealing with the isolation of the inhibitory molecules [98]. Table 4. Summary of the preliminary characterization of the porcine APN inhibitory activity detected in crude extracts of marine invertebrate species (screening in the period 2011–2015) (adapted from Pascual et al. [125]). pAPN Inhibitory pAPN Specific Pre-Incubation Species Phylum Activity Inhibitory Activity IC50 (mg/mL) Time (min) (U/mL) (U/mg) Phallusia nigra Chordata 2.3060 0.8363 - - Lisoclinum verrilli Chordata 7.8770 0.7361 1 0.11 ± 0.06 Ascidia sidneyensis Chordata 3.1760 0.6481 - - Microcosmus Chordata 7.1368 0.5366 - - guanus Esteinacidia Chordata 3.6340 0.5344 - - turbinata Diplosoma Chordata 8.1300 0.4394 30 0.11 ± 0.26 listerianum Poticlenum Chordata 3.2230 0.2984 - - constellatum Eucidaris tribuloides Echinodermata 6.2526 0.3206 5 1.35 ± 0.19 Ophiocoma echinata Echinodermata 8.2337 0.0874 60 2.39 ± 1.09 Lebrunia danae Echinodermata - - - - Bryozoo sp1 Bryozoa 5.7240 0.0560 - - Bryozoo sp2 Bryozoa 6.7600 1.1266 30 0.29 ± 0.05 Hermodice Annelida - - - - carunculata Mar. Mar. Drugs 2023, 21, Drugs 2023, 21, 279 x 20 of 21 of 37 37 Figure 15. Some of the marine invertebrates screened for inhibitory activity of porcine APN. (A) Figure 15. Some of the marine invertebrates screened for inhibitory activity of porcine APN. Eucidiaris tribuloides, (B) Ophiocoma echinata, (C) Lebrunia danae, (D) Ascidia sydneiensis, (E) Esteinaci- (A) Eucidiaris and dia turbinata, tribuloides, (B) Ophiocoma (F) Phallusia echinata, nigra. Pictures (C) Lebrunia courtesy danae, (D) of Professor JoséAscidia sydneiensis, Espinosa, PhD from Es- (E)ICI- teinacidia MAR, CITMA,turbinata, Cuba.and (F) Phallusia nigra. Pictures courtesy of Professor José Espinosa, PhD from ICIMAR, CITMA, Cuba. In a second study performed in 2015–2019, aqueous extracts from species belonging to the phyla Mollusca, Poriphera, Echinodermata, and Cnidaria (Figure 16) were screened using human placental APN as the target (Table 5). The initial evaluations allowed detection of an inhibitory activity of hAPN only from the species Cenchritis muricatus and Isostichopus Mar. Drugs 2023, 21, 279 21 of 37 badionotus. Increased L-Leu-AMC hydrolysis rates over the control value are found instead of inhibitory activities for the rest of the species. These results suggest either the presence of an activator of the target enzymes used in the assays or neutral aminopeptidase-like enzymes hydrolyzing L-Leu-AMC in the corresponding aqueous extracts. The clarification of all aqueous crude extracts (2.5% TCA treatment) increased, in all cases, the recovery of specific inhibitory activities as compared to their detection in positive crude extracts. The treatment also allowed the identification of inhibitory activities from species that were negative after screening using aqueous crude extracts. This result indicated that clarification eliminates contaminants and/or induces dissociation from endogenous inhibitor–target complexes that do not allow the detection of inhibitory components in crude extracts. Table 5. Summary of the screening of phyla Mollusca, Poriphera, Echinodermata, and Cnidaria for inhibitory activity against human APN (hAPN) (see note below). Adapted from [98]. 2.5% TCA Treated NA-like Activity Crude Extracts hAPN Species Phylum Extracts hAPN sIA (×104 U/mg) sIA (U/mg) (U/mg) Cenchritis muricatus Mollusca ND 0.46 5.20 Nerita peloronta Mollusca 6.95 ± 2.84 ND 5.06 Nerita versicolor Mollusca 12.56 ± 1.70 ND 2.21 Lissodendoryx (Lissodendoryx) Porifera 1.51 ± 0.49 ND 171.92 isodictyalis Tripneustes ventricosus Echinodermata 6.39 ± 0.07 ND 56.86 Echinaster (Othilia) Echinodermata 58.87 ± 12.62 ND 10.82 echinophorus Isostichopus badionotus Echinodermata ND 1.56 33.81 Stichodactyla helianthus Cnidaria 9.63 ± 2.68 ND 32.81 Bunodosoma Cnidaria 39.44 ± 5.45 ND 4.31 granuliferum Physalia physalis Cnidaria 2.04 ± 0.52 ND 13.13 NOTE: hAPN inhibitory activities in aqueous crude and 2.5% TCA extracts are expressed as specific Inhibitory Activity (sIA) in U/mg. One unit of enzyme activity was defined as the amount of enzyme needed to produce one arbitrary unit of fluorescence per minute, and inhibitory activities are expressed per mg of extract. The first column indicates tested species, the second column shows neutral aminopeptidase-like activity (NA) detected in aqueous crude extracts using L-Leu-AMC as substrate, and the remaining columns refer to sIA. (ND): not detected. The clarified extracts (2.5% TCA) from the species Cenchritis muricatus, Nerita peloronta, Nerita versicolor, Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventricosus, Echinaster (Othilia) echinophorus, Isostichopus badionotus, Stichodactyla helianthus, Bunodosoma gran- uliferum, and Physalia physalis were used to continue the inhibition studies vs. human APN (hAPN) [98]. Additionally, the enhanced activities over the control detected in some extracts were lost, in all cases, after the 2.5% TCA treatments, suggesting susceptibility of the active molecules to the chaotropic agent and/or to the acidic pH of the molecule(s) responsible for these effects (Table 5). To test the presence of neutral aminopeptidase-like enzymes in aqueous crude extracts, preliminary enzymatic assays were performed using different amounts of the samples (in absence of the initial target hAPN) in the presence of L-Leu-AMC. A linear dependence of the initial rate versus the amount of crude extract in the assays was detected for the species Nerita peloronta, Nerita versicolor, Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventri- cosus, Echinaster (Othilia) echinophorus, Stichodactyla helianthus, Bunodosoma granuliferum, and Physalia physalis. These neutral aminopeptidase-like activities were recently characterized by a kinetic approach combining substrates, inhibitors, and cations, showing for the first time a biochemical behavior indicative of the presence of M1 and M17 enzymes in these species [30]. Mar. Drugs 2023, 21, 279 22 of 37 Mar. Drugs 2023, 21, x 23 of 37 Figure 16. Some of the marine invertebrates which show inhibitory activity of pAPN, pAPA, Figure hAPN, Some 16.and of the marine rPfA-M17. invertebrates (A) Cenchritis which(B) muricatus, show inhibitory Nerita activity versicolor, of pAPN, pAPA, (C) Lissodendoryx hAPN, (Lissoden- doryx) isodictyalis, (D) Tripneustes ventricosus, (E) Echinaster (Othilia) echinophorus, (F) Isostichopus iso- and rPfA-M17. (A) Cenchritis muricatus, (B) Nerita versicolor, (C) Lissodendoryx (Lissodendoryx) dictyalis, Tripneustes (D) (G) badionotus, ventricosus, Stichodactyla Echinaster (E)and helianthus, (Othilia) granuliferum. (H) Bunodosoma echinophorus,Pictures (F) Isostichopus courtesybadionotus, of Pro- fessor José Espinosa, PhD from ICIMAR, CITMA, Cuba. (G) Stichodactyla helianthus, and (H) Bunodosoma granuliferum. Pictures courtesy of Professor José Espinosa, PhD from ICIMAR, CITMA, Cuba. Mar. Drugs 2023, 21, 279 23 of 37 The clarified extracts inhibit hAPN activity in a dose-dependent manner, and the inhibition was characterized by a concave behavior, indicating the reversibility of the inhibition and corroborating the presence of inhibitory molecules in the samples (and not artifacts interfering with the enzyme activity). The IC50 values are in the range of 11.7–567.6 µg/mL [98]. As a promissory result, hAPN is inhibited with IC50 values around or less than 100 µg/mL in four of the ten species tested (Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventricosus, Isostichopus badionotus, and Stichodactyla helianthus); this inhibition is stronger than that produced by bestatin or amastatin (pure compounds) assayed in parallel as controls (Table 5) [98]. Taking into account that the effect of the inhibition of hAPN was corroborated, the effect of each treated extract on the viability of two APN+ cancer cell lines PC3 and 3LL was evaluated [98]. All treated extracts, and bestatin used as a positive control, have a dose- dependent effect on PC3 and 3LL cell viability. The higher effects on both cell lines, with IC50 values below 100 µg/mL, are observed for the species showing the strongest hAPN inhibition. An IC50 value under 5 µg/mL for L. isodictyalis extract vs. both cancer cells lines, similar to the effect displayed by bestatin, indicates that this species is promissory for the isolation of hAPN inhibitors. In this work, the IC50 values for cell viability are in good agreement with the IC50 values for hAPN inhibition, including the bestatin results. To the best of our knowledge, this work was the first to show concomitantly natural inhibitor potency on hAPN and indications of activity on hAPN-expressing cells [98]. Inhibitors of Aminopeptidase A Isolated from Marine Organisms Membrane glutamyl aminopeptidase, also known as acidic aminopeptidase (APA, EC 3.4.11.7), is a type II membrane protein of the M1 family, MA subclade (E), of met- allopeptidases [8] (Figure 1B). This enzyme is widely distributed in mammalian tissues. Aminopeptidase A (APA) has been reported to have molecular weights around 109 kDa for the human and 108 kDa for the porcine enzyme [1]. APA’s S1 pocket accommodates acid residue side chains, whereby this enzyme hydrolyzes aspartic and glutamic residues from the peptide N-terminus [77]. APA performs fundamental functions in a wide range of physiological processes, since it participates in the metabolism of angiotensin II, involved in the renin–angiotensin system in the central nervous system and other anatomical locations, making it an important regulator of blood pressure (Figure 17) [126]. In addition, it is involved in the development of Alzheimer’s disease and glomerulosclerosis and in the progression of cancer. It is associated with the development of renal neoplasms, malignant trophoblasts, renal choriocarcinoma, and colorectal cancer [126–130]. APA plays a key role in blood pressure regulation, which has made it a promising therapeutic target for the development of antihypertensive agents (Figure 17) [30,126,131]. Recently, we extended the screening of marine organism extracts to porcine aminopep- tidase A (pAPA). We observed that extracts from the species Nerita peloronta, Nerita versicolor, Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventricosus, Echinaster (Othilia) echinopho- rus, Isostichopus badionotus, and Stichodactyla helianthus displayed dose-dependent inhibition of porcine APA activity, with IC50 values in the range of 11.00–1005.00 µg/mL (Table 6), showing that Nerita versicolor has a certain selectivity for pAPA rather than for hAPN. These results strongly support the exploration of marine fauna of invertebrates as promissory sources of inhibitors of M1 family enzymes with potential biomedical applications, such as APN and APA. ta versicolor, Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventricosus, Echinaster (Othilia) echinophorus, Isostichopus badionotus, and Stichodactyla helianthus displayed dose- dependent inhibition of porcine APA activity, with IC50 values in the range of 11.00– 1005.00 µg/mL (Table 6), showing that Nerita versicolor has a certain selectivity for pAPA rather than for hAPN. These results strongly support the exploration of marine fauna of Mar. Drugs 2023, 21, 279 24 of 37 invertebrates as promissory sources of inhibitors of M1 family enzymes with potential biomedical applications, such as APN and APA. Asp-Arg-Val—Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr Angiotensinogen Renin Asp-Arg-Val—Tyr-Ile-His-Pro-Phe-His-Leu Angiotensin I Bradykinin ACE Asp-Arg-Val—Tyr-Ile-His-Pro-Phe Inactive kinins Angiotensin II APA Major effector Arg-Val-Tyr-Ile-His-Pro-Phe peptide in Angiotensin III systemic RAS Major effector peptide in Central Nervous System RAS Vascular smooth Aldosterone muscle contraction secretion APN Val-Tyr-Ile-His-Pro-Phe Increased extracelular fluid Angiotensin IV Increased peripheral volume by increased Na+ vascular resistance and water retention HYPERTENSION Figure Figure17. 17.Role Roleofofaminopeptidase aminopeptidaseAAand andaminopeptidase aminopeptidaseNNininthe therenin–angiotensin renin–angiotensinsystems. systems. 2.5. Clan MF: Family M17 Clan MF contains aminopeptidases that require cocatalytic metal ions for activity. The clan contains only the single family M17, a family of leucyl aminopeptidases [1], sum- marized in Table 7. The M17 aminopeptidases utilize two divalent metal ion cofactors to catalyze the removal of selected N-terminal amino acids from short peptide chains. M17 aminopeptidases are found in all kingdoms (Figure 18), wherein they possess a charac- teristic homo-hexameric three-dimensional arrangement of their monomers (Figure 19) and play roles in a wide range of cellular processes [9]. The proteolytic reaction con- tributes to intracellular protein turnover, a fundamental housekeeping process across all living organisms [132] (Figure 18). However, a wide range of additional functions be- yond aminopeptidase activity have also been attributed to M17 family members. M17 aminopeptidases from plants possess chaperone activity [133], which might contribute to their function in the stress response pathway [134], while in bacteria they play roles in site-specific DNA recombination [135], and further, can moderate transcription of key virulence factors [136]. Therefore, the family of M17 aminopeptidases is multifunctional, capable of performing diverse organism-specific functions far beyond peptide hydrolysis (Figure 18). Mar. Drugs 2023, 21, 279 25 of 37 Table 6. Summary of the preliminary characterization of inhibitory activities against porcine APA, porcine APN, and human APN in TCA 2.5% treated crude extracts from marine invertebrates (screening in the period 2015–2019). Effect on 3LL and PC3 tumor cell viability (adapted from [98]). ND: not determined. IC50 vs. 3LL IC50 vs. PC3 IC50 vs. pAPA IC50 vs. pAPN IC50 vs. hAPN Species Phylum Viability Viability (µg/mL) (µg/ML) (µg/mL) (µg/mL) (µg/mL) Cenchritis Mollusca ND ND 450.20 ± 77.40 214.00 ± 46.50 352.90 ± 65.00 muricatus Nerita peloronta Mollusca 487 ± 11.03 287.03 ± 12.00 237.80 ± 20.90 273.30 ± 78.80 299.10 ± 31.70 Nerita versicolor Mollusca 92.23 ± 12.34 132.11 ± 22.05 370.00 ± 50.00 358.80 ± 70.20 289.70 ± 39.70 Lissodendoryx (Lissodendoryx) Porifera 659.87 ± 10.65 613.24 ± 10.76 11.70 ± 2.70 <5.00 <5.00 isodictyalis Tripneustes Echinodermata 11.03 ± 0.12 13.35 ± 3.91 25.00 ± 3.10 39.90 ± 2.00 77.00 ± 3.90 ventricosus Echinaster (Othilia) Echinodermata 182.01 ± 67.12 112.55 ± 23.21 198.20 ± 27.20 265.70 ± 29.60 405.60 ± 50.40 echinophorus Isostichopus Echinodermata 1005.12 ± 293.32 11.08 ± 0.27 69.70 ± 10.00 57.10 ± 2.70 83.10 ± 3.00 badionotus Stichodactyla Cnidaria 256.3 ± 10.00 136.56 ± 22.87 103.60 ± 20.60 110.80 ± 13.20 58.10 ± 7.50 helianthus Bunodosoma Cnidaria ND 98.02 ± 18.05 567.60 ± 88.00 786.80 ± 37.10 711.30 ± 29.30 granuliferum Physalia physalis Cnidaria ND 198.92 ± 10.76 123.10 ± 21.30 257.30 ± 6.70 234.50 ± 5.00 Bestatin (positive - 25.50 ± 2.35 6.54 ± 0.82 6.70 ± 1.90 0.54 ± 0.01 3.15 ± 0.72 control) Amastatin (positive - 75.45 ± 4.55 58.32 ± 3.34 63.45 ± 7.61 ND ND control) Table 7. Some members of the M17 family of the MF clan of metallopeptidases. (Compiled from https://www.ebi.ac.uk/merops/cgi-bin/, accessed on 11 January 2023). IUBMB * Enzyme Merops ID Sources Nomenclature Homo sapiens, Haemaphysalis Leucyl aminopeptidase 3 3.4.11.1 M17.001 longicornis, Mus musculus Leucyl aminopeptidase (plant-type) 3.4.11.1 M17.002 Solanum lycopersicum Escherichia coli, Pseudomonas PepA aminopeptidase 3.4.11.10 M17.003 aeruginosa Escherichia coli, Salmonella PepB aminopeptidase 3.4.11.23 M17.004 typhimurium Mername-AA040 peptidase - M17.005 Homo sapiens Leucyl aminopeptidase-1 (Caenorhabditis-type) - M17.006 Caenorhabditis elegans M17 aminopeptidase (Plasmodium spp.) 3.4.11.1 M17.008 Plasmodium spp. Aminopeptidase yspII (Schizosaccharomyces sp.) - M17.009 Schizosaccharomyces sp. Leucyl aminopeptidase (Bacillus-type) - M17.010 Geobacillus kaustophilus Leucyl aminopeptidase (Fasciola-type) - M17.011 Fasciola hepática PwLAP aminopeptidase - M17.012 Paragonimus westermani Cysteinylglycinase (Treponema denticola)-like peptidase - M17.013 Treponema denticola Trypanosoma cruzi, Leishmania LAPTc aminopeptidase - M17.014 major Aminopeptidase pepZ (Staphylococcus sp.) - M17.015 Staphylococcus aureus Aminopeptidase A/I (Helicobacter-type) - M17.016 Helicobacter pylori similar to cytosol aminopeptidase (Rattus norvegicus) - M17.950 Rattus norvegicus At4g30920 (Arabidopsis thaliana) - M17.A01 Arabidopsis thaliana At4g30910 (Arabidopsis thaliana) - M17.A02 Arabidopsis thaliana At2g24200 (Arabidopsis thaliana) - M17.A03 Arabidopsis thaliana CG7340 g.p. (Drosophila melanogaster) - M17.A04 Drosophila melanogaster ZK353.6 (Caenorhabditis elegans) - M17.A05 Caenorhabditis elegans * IUBMB: International Union of Biochemistry and Molecular Biology. similar to cytosol aminopeptidase (Rattus norvegicus) - M17.950 Rattus norvegicus At4g30920 (Arabidopsis thaliana) - M17.A01 Arabidopsis thaliana At4g30910 (Arabidopsis thaliana) - M17.A02 Arabidopsis thaliana At2g24200 (Arabidopsis thaliana) - M17.A03 Arabidopsis thaliana CG7340 g.p. (Drosophila melanogaster) - M17.A04 Drosophila melanogaster Mar. Drugs 2023, 21, 279 26 of 37 ZK353.6 (Caenorhabditis elegans) - M17.A05 Caenorhabditis elegans * IUBMB: International Union of Biochemistry and Molecular Biology. Mar. Drugs 2023, 21, x 28 of 37 Figure Figure 18. 18. Functions Functions of of M17 M17 aminopeptidases aminopeptidases from from different life groups. different life groups. Leucyl aminopeptidases are also distributed in Apicomplexan protist parasites such as Plasmodium falciparum, the main agent of malaria in humans. The most important clin- ical stage of the complex P. falciparum life cycle [137], which has attracted the highest at- tention for the development of antimalarials, takes place in the human erythrocyte, where significant hemoglobin degradation occurs under the concerted action of endo- and exopeptidases [138]. PfA-M17 is involved in the final steps of hemoglobin digestion [139] and is currently a promising chemotherapeutic target as its inhibitors can kill para- sites in vitro and in vivo (Figure 1) [9,140,141]. Figure19. Figure 19. Cartoon Cartoon representation representation of of different different M17 M17 family family aminopeptidases: aminopeptidases:(A) (A)Bos Bostaurus taurusleucyl leucyl aminopeptidase 3 (PDB ID: 1bll), (B) Escherichia coli PepA aminopeptidase (PDB ID: 1gyt), aminopeptidase 3 (PDB ID: 1bll), (B) Escherichia coli PepA aminopeptidase (PDB ID: 1gyt), and (C) and ZK353.6 (Caenorhabditis elegans) (PDB ID: 42hc9). Colors: alpha-helices (cyan), beta sheets (warm (C) ZK353.6 (Caenorhabditis elegans) (PDB ID: 42hc9). Colors: alpha-helices (cyan), beta sheets (warm pink), and loops (salmon). The zinc atoms are shown as gray spheres highlighted in a yellow box. pink), and loops (salmon). The zinc atoms are shown as gray spheres highlighted in a yellow box. Through Leucyl the techniqueare aminopeptidases ofalsocrystallography and X-ray diffraction, distributed in Apicomplexan the three- protist parasites such dimensional structure of several members of this family has been as Plasmodium falciparum, the main agent of malaria in humans. The most important elucidated, such as leucyl aminopeptidase clinical 3 fromP.Bos stage of the complex taurus, life falciparum leucyl aminopeptidase cycle [137], which has(plant-type) attracted from Sola- the highest num lycopersicum, PepA aminopeptidasa from Escherichia coli and other attention for the development of antimalarials, takes place in the human erythrocyte, bacteria, and where significant hemoglobin degradation occurs under the concerted action of endo- the PfA-M17 from Plasmodium falciparum among others, being PfA-M17, joint with and mammalian enzyme, exopeptidases the more [138]. PfA-M17 representative is involved in the in tridimensional final structuredigestion steps of hemoglobin available[139] at PDB, supporting the relevance of the search for new inhibitors as potential and is currently a promising chemotherapeutic target as its inhibitors can kill parasites new antima- larial in vitrochemotherapy agents 1) and in vivo (Figure (Table 8, Figure 19). [9,140,141]. Natural inhibitors of leucine aminopeptidases are scarce and have mainly been de- scribed from microorganisms sharing unspecific inhibition of APN and other M1 family inhibitors, such as actinonin, amastatin, bestatin, and various bestatin derivaties (Figures 12 and 20) [18,139,142]. Several compounds from marine organisms have been described with anticancer and antiplasmodial activities [103,106,143–146]; however, only two of Mar. Drugs 2023, 21, 279 27 of 37 Mar. Drugs 2023, 21, x 28 of 37 Through the technique of crystallography and X-ray diffraction, the three-dimensional structure of several members of this family has been elucidated, such as leucyl aminopepti- dase 3 from Bos taurus, leucyl aminopeptidase (plant-type) from Solanum lycopersicum, PepA aminopeptidasa from Escherichia coli and other bacteria, and PfA-M17 from Plasmodium falciparum among others, being PfA-M17, joint with the mammalian enzyme, the more representative in tridimensional structure available at PDB, supporting the relevance of the search for new inhibitors as potential new antimalarial chemotherapy agents (Table 8, Figure 19). Table 8. Crystallographic structures reported for members of the M17 family of metallopeptidases (Compiled from https://www.ebi.ac.uk/merops/cgi-bin/, accessed on 11 January 2023). Crystallographic Codes of the Enzyme Source Structures at Protein Data Bank (PDB) 1BLL, 1BPM, 1BPN, 1LAM, 1LAN, 1LAP, Leucyl aminopeptidase 3 Bos taurus Figure 19. Cartoon representation of different M17 family1LCP,aminopeptidases: 2EWB, 2J9A (A) Bos taurus leucyl Leucyl aminopeptidase (plant-type) Solanum lycopersicum 4KSI, 5D8N aminopeptidase 3 (PDB ID: 1bll), (B) Escherichia coli PepA aminopeptidase (PDB ID: 1gyt), and (C) Escherichia colielegans) (PDB ID: 42hc9). Colors: ZK353.6 (Caenorhabditis 1GYT alpha-helices (cyan), beta sheets (warm Francisella tularensis 3PEI PepA aminopeptidase pink), and loops (salmon). The zinc atoms are shown as gray spheres highlighted in a yellow box. Pseudomonas putida 3H8E, 3H8F, 3H8G Xanthomonas oryzae 3JRU Through the coli Escherichia technique of crystallography 6OV8 and X-ray diffraction, the three- PepB aminopeptidase dimensional structure Yersinia pestis of several members of this 6CXDfamily has been elucidated, such as leucyl aminopeptidase 3 from Bos taurus, leucyl aminopeptidase 3KQX, 3KQZ, 3KR4, (plant-type) 3KR5, 3T8W, from Sola- 4K3N, M17 aminopeptidase (Plasmodium spp.) Plasmodium falciparum num lycopersicum, PepA aminopeptidasa from Escherichia 4R6T, 4R7M,coli 4X2Tand other bacteria, and LAPTc aminopeptidase PfA-M17 Leishmania major from Plasmodium 5NTH being PfA-M17, joint with the falciparum among others, Trypanosoma brucei 5NSK, 5NSM, 5NSQ, 5NTD mammalian enzyme, the more representative in tridimensional structure available at Aminopeptidase A/I (Helicobacter-type) Helicobacter pylori 4ZI6, 4ZLA ZK353.6 (Caenorhabditis elegans) PDB, supporting the relevance Caenorhabditis elegans of the search for new2HB6, inhibitors 2HC9 as potential new antima- larial chemotherapy agents (Table 8, Figure 19). Natural inhibitors of leucine aminopeptidases are scarce and have mainly been de- scribed frominhibitors Natural of leucine microorganisms aminopeptidases sharing are scarce unspecific inhibition ofand APNhave andmainly other M1 been de- family scribed from microorganisms sharing unspecific inhibition of APN and other M1 inhibitors, such as actinonin, amastatin, bestatin, and various bestatin derivaties (Figures fam- ily 12 inhibitors, such as actinonin, and 20) [18,139,142]. amastatin,from Several compounds bestatin, marine and various have organisms bestatin beenderivaties described (Figures 12 and 20) [18,139,142]. Several compounds from marine organisms with anticancer and antiplasmodial activities [103,106,143–146]; however, only havetwobeenof described them have been linked with LAP inhibition or interaction [102,104,105]. In the nextonly with anticancer and antiplasmodial activities [103,106,143–146]; however, sec- two of them have been linked with LAP inhibition or interaction [102,104,105]. tion, we review the information available regarding marine organisms as a promissory In the next section, and stillwe review thesource unexplored information available of inhibitors regarding of M17 enzymemarine organisms inhibitors as a promis- with biomedical rele- sory and still unexplored source vance, mainly in cancer and malaria. of inhibitors of M17 enzyme inhibitors with biomedical relevance, mainly in cancer and malaria. Figure20. Figure 20.Examples Examplesofofnatural naturalinhibitors inhibitorsofofM17 M17enzymes. enzymes. 2.6. Inhibitors Table of M17 Leucyl 8. Crystallographic Aminopeptidases structures from reported for MarineofOrganisms members the M17 family of metallopeptidases (Compiled In the from workhttps://www.ebi.ac.uk/merops/cgi-bin/, of Pascual et al. [98], the screening accessed on 11 January of inhibitory 2023.in aqueous activities extracts from species belonging to the phyla Mollusca, Poriphera, Echinodermata, and Crystallographic Codes of the Structures Enzyme from the Cuban coastline Cnidaria Sourceinvolved human LAP (hLAP) and a recombinant form at Protein Data Bank (PDB) of PfA-M17 (rPfA-M17), both leucil aminopeptidases. As a result of preliminary assays, 1BLL, 1BPM, 1BPN, 1LAM, 1LAN, 1LAP, Leucyl aminopeptidase inhibitory 3 activity vs. hLAP Bosdetected was taurus in the species Cenchritis muricatus, Lissodendoryx 1LCP, 2EWB, 2J9A (Lissodendoryx) isodyctialis, Leucyl aminopeptidase Isostichopus badionotus, and Stichodactyla helianthus. Inhibitory ac- (plant- Solanum lycopersicum 4KSI, 5D8N type)against rPfA-M17 was detected in the species Cenchritis muricatus, Echinaster (Othilia) tivity echinophorus, Isostichopus badionotus, Physalia Escherichia coli physalis, Stichodactyla helianthus, and Buno- 1GYT PepA aminopeptidase Francisella tularensis 3PEI Pseudomonas putida 3H8E, 3H8F, 3H8G Mar. Drugs 2023, 21, 279 28 of 37 dosoma granuliferum (specific inhibitory activity values are summarized in Table 9, Figure 16), i.e., in four different phyla. The clarification of all aqueous crude extracts with a 2.5% TCA treatment increased the recovery of specific inhibitory activities as compared to their detec- tion in positive crude extracts. The treatment also allowed the identification of inhibitory activities from species that were negative after screening using aqueous crude extracts. This result indicated that this clarification step was useful in the elimination of contaminants and/or induced dissociation from endogenous inhibitor–target complexes that did not allow the detection of inhibitory components in crude extracts. Table 9. Summary of the screening of inhibitory activity against malarial rPfA-M17 and hLAP in aqueous extracts from marine invertebrates from the Cuban coastline (see note below). (adapted from Pascual et al. [98]). Crude Extract 2.5% TCA Treated 2.5% TCA Treated Crude Extract Species Phyla rPfA-M17 Extracts rPfA-M17 Extracts hLAP sIA hLAP sIA (U/mg) sIA (U/mg) sIA (U/mg) (U/mg) Cenchritis muricatus Mollusca 0.40 0.16 7.56 4.30 Nerita peloronta Mollusca ND ND 10.61 6.48 Nerita versicolor Mollusca ND ND 7.17 5.58 Lissodendoryx (Lissodendoryx) Porifera ND 0.27 256.13 312.01 isodictyalis Tripneustes Equinodermata ND ND 41.85 43.31 ventricosus Echinaster (Othilia) Equinodermata 0.11 ND 9.10 7.85 echinophorus Isostichopus Equinodermata 0.18 1.24 55.83 27.86 badionotus Stichodactyla Cnidaria 0.55 0.68 38.15 18.67 helianthus Bunodosoma Cnidaria 0.19 ND 5.45 4.08 granuliferum Physalia physalis Cnidaria 0.40 ND 19.21 11.00 NOTE: Inhibitory activities found in aqueous crude and 2.5% TCA extracts are expressed as specific Inhibitory Activity (sIA) in U/mg. One unit of enzyme activity was defined as the amount of enzyme needed to produce one arbitrary unit of fluorescence (AUF) per minute, and inhibitory activities are expressed per mg of extracts. The first column indicates the species, and the remaining columns refer to specific sIA. (ND): not detected. All the clarified extracts showed inhibitory activity against both peptidases: malarial rPfA-M17 and native human LAP (Table 10, Figure 16). These activities have a concave dose-response behavior, corroborating the presence of reversible inhibitory molecules with IC50 values in µg/mL for both enzymes tested (Table 10). Inhibition of rPfA-M17 with IC50 values up to ~100 µg/mL is detected for 6 of the 10 extracts (those of Cenchritis muricatus, Nerita perolonta, Lissodendoryx (Lissodendoryx) isodictyalis, Tripneustes ventricosus, Isostichopus badionotus, and Stichodactyla helianthus). Comparing the inhibitions on rPfA-M17 and hLAP, in all cases, the plasmodial enzyme was more susceptible than its human counterpart, with ratios of selectivity between 1.87 and 60 times. The most selective extract was from Nerita versicolor, an attractive result even if its IC50 value for the malarial enzyme is moderate. All of the treated extracts displayed a dose-dependent effect on a chloroquine-resistant Plasmodium falciparum strain (FcB1) cell’s viability, with the exception of Cenchritis muricatus, Stichodactyla helianthus, and Bunodosoma granuliferum. The best effects were obtained for Tripneustes ventricosus and Lissodendoryx (Lissodendoryx) isodictyalis. Particularly attractive were the T. ventricosus extracts that displayed a 300 times more potent effect on the FcB1 strain of P. falciparum than the human cancer cells, indicating parasite effect specificity. Additionally, this effect of the T. ventricosus extract on the FcB1 strain was stronger than the effect of bestatin (a pure compound). Due to the fact that IC50 for L. isodyctialis and T. ventricosus were lower on the FcB1 strain of P. falciparum than on the PfA-M17 recombinant Mar. Drugs 2023, 21, 279 29 of 37 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 IC50 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 IC50 in the same order of enzyme inhibition, indicating that this species is also attractive. These results are the first and still only report of inhibition of M17 enzymes (human and plasmodial) by marine invertebrate species aqueous extracts and supports that sponges as well as marine invertebrates from other phyla such as mollusks, echinoderms, and cnidarians are a good and still underexplored source of potential anticancer and antimalarials associated with the inhibition of neutral aminopeptidases from the M1 and M17 families involved in these human pathologies. Table 10. Summary of the IC50 determination for the 2.5% TCA clarified extracts against rPfA- M17 and hLAP. Preliminarndicates position with high degree of conservation of the resy inhibitory analyses on the growth of Plasmodium falciparum FcB1 strain are included in the last column (adapted from Pascual et al. [98]). IC50 Value vs. IC50 hLAP/ Species IC50 vs. hLAP (µg/mL) IC50 Pf FcB1 (µg/mL) rPfA-M17 (µg/mL) IC50 rPfA-M17 Cenchritis muricatus 113.40 ± 3.00 341.00 ± 110.00 3.00 >400 Nerita peloronta 22.20 ± 2.70 329.50 ± 100.00 14.80 291.80 ± 38.50 Nerita versicolor 207.00 ± 30.60 12 429.60 ± 633.00 60.00 325.50 ± 0.80 Lissodendoryx (Lissodendoryx) 27.30 ± 9.40 66.30 ± 27.60 2.42 2.60 ± 0.60 isodictyalis Tripneustes ventricosus 84.80 ± 7.30 607.70 ± 300.50 7.16 0.24 ± 0.01 Echinaster (Othilia) 127.50 ± 82.10 308.70 ± 100.00 2.42 201.60 ± 162.10 echinophorus Isostichopus badionotus 86.70 ± 32.60 272.70 ± 63.50 3.13 183.70 ± 155.70 Stichodactyla helianthus 15.30 ± 6.20 234.90 ± 34.60 15.35 >400 Bunodosoma granuliferum 509.20 ± 100.90 1171.00 ± 92.10 2.29 >400 Physalia physalis 293.70 ± 100.00 550.00 ± 85.00 1.87 206.00 ± 84.00 Bestatin (positive control) 0.15 ± 0.02 11.83 ± 2.61 78.86 1.14 ± 0.27 Amastatin (positive control) 60.70 ± 19.84 158.05 ± 28.44 2.60 ND M17 enzymes are not only a very well established target for malaria and other parasitic diseases but also for bacterial infections and chronic pathologies such as cancer. Bacterial LAPs (from gram-negative or -positive bacteria such as Escherichia coli, Aeromonas prote- olytica, Streptomyces lividans, and Pseudomonas aeruginosa, among others) are important virulence factors [147]. Leucine aminopeptidase 3 in humans (LAP3) is associated with various diseases and cancers, such as breast cancer and ovarian cancer [148]. Recently, Yang et al. [149] identified two compounds named compounds 5 and 6 (Figure 21) from 43 natural marine products screened as new inhibitors of LAP3 (from K562 cells with overexpression of LAP3). The inhibition of LAP3 at 30 µM by these two compounds was stronger than that of bestatin used as a control of inhibition in the same conditions. The authors explored the anticancer properties of these new compounds in different models of breast cancer. The results showed that compounds 5 and 6 displayed stronger antipro- liferative activity of the breast cancer tumor cells MDA-MB-231 at 30 µM than bestatin. Additionally, both compounds 5 and 6 displayed a more potent suppression effect on the migration of MDA-MB-231 cells than bestatin (the effect of compound 5 was stronger than that of compound 6). It is well established that LAP3 plays an important role in the metastasis of breast cancer; hence LAP3 inhibitors may have a remarkable effect on the treatment of breast cancer [149]. an important role in the metastasis of breast remarkable effect on the treatment of breast c Mar. Drugs 2023, 21, 279 30 of 37 Figure 21. Compounds 5 and 6 as new inhibitors of LAP3 (adapted from Yang et al. [149]). Figure3.21. Compounds 5 and 6 as new inhibitors o Conclusions Marine biodiversity is an important and promising source of inhibitors of metalloex- opeptidases from different families, in particular M1 and M17 enzymes with biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further fundamental applicative studies with inhibitors isolated from ma- 3. Conclusions rine species in different biomedical models associated with the activity of these families of exopeptidases. Marine biodiversity is an important an Author Contributions: Conceptualization, I.P.A. and F.X.A.; writing—original draft preparation I.P.A., F.A.G., M.E.V.T., Y.A.S., D.O.d.S., B.S.R., I.F., M.S. and F.X.A.; writing—review and editing, I.P.A., F.A.G. and F.X.A.; visualization, I.P.A. and M.E.V.T. All authors have read and agreed to the loexopeptidases from different families, in p published version of the manuscript. Funding: This research was funded by the French ANR-12-BS07-0020 project MAMMAMIA: “design medical applications in human diseases. Th of potential anti MAlarial M1/M17 AMinopeptiIdase Agents” and UH-CIM project: “New inhibitors of aminopeptidases with potential applications in cancer” (2020–2023). tion support and encourage further fundame Data Availability Statement: Not applicable. Acknowledgments: This contribution is dedicated to María de los Angeles Chávez, renowned Cuban lated from marine species in different biome Biochemistry Professor, founder of the teaching of Biochemistry in Cuba and the Center for Protein Studies at the University of Havana. She significantly contributed to the knowledge in the field of peptidase inhibitors of different mechanistic classes isolated from Cuban marine biodiversity. To these families of exopeptidases. Jean Louis Charli, IBT-UNAM, Cuernavaca, Mexico, for the revision of the manuscript and useful comments. To Aymara Cabrera Munoz, from the Center for Protein Studies, Faculty of Biology, University of Havana, who kindly supplied images for Figure 4. To José Espinosa, PhD from ICIMAR, CITMA, Cuba, who kindly supplied the pictures of the marine species. To IFS-OPCW research grants 3276/1, 3276/2, 3276/3, to Isel Pascual Alonso. To IUBMB mid-career fellowship program for Author Contributions: Conceptualization, I.P.A. supporting the research stay of Isel Pascual Alonso at Laboratory MCAM, UMR 7245, MNHN, Paris, in 2014 and in Instituto de Biotecnología, UNAM, Cuernavaca, Mexico, in 2017. To the French ANR- 12-BS07-0020 project MAMMAMIA: “design of potential anti MAlarial M1/M17 AMinopeptiIdase I.P.A., F.A.G., M.E.V.T., Y.A.S., D.O.d.S., B.S.R., I.F Agents”. To UH-CIM project: “New inhibitors of aminopeptidases with potential applications in cancer” (2020–2023). I.P.A., F.A.G., and F.X.A.; visualization, I.P.A. an Conflicts of Interest: The authors declare no conflict of interest. References 1. the published version of the manuscript. Rawlings, N.D.; Barrett, A.J.; Thomas, P.D.; Huang, X.; Bateman, A.; Finn, R.D. The Merops Database of Proteolytic Enzymes, Their Substrates and Inhibitors in 2017 and a Comparison with Peptidases in the Panther Database. Nucleic Acids Res. 2018, 46, D624–D632. [CrossRef] [PubMed] 2. Funding: This research was funded by the French Drag, M.; Salvesen, G.S. Emerging Principles in Protease-Based Drug Discovery. Nat. Rev. Drug Discov. 2010, 9, 690–701. [CrossRef] [PubMed] sign of potential anti MAlarial M1/M17 AMinopep hibitors of aminopeptidases with potential applica Mar. Drugs 2023, 21, 279 31 of 37 3. Deu, E.; Verdoes, M.; Bogyo, M. New Approaches for Dissecting Protease Functions to Improve Probe Development and Drug Discovery. Nat. Struct. Mol. Biol. 2012, 19, 9–16. [CrossRef] [PubMed] 4. Kryvalap, Y.; Czyzyk, J. The Role of Proteases and Serpin Protease Inhibitors in B-Cell Biology and Diabetes. Biomolecules 2022, 12, 67. [CrossRef] 5. Rai, M.; Curley, M.; Coleman, Z.; Demontis, F. Contribution of Proteases to the Hallmarks of Aging and to Age-Related Neurodegeneration. Aging Cell 2022, 21, e13603. [CrossRef] 6. Verhulst, E.; Garnier, D.; De Meester, I.; Bauvois, B. Validating Cell Surface Proteases as Drug Targets for Cancer Therapy: What Do We Know, and Where Do We Go? Cancers 2022, 14, 624. [CrossRef] 7. Newman, D.J.; Cragg, G.M. Drugs and Drug Candidates from Marine Sources: An Assessment of the Current State of Play. Planta Med. 2016, 82, 775–789. [CrossRef] 8. Drinkwater, N.; Lee, J.; Yang, W.; Malcolm, T.R.; McGowan, S. M1 Aminopeptidases as Drug Targets: Broad Applications or Therapeutic Niche? FEBS J. 2017, 284, 1473–1488. [CrossRef] 9. Drinkwater, N.; Malcolm, T.R.; McGowan, S. M17 Aminopeptidases Diversify Function by Moderating Their Macromolecular Assemblies and Active Site Environment. Biochimie 2019, 166, 38–51. [CrossRef] 10. Hammers, D.; Carothers, K.; Lee, S. The Role of Bacterial Proteases in Microbe and Host-Microbe Interactions. Curr. Drug Targets 2022, 23, 222–239. [CrossRef] 11. Carvalho, L.A.; Bernardes, G.J. The Impact of Activity-Based Protein Profiling in Malaria Drug Discovery. ChemMedChem 2022, 17, e202200174. [CrossRef] [PubMed] 12. Beltran-Hortelano, I.; Alcolea, V.; Font, M.; Pérez-Silanes, S. Examination of Multiple Trypanosoma cruzi Targets in a New Drug Discovery Approach for Chagas Disease. Bioorganic Med. Chem. 2022, 58, 116577. [CrossRef] [PubMed] 13. Vermelho, A.B.; Cardoso, V.; Mansoldo, F.R.P.; Supuran, C.T.; Cedrola, S.M.L.; Rodrigues, I.A.; Rodrigues, G.C. Chagas Disease: Drug Development and Parasite Targets. In Antiprotozoal Drug Development and Delivery; Springer: Berlin/Heidelberg, Germany, 2022; pp. 49–81. 14. Mukherjee, R.; Dikic, I. Regulation of Host-Pathogen Interactions Via the Ubiquitin System. Annu. Rev. Microbiol. 2022, 76, 211–233. [CrossRef] [PubMed] 15. Salomon, E.; Schmitt, M.; Marapaka, A.K.; Stamogiannos, A.; Revelant, G.; Schmitt, C.; Alavi, S.; Florent, I.; Addlagatta, A.; Stratikos, E.; et al. Aminobenzosuberone Scaffold as a Modular Chemical Tool for the Inhibition of Therapeutically Relevant M1 Aminopeptidases. Molecules 2018, 23, 2607. [CrossRef] [PubMed] 16. Albiston, A.L.; Ye, S.; Chai, S.Y. Membrane Bound Members of the M1 Family: More Than Aminopeptidases. Protein Pept. Lett. 2004, 11, 491–500. [CrossRef] [PubMed] 17. Carl-McGrath, S.; Lendeckel, U.; Ebert, M.; Röcken, C. Ectopeptidases in Tumour Biology: A Review. Histol. Histopathol. 2006, 12, 1339–1353. 18. Mucha, A.; Drag, M.; Dalton, J.P.; Kafarski, P. Metallo-Aminopeptidase Inhibitors. Biochimie 2010, 92, 1509–1529. [CrossRef] [PubMed] 19. Amin, S.A.; Adhikari, N.; Jha, T. Design of Aminopeptidase N Inhibitors as Anti-Cancer Agents. J. Med. Chem. 2018, 61, 6468–6490. [CrossRef] 20. Alvarez, C.; Pazos, F.; Soto, C.; Laborde, R.; Lanio, M.E. Pore-Forming Toxins from Sea Anemones: From Protein-Membrane Interaction to Its Implications for Developing Biomedical Applications. In Advances in Biomembranes and Lipid Self-Assembly; Elsevier: Amsterdam, The Netherlands, 2020; pp. 129–183. 21. Alvarez, C.; Soto, C.; Cabezas, S.; Alvarado-Mesén, J.; Laborde, R.; Pazos, F.; Ros, U.; Hernández, A.M.; Lanio, M.E. Panorama of the Intracellular Molecular Concert Orchestrated by Actinoporins, Pore-Forming Toxins from Sea Anemones. Toxins 2021, 13, 567. [CrossRef] 22. Martell, E.M.; González-Garcia, M.; Ständker, L.; Otero-González, A.J. Host Defense Peptides as Immunomodulators: The Other Side of the Coin. Peptides 2021, 146, 170644. [CrossRef] 23. Rodríguez, A.A.; Otero-González, A.; Ghattas, M.; Ständker, L. Discovery, Optimization, and Clinical Application of Natural Antimicrobial Peptides. Biomedicines 2021, 9, 1381. [CrossRef] [PubMed] 24. Riccio, G.; Ruocco, N.; Mutalipassi, M.; Costantini, M.; Zupo, V.; Coppola, D.; De Pascale, D.; Lauritano, C. Ten-Year Research Update Review: Antiviral Activities from Marine Organisms. Biomolecules 2020, 10, 1007. [CrossRef] [PubMed] 25. Nakao, Y.; Fusetani, N. Enzyme Inhibitors from Marine Invertebrates. J. Nat. Prod. 2007, 70, 689–710. [CrossRef] 26. Pujiastuti, D.Y.; Amin, M.N.G.; Alamsjah, M.A.; Hsu, J.-L. Marine Organisms as Potential Sources of Bioactive Peptides That Inhibit the Activity of Angiotensin I-Converting Enzyme: A Review. Molecules 2019, 24, 2541. [CrossRef] 27. Tischler, D. A Perspective on Enzyme Inhibitors from Marine Organisms. Mar. Drugs 2020, 18, 431. [CrossRef] [PubMed] 28. Moodie, L.W.K.; Sepčić, K.; Turk, T.; Frangež, R.; Svenson, J. Natural Cholinesterase Inhibitors from Marine Organisms. Nat. Prod. Rep. 2019, 36, 1053–1092. [CrossRef] 29. Alonso-del-Rivero, M.; Trejo, S.A.; Rodriguez de la Vega, M.; González, Y.; Bronsoms, S.; Canals, F.; Delfín, J.; Diaz, J.; Aviles, F.X.; Chávez, M.A. A Novel Metallocarboxypeptidase-Like Enzyme from the Marine Annelid Sabellastarte magnifica–a Step into the Invertebrate World of Proteases. FEBS J. 2009, 276, 4875–4890. [CrossRef] Mar. Drugs 2023, 21, 279 32 of 37 30. Alonso, I.P.; Méndez, L.R.; Almeida, F.; Tresano, M.E.V.; Sánchez, Y.A.; Hernández-Zanuy, A.; Álvarez-Lajonchere, L.; Díaz, D.; Sánchez, B.; Florent, I. Marine Organisms: A Source of Biomedically Relevant Metallo M1, M2 and M17 Exopeptidase Inhibitors. Rev. Cuba. Cienc. Biológicas 2020, 8, 1–36. 31. Chávez, M.; Delfín, J.; Díaz, J.; Pérez, U.; Martínez, J.; González, J.; Márquez, M.; Más, R. Caracterización de un Inhibidor de Proteasas Obtenido de la Anémona S. helianthus. Rev. CENIC 1988, 19, 82. 32. Delfin, J.; Gonzalez, Y.; Diaz, J.; Chavez, M. Proteinase Inhibitor from Stichodactyla helianthus: Purification, Characterization and Immobilization. Arch. Med. Res. 1994, 25, 199–204. 33. Delfin, J.; Martinez, I.; Antuch, W.; Morera, V.; Gonzalez, Y.; Rodriguez, R.; Marquez, M.; Saroyan, A.; Larionova, N.; Diaz, J. Purification, Characterization and Immobilization of Proteinase Inhibitors from Stichodactyla helianthus. Toxicon 1996, 34, 1367–1376. [CrossRef] [PubMed] 34. Lenarčič, B.; Ritonja, A.; Štrukelj, B.; Turk, B.; Turk, V. Equistatin, a New Inhibitor of Cysteine Proteinases from Actinia equina, Is Structurally Related to Thyroglobulin Type-1 Domain. J. Biol. Chem. 1997, 272, 13899–13903. [CrossRef] [PubMed] 35. González, Y.; Araujo, M.; Oliva, M.; Sampaio, C.; Chávez, M. Purification and Preliminary Characterization of a Plasma Kallikrein Inhibitor Isolated from Sea Hares Aplysia dactylomela Rang, 1828. Toxicon 2004, 43, 219–223. [CrossRef] [PubMed] 36. Reytor, M.L.; González, Y.; Pascual, I.; Hernández, A.; Chávez M, Á.; Alonso del Rivero, M. Screening of Protease Inhibitory Activity in Extracts of Five Ascidian Species from Cuban Coasts. Biotecnol. Apl. 2011, 28, 77–82. 37. Alonso-Del-Rivero, M.; Trejo, S.; Reytor, M.L.; Rodriguez-De-La-Vega, M.; Delfin, J.; Diaz, J.; Gonzalez, M.L.R.; Canals, F.; Chavez, M.A.; Aviles, F.X. Tri-Domain Bifunctional Inhibitor of Metallocarboxypeptidases a and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica. J. Biol. Chem. 2012, 287, 15427–15438. [CrossRef] [PubMed] 38. Salas-Sarduy, E.; Cabrera-Muñoz, A.; Cauerhff, A.; González-González, Y.; Trejo, S.A.; Chidichimo, A.; de los Angeles Chávez- Planes, M.; José Cazzulo, J. Antiparasitic Effect of a Fraction Enriched in Tight-Binding Protease Inhibitors Isolated from the Caribbean Coral Plexaura homomalla. Exp. Parasitol. 2013, 135, 611–622. [CrossRef] 39. González, L.; Sánchez, R.E.; Rojas, L.; Pascual, I.; García-Fernández, R.; Chávez, M.A.; Betzel, C. Screening of Protease Inhibitory Activity in Aqueous Extracts of Marine Invertebrates from Cuban Coast. Am. J. Anal. Chem. 2016, 7, 319–331. [CrossRef] 40. Salas-Sarduy, E.; Guerra, Y.; Covaleda Cortés, G.; Avilés, F.X.; Chávez Planes, M.A. Identification of Tight-Binding Plasmepsin Ii and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays. Mar. Drugs 2017, 15, 123. [CrossRef] 41. Covaleda, G.; Trejo, S.A.; Salas-Sarduy, E.; del Rivero, M.A.; Chavez, M.A.; Aviles, F.X. Intensity Fading Maldi-Tof Mass Spectrometry and Functional Proteomics Assignments to Identify Protease Inhibitors in Marine Invertebrates. J. Proteom. 2017, 165, 75–92. [CrossRef] 42. Hong, T.T.; Dat, T.T.H.; Cuong, P.V.; Cuc, N.T.K. Protease Inhibitors from Marine Sponge and Sponge-Associated Microorganisms. Vietnam. J. Sci. Technol. 2018, 56, 405–423. [CrossRef] 43. Ikegami, S.; Kobayashi, H.; Myotoishi, Y.; Ohta, S.; Kato, K.H. Selective Inhibition of Exoplasmic Membrane Fusion in Echinoderm Gametes with Jaspisin, a Novel Antihatching Substance Isolated from a Marine Sponge. J. Biol. Chem. 1994, 269, 23262–23267. [CrossRef] [PubMed] 44. Kato, K.H.; Takemoto, K.; Kato, E.; Miyazaki, K.; Kobayashi, H.; Ikegami, S. Inhibition of Sea Urchin Fertilization by Jaspisin, a Specific Inhibitor of Matrix Metalloendoproteinase. Dev. Growth Differ. 1998, 40, 221–230. [CrossRef] [PubMed] 45. Fusetani, N.; Fujita, M.; Nakao, Y.; Matsunaga, S.; van Soest, R.W. Tokaramide a, a New Cathepsin B Inhibitor from the Marine Sponge Theonella Aff. mirabilis. Bioorganic Med. Chem. Lett. 1999, 9, 3397–3402. [CrossRef] [PubMed] 46. Fujita, M.; Nakao, Y.; Matsunaga, S.; van Soest, R.W.; Itoh, Y.; Seiki, M.; Fusetani, N. Callysponginol Sulfate a, an Mt1-Mmp Inhibitor Isolated from the Marine Sponge Callyspongia tuncata. J. Nat. Prod. 2003, 66, 569–571. [CrossRef] [PubMed] 47. Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.; Yamashita, J.; Van Soest, R.W.; Fusetani, N. Ageladine A: An Antiangiogenic Matrixmetalloproteinase Inhibitor from the Marine Sponge Agelas N Akamurai. J. Am. Chem. Soc. 2003, 125, 15700–15701. [CrossRef] 48. Shim, J.S.; Lee, H.-S.; Shin, J.; Kwon, H.J. Psammaplin a, a Marine Natural Product, Inhibits Aminopeptidase N and Suppresses Angiogenesis in Vitro. Cancer Lett. 2004, 203, 163–169. [CrossRef] 49. Pascual, I.; Gil-Parrado, S.; Cisneros, M.; Joseph-Bravo, P.; Dıaz, J.; Possani, L.D.; Charli, J.L.; Chávez, M. Purification of a Specific Inhibitor of Pyroglutamyl Aminopeptidase Ii from the Marine Annelide Hermodice carunculata: In Vivo Effects in Rodent Brain. Int. J. Biochem. Cell Biol. 2004, 36, 138–152. [CrossRef] 50. Thomas, N.V.; Kim, S.-K. Metalloproteinase Inhibitors: Status and Scope from Marine Organisms. Biochem. Res. Int. 2010, 2010, 845975. [CrossRef] 51. del Rivero, M.A.; Reytor, M.L.; Trejo, S.A.; Chávez, M.A.; Avilés, F.X.; Reverter, D. A Noncanonical Mechanism of Carboxypep- tidase Inhibition Revealed by the Crystal Structure of the Tri-Kunitz Smci in Complex with Human Cpa4. Structure 2013, 21, 1118–1126. 52. Covaleda, G.; del Rivero, M.A.; Chávez, M.A.; Avilés, F.X.; Reverter, D. Crystal Structure of Novel Metallocarboxypeptidase Inhibitor from Marine Mollusk Nerita versicolor in Complex with Human Carboxypeptidase A4. J. Biol. Chem. 2012, 287, 9250–9258. [CrossRef] Mar. Drugs 2023, 21, 279 33 of 37 53. Cabrera-Muñoz, A.; Valiente, P.A.; Rojas, L.; Antigua, M.A.D.R.; Pires, J.R. Nmr Structure of Cmpi-Ii, a Non-Classical Kazal Protease Inhibitor: Understanding Its Conformational Dynamics and Subtilisin a Inhibition. J. Struct. Biol. 2019, 206, 280–294. [CrossRef] [PubMed] 54. Pascual Alonso, I.; Rivera Méndez, L.; Valdés-Tresanco, M.E.; Bounaadja, L.; Schmitt, M.; Arrebola Sánchez, Y.; Alvarez Lajonchere, L.; Charli, J.L.; Florent, I. Biochemical Evidences for M1-, M17- and M18-Like Aminopeptidases in Marine Invertebrates from Cuban Coastline. Z. Für Nat. C 2020, 75, 397–407. [CrossRef] [PubMed] 55. Sarfaraj, H.M.; Sheeba, F.; Saba, A.; Khan, M. Marine Natural Products: A Lead for Anti-Cancer; NISCAIR-CSIR: New Delhi, India, 2012. 56. Nakao, Y.; Oku, N.; Matsunaga, S.; Fusetani, N. Cyclotheonamides E2 and E3, New Potent Serine Protease Inhibitors from the Marine Sponge of the Genus Theonella. J. Nat. Prod. 1998, 61, 667–670. [CrossRef] [PubMed] 57. Hanessian, S.; Tremblay, M.; Petersen, J.F. The N-Acyloxyiminium Ion Aza-Prins Route to Octahydroindoles: Total Synthesis and Structural Confirmation of the Antithrombotic Marine Natural Product Oscillarin. J. Am. Chem. Soc. 2004, 126, 6064–6071. [CrossRef] 58. Gunasekera, S.P.; McCarthy, P.J.; Longley, R.E.; Pomponi, S.A.; Wright, A.E.; Lobkovsky, E.; Clardy, J. Discorhabdin P, a New Enzyme Inhibitor from a Deep-Water Caribbean Sponge of the Genus Batzella. J. Nat. Prod. 1999, 62, 173–175. [CrossRef] 59. Gunasekera, S.P.; McCarthy, P.J.; Longley, R.E.; Pomponi, S.A.; Wright, A.E. Secobatzellines a and B, Two New Enzyme Inhibitors from a Deep-Water Caribbean Sponge of the Genus Batzella. J. Nat. Prod. 1999, 62, 1208–1211. [CrossRef] [PubMed] 60. Hu, J.F.; Schetz, J.A.; Kelly, M.; Peng, J.N.; Ang, K.K.; Flotow, H.; Leong, C.Y.; Ng, S.B.; Buss, A.D.; Wilkins, S.P. New Antiinfective and Human 5-Ht2 Receptor Binding Natural and Semisynthetic Compounds from the Jamaican Sponge Smenospongia aurea. J. Nat. Prod. 2002, 65, 476–480. [CrossRef] [PubMed] 61. Fujita, M.; Nakao, Y.; Matsunaga, S.; Nishikawa, T.; Fusetani, N. Sodium 1-(12-Hydroxy) Octadecanyl Sulfate, an Mmp2 Inhibitor, Isolated from a Tunicate of the Family Polyclinidae. J. Nat. Prod. 2002, 65, 1936–1938. [CrossRef] 62. Joe, M.-J.; Kim, S.-N.; Choi, H.-Y.; Shin, W.-S.; Park, G.-M.; Kang, D.-W.; Kim, Y.K. The Inhibitory Effects of Eckol and Dieckol from Ecklonia stolonifera on the Expression of Matrix Metalloproteinase-1 in Human Dermal Fibroblasts. Biol. Pharm. Bull. 2006, 29, 1735–1739. [CrossRef] 63. Harper, J.W.; Powers, J.C. Inhibitors of Metallo-Proteases. In Proteinase Inhibitors; Elsevier: Amsterdam, The Netherlands, 1986; pp. 219–298. 64. Rawlings, N.D.; Barrett, A.J. Evolutionary Families of Peptidases. Biochem. J. 1993, 290, 205–218. [CrossRef] 65. Rawlings, N.D.; Barrett, A.J. [13] Evolutionary Families of Metallopeptidases. In Methods in Enzymology; Elsevier: Amsterdam, The Netherlands, 1995; pp. 183–228. 66. Reeck, G.R.; de Haën, C.; Teller, D.C.; Doolittle, R.F.; Fitch, W.M.; Dickerson, R.E.; Chambon, P.; McLachlan, A.D.; Margoliash, E.; Jukes, T.H.; et al. Homology in Proteins and Nucleic Acids: A Terminology Muddle and a Way out of It. Cell 1987, 50, 667. [CrossRef] [PubMed] 67. Haeggström, J.Z.; Nordlund, P.; Thunnissen, M.M. Functional Properties and Molecular Architecture of Leukotriene A4 Hydrolase, a Pivotal Catalyst of Chemotactic Leukotriene Formation. Sci. World J. 2002, 2, 1734–1749. [CrossRef] [PubMed] 68. Fukasawa, K.M.; Fukasawa, K.; Harada, M.; Hirose, J.; Izumi, T.; Shimizu, T. Aminopeptidase B Is Structurally Related to Leukotriene-A4 Hydrolase but Is Not a Bifunctional Enzyme with Epoxide Hydrolase Activity. Biochem. J. 1999, 339, 497–502. [CrossRef] [PubMed] 69. Klinke, T.; Rump, A.; Pönisch, R.; Schellenberger, W.; Müller, E.C.; Otto, A.; Klimm, W.; Kriegel, T.M. Identification and Characterization of Caape2–a Neutral Arginine/Alanine/Leucine-Specific Metallo-Aminopeptidase from Candida albicans. FEMS Yeast Res. 2008, 8, 858–869. [CrossRef] 70. Wong, A.H.M.; Zhou, D.; Rini, J.M. The X-Ray Crystal Structure of Human Aminopeptidase N Reveals a Novel Dimer and the Basis for Peptide Processing. J. Biol. Chem. 2012, 287, 36804–36813. [CrossRef] 71. Thunnissen, M.M.; Nordlund, P.; Haeggström, J.Z. Crystal Structure of Human Leukotriene A4 Hydrolase, a Bifunctional Enzyme in Inflammation. Nat. Struct. Biol. 2001, 8, 131–135. [CrossRef] 72. Kyrieleis, O.J.; Goettig, P.; Kiefersauer, R.; Huber, R.; Brandstetter, H. Crystal Structures of the Tricorn Interacting Factor F3 from Thermoplasma Acidophilum, a Zinc Aminopeptidase in Three Different Conformations. J. Mol. Biol. 2005, 349, 787–800. [CrossRef] 73. Ito, K.; Nakajima, Y.; Onohara, Y.; Takeo, M.; Nakashima, K.; Matsubara, F.; Ito, T.; Yoshimoto, T. Crystal Structure of Aminopepti- dase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition. J. Biol. Chem. 2006, 281, 33664–33676. [CrossRef] 74. McGowan, S.; Porter, C.J.; Lowther, J.; Stack, C.M.; Golding, S.J.; Skinner-Adams, T.S.; Trenholme, K.R.; Teuscher, F.; Donnelly, S.M.; Grembecka, J.; et al. Structural Basis for the Inhibition of the Essential Plasmodium Falciparum M1 Neutral Aminopeptidase. Proc. Natl. Acad. Sci. USA 2009, 106, 2537–2542. [CrossRef] 75. Harbut, M.B.; Velmourougane, G.; Dalal, S.; Reiss, G.; Whisstock, J.C.; Onder, O.; Brisson, D.; McGowan, S.; Klemba, M.; Green- baum, D.C. Bestatin-Based Chemical Biology Strategy Reveals Distinct Roles for Malaria M1-and M17-Family Aminopeptidases. Proc. Natl. Acad. Sci. USA 2011, 108, E526–E534. [CrossRef] Mar. Drugs 2023, 21, 279 34 of 37 76. Salomon, E.; Schmitt, M.; Mouray, E.; McEwen, A.G.; Bounaadja, L.; Torchy, M.; Poussin-Courmontagne, P.; Alavi, S.; Tarnus, C.; Cavarelli, J.; et al. Aminobenzosuberone Derivatives as Pfa-M1 Inhibitors: Molecular Recognition and Antiplasmodial Evaluation. Bioorganic Chem. 2020, 98, 103750. [CrossRef] [PubMed] 77. Yang, Y.; Liu, C.; Lin, Y.-L.; Li, F. Structural Insights into Central Hypertension Regulation by Human Aminopeptidase A. J. Biol. Chem. 2013, 288, 25638–25645. [CrossRef] [PubMed] 78. Kochan, G.; Krojer, T.; Harvey, D.; Fischer, R.; Chen, L.; Vollmar, M.; von Delft, F.; Kavanagh, K.L.; Brown, M.A.; Bowness, P.; et al. Crystal Structures of the Endoplasmic Reticulum Aminopeptidase-1 (Erap1) Reveal the Molecular Basis for N-Terminal Peptide Trimming. Proc. Natl. Acad. Sci. USA 2011, 108, 7745–7750. [CrossRef] [PubMed] 79. Chen, J.; Wang, Y.; Zhong, Q.; Wu, Y.; Xia, W. Purification and Characterization of a Novel Angiotensin-I Converting Enzyme (Ace) Inhibitory Peptide Derived from Enzymatic Hydrolysate of Grass Carp Protein. Peptides 2012, 33, 52–58. [CrossRef] [PubMed] 80. Gago, F. Computational Approaches to Enzyme Inhibition by Marine Natural Products in the Search for New Drugs. Mar. Drugs 2023, 21, 100. [CrossRef] [PubMed] 81. Omar, A.M.; Mohammad, K.A.; Sindi, I.A.; Mohamed, G.A.; Ibrahim, S.R.M. Unveiling the Efficacy of Sesquiterpenes from Marine Sponge Dactylospongia elegans in Inhibiting Dihydrofolate Reductase Using Docking and Molecular Dynamic Studies. Molecules 2023, 28, 1292. [CrossRef] [PubMed] 82. Joseph-Bravo, P.; Jaimes-Hoy, L.; Uribe, R.M.; Charli, J.L. 60 Years of Neuroendocrinology: TRH, the First Hypophysiotropic Releasing Hormone Isolated: Control of the Pituitary–Thyroid Axis. J. Endocrinol. 2015, 226, T85–T100. [CrossRef] 83. Kronenberg, H.M.; Shlomo Melmed, M.D.; Polonsky, K.S.; Wilson, J.D.; Foster, D.W.; Kronenberg, H.M. Williams Textbook of Endocrinology; Saunders: Philadelphia, PA, USA, 2002. 84. Charli, J.L.; Rodríguez-Rodríguez, A.; Hernández-Ortega, K.; Cote-Vélez, A.; Uribe, R.M.; Jaimes-Hoy, L.; Joseph-Bravo, P. The Thyrotropin-Releasing Hormone-Degrading Ectoenzyme, a Therapeutic Target? Front. Pharm. 2020, 11, 640. [CrossRef] 85. A Kelly, J. Thyrotropin-Releasing Hormone: Basis and Potential for Its Therapeutic Use. Essays Biochem. 1995, 30, 133. 86. Charli, J.-L.; Mendez, M.; Vargas, M.-A.; Cisneros, M.; Assai, M.; Joseph-Bravo, P.; Wilk, S. Pyroglutamyl Peptidase Ii Inhibition Specifically Increases Recovery of Trh Released from Rat Brain Slices. Neuropeptides 1989, 14, 191–196. [CrossRef] 87. Kelly, J.A.; Slator, G.R.; Tipton, K.F.; Williams, C.H.; Bauer, K. Kinetic Investigation of the Specificity of Porcine Brain Thyrotropin- Releasing Hormone-Degrading Ectoenzyme for Thyrotropin-Releasing Hormone-Like Peptides. J. Biol. Chem. 2000, 275, 16746–16751. [CrossRef] [PubMed] 88. Kelly, J.A.; Scalabrino, G.A.; Slator, G.R.; Cullen, A.A.; Gilmer, J.F.; Lloyd, D.G.; Bennett, G.W.; Bauer, K.; Tipton, K.F.; Williams, C.H. Structure–Activity Studies with High-Affinity Inhibitors of Pyroglutamyl-Peptidase Ii. Biochem. J. 2005, 389, 569–576. [CrossRef] [PubMed] 89. Cruz, R.; Vargas, M.A.; Uribe, R.M.; Pascual, I.; Lazcano, I.; Yiotakis, A.; Matziari, M.; Joseph-Bravo, P.; Charli, J.-L. Anterior Pituitary Pyroglutamyl Peptidase Ii Activity Controls Trh-Induced Prolactin Release. Peptides 2008, 29, 1953–1964. [CrossRef] [PubMed] 90. Sinko, R.; Mohácsik, P.; Kővári, D.; Penksza, V.; Wittmann, G.; Mácsai, L.; Fonseca, T.L.; Bianco, A.C.; Fekete, C.; Gereben, B. Different Hypothalamic Mechanisms Control Decreased Circulating Thyroid Hormone Levels in Infection and Fasting-Induced Non-Thyroidal Illness Syndrome in Male Thyroid Hormone Action Indicator Mice. Thyroid 2023, 33, 109–118. [CrossRef] 91. Barnieh, F.M.; Loadman, P.M.; Falconer, R.A. Is Tumour-Expressed Aminopeptidase N (Apn/Cd13) Structurally and Functionally Unique? Biochim. Biophys. Acta (BBA)-Rev. Cancer 2021, 1876, 188641. [CrossRef] 92. Wickström, M.; Larsson, R.; Nygren, P.; Gullbo, J. Aminopeptidase N (Cd13) as a Target for Cancer Chemotherapy. Cancer Sci. 2011, 102, 501–508. [CrossRef] 93. Ni, J.; Wang, X.; Shang, Y.; Li, Y.; Chen, S. Cd13 Inhibition Augments Dr4-Induced Tumor Cell Death in a P-Erk1/2-Independent Manner. Cancer Biol. Med. 2021, 18, 569. [CrossRef] 94. Schreiter, A.; Gore, C.; Labuz, D.; Fournie-Zaluski, M.C.; Roques, B.P.; Stein, C.; Machelska, H. Pain Inhibition by Blocking Leukocytic and Neuronal Opioid Peptidases in Peripheral Inflamed Tissue. FASEB J. 2012, 26, 5161–5171. [CrossRef] 95. Bonnard, E.; Poras, H.; Nadal, X.; Maldonado, R.; Fournié-Zaluski, M.C.; Roques, B.P. Long-Lasting Oral Analgesic Effects of N-Protected Aminophosphinic Dual Enk Ephalinase Inhibitors (Denki S) in Peripherally Controlled Pain. Pharmacol. Res. Perspect. 2015, 3, e0116. [CrossRef] 96. Arrebola, Y.; Rivera, L.; Pedroso, A.; McGuire, R.; Tresanco, M.E.V.; Bergado, G.; Charli, J.-L.; Sánchez, B.; Alonso, I.P. Bacitracin Is a Non-Competitive Inhibitor of Porcine M1 Family Neutral and Glutamyl Aminopeptidases. Nat. Prod. Res. 2021, 35, 2958–2962. [CrossRef] 97. Melzig, M.F.; Bormann, H. Betulinic Acid Inhibits Aminopeptidase N Activity. Planta Med. 1998, 64, 655–657. [CrossRef] [PubMed] 98. Pascual Alonso, I.; Bounaadja, L.; Sánchez, L.; Rivera, L.; Tarnus, C.; Schmitt, M.; Garcia, G.; Diaz, L.; Hernandez-Zanuy, A.; Sánchez, B.; et al. Aqueous Extracts of Marine Invertebrates from Cuba Coastline Display Neutral Aminopeptidase Inhibitory Activities and Effects on Cancer Cells and Plasmodium Falciparum Parasites. Indian J. Nat. Prod. Resour. 2017, 8, 107–119. 99. Pascual, I.; Valiente, P.A.; García, G.; Valdés-Tresanco, M.E.; Arrebola, Y.; Díaz, L.; Bounaadja, L.; Uribe, R.M.; Pacheco, M.C.; Florent, I.; et al. Discovery of Novel Non-Competitive Inhibitors of Mammalian Neutral M1 Aminopeptidase (Apn). Biochimie 2017, 142, 216–225. [CrossRef] Mar. Drugs 2023, 21, 279 35 of 37 100. Pascual-Alonso, I.; Alonso-Bosch, R.; Cabrera-Muñoz, A.; Perera, W.H.; Charli, J.L. Methanolic Extracts of Paratoid Gland Secretions from Cuban Peltophryne Toads Contain Inhibitory Activities against Peptidases with Biomedical Relevance. Biotecnol. Apl. 2019, 36, 2221–2227. 101. Alonso, I.P.; Méndez, L.R.; García, F.A.; Valdés-Tresanco, M.E.; Bosch, R.A.; Perera, W.H.; Sánchez, Y.A.; Bergado, G.; Ramírez, B.S.; Charli, J.-L. Bufadienolides Preferentially Inhibit Aminopeptidase N among Mammalian Metallo-Aminopeptidases; Relationship with Effects on Human Melanoma Mewo Cells. Int. J. Biol. Macromol. 2023, 229, 825–837. [CrossRef] [PubMed] 102. Wang, E.; Sorolla, M.A.; Krishnan, P.D.G.; Sorolla, A. From Seabed to Bedside: A Review on Promising Marine Anticancer Compounds. Biomolecules 2020, 10, 248. [CrossRef] [PubMed] 103. Malla, R.R.; Farran, B.; Nagaraju, G.P. Understanding the Function of the Tumor Microenvironment, and Compounds from Marine Organisms for Breast Cancer Therapy. World J. Biol. Chem. 2021, 12, 15. [CrossRef] 104. Aldrich, L.N.; Burdette, J.E.; de Blanco, E.C.; Coss, C.C.; Eustaquio, A.S.; Fuchs, J.R.; Kinghorn, A.D.; MacFarlane, A.; Mize, B.K.; Oberlies, N.H.; et al. Discovery of Anticancer Agents of Diverse Natural Origin. J. Nat. Prod. 2022, 85, 702–719. [CrossRef] 105. Nuzzo, G.; Senese, G.; Gallo, C.; Albiani, F.; Romano, L.; D’ippolito, G.; Manzo, E.; Fontana, A. Antitumor Potential of Immunomodulatory Natural Products. Mar. Drugs 2022, 20, 386. [CrossRef] 106. Saeed, A.F.; Su, J.; Ouyang, S. Marine-Derived Drugs: Recent Advances in Cancer Therapy and Immune Signaling. Biomed. Pharmacother. 2021, 134, 111091. [CrossRef] 107. Jung, J.H.; Sim, C.J.; Lee, C.-O. Cytotoxic Compounds from a Two-Sponge Association. J. Nat. Prod. 1995, 58, 1722–1726. [CrossRef] [PubMed] 108. Shin, J.; Lee, H.-S.; Seo, Y.; Rho, J.-R.; Cho, K.W.; Paul, V.J. New Bromotyrosine Metabolites from the Sponge Aplysinella rhax. Tetrahedron 2000, 56, 9071–9077. [CrossRef] 109. Pina, I.C.; Gautschi, J.T.; Wang, G.Y.S.; Sanders, M.L.; Schmitz, F.J.; France, D.; Cornell-Kennon, S.; Sambucetti, L.C.; Remiszewski, S.W.; Perez, L.B.; et al. Psammaplins from the Sponge Pseudoceratina P Urpurea: Inhibition of Both Histone Deacetylase and DNA Methyltransferase. J. Org. Chem. 2003, 68, 3866–3873. [CrossRef] [PubMed] 110. Park, Y.; Liu, Y.; Hong, J.; Lee, C.O.; Cho, H.; Kim, D.K.; Im, K.S.; Jung, J.H. New Bromotyrosine Derivatives from an Association of Two Sponges, Jaspis W Ondoensis and Poecillastra W Ondoensis. J. Nat. Prod. 2003, 66, 1495–1498. [CrossRef] [PubMed] 111. Yang, Q.; Liu, D.; Sun, D.; Yang, S.; Hu, G.; Wu, Z.; Zhao, L. Synthesis of the Marine Bromotyrosine Psammaplin F and Crystal Structure of a Psammaplin a Analogue. Molecules 2010, 15, 8784–8795. [CrossRef] 112. Mujumdar, P.; Teruya, K.; Tonissen, K.F.; Vullo, D.; Supuran, C.T.; Peat, T.S.; Poulsen, S.A. An Unusual Natural Product Primary Sulfonamide: Synthesis, Carbonic Anhydrase Inhibition, and Protein X-Ray Structures of Psammaplin C. J. Med. Chem. 2016, 59, 5462–5470. [CrossRef] 113. Kim, D.; Lee, I.S.; Jung, J.H.; Yang, S.I. Psammaplin a, a Natural Bromotyrosine Derivative from a Sponge, Possesses the Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus and the DNA Gyrase-Inhibitory Activity. Arch. Pharmacal Res. 1999, 22, 25–29. [CrossRef] 114. TTabudravu, J.; Eijsink, V.; Gooday, G.; Jaspars, M.; Komander, D.; Legg, M.; Synstad, B.; van Aalten, D. Psammaplin a, a Chitinase Inhibitor Isolated from the Fijian Marine Sponge Aplysinella rhax. Bioorganic Med. Chem. 2002, 10, 1123–1128. [CrossRef] 115. Revelant, G.; Al-Lakkis-Wehbe, M.; Schmitt, M.; Alavi, S.; Schmitt, C.; Roux, L.; Al-Masri, M.; Schifano-Faux, N.; Maiereanu, C.; Tarnus, C.; et al. Exploring S1 Plasticity and Probing S10 Subsite of Mammalian Aminopeptidase N/Cd13 with Highly Potent and Selective Aminobenzosuberone Inhibitors. Bioorganic Med. Chem. 2015, 23, 3192–3207. [CrossRef] 116. Kim, T.H.; Kim, H.S.; Kang, Y.J.; Yoon, S.; Lee, J.; Choi, W.S.; Jung, J.H.; Kim, H.S. Psammaplin a Induces Sirtuin 1-Dependent Autophagic Cell Death in Doxorubicin-Resistant Mcf-7/Adr Human Breast Cancer Cells and Xenografts. Biochim. Biophys. Acta (BBA)-Gen. Subj. 2015, 1850, 401–410. [CrossRef] 117. Ratovitski, E.A. Tumor Protein (Tp)-P53 Members as Regulators of Autophagy in Tumor Cells Upon Marine Drug Exposure. Mar. Drugs 2016, 14, 154. [CrossRef] [PubMed] 118. Zhou, Y.-D.; Li, J.; Du, L.; Mahdi, F.; Le, T.P.; Chen, W.-L.; Swanson, S.M.; Watabe, K.; Nagle, D.G. Biochemical and Anti-Triple Negative Metastatic Breast Tumor Cell Properties of Psammaplins. Mar. Drugs 2018, 16, 442. [CrossRef] [PubMed] 119. Ahn, M.Y.; Jung, J.H.; Na, Y.J.; Kim, H.S. A Natural Histone Deacetylase Inhibitor, Psammaplin a, Induces Cell Cycle Arrest and Apoptosis in Human Endometrial Cancer Cells. Gynecol. Oncol. 2008, 108, 27–33. [CrossRef] [PubMed] 120. Kim, H.J.; Kim, J.H.; Chie, E.K.; Da Young, P.; Kim, I.A.; Kim, I.H. Dnmt (DNA Methyltransferase) Inhibitors Radiosensitize Human Cancer Cells by Suppressing DNA Repair Activity. Radiat. Oncol. 2012, 7, 1–10. [CrossRef] 121. Kim, D.H.; Shin, J.; Kwon, H.J. Psammaplin a Is a Natural Prodrug That Inhibits Class I Histone Deacetylase. Exp. Mol. Med. 2007, 39, 47–55. [CrossRef] 122. Jing, Q.; Hu, X.; Ma, Y.; Mu, J.; Liu, W.; Xu, F.; Li, Z.; Bai, J.; Hua, H.; Li, D. Marine-Derived Natural Lead Compound Disulfide-Linked Dimer Psammaplin A: Biological Activity and Structural Modification. Mar. Drugs 2019, 17, 384. [CrossRef] [PubMed] 123. Nicolaou, K.C.; Hughes, R.; Pfefferkorn, J.A.; Barluenga, S.; Roecker, A.J. Combinatorial Synthesis through Disulfide Exchange: Discovery of Potent Psammaplin a Type Antibacterial Agents Active against Methicillin-Resistant Staphylococcus aureus (MRSA). Chem. –A Eur. J. 2001, 7, 4280–4295. [CrossRef] Mar. Drugs 2023, 21, 279 36 of 37 124. Nicolaou, K.C.; Hughes, R.; Pfefferkorn, J.A.; Barluenga, S. Optimization and Mechanistic Studies of Psammaplin a Type Antibacterial Agents Active against Methicillin-Resistant Staphylococcus aureus (MRSA). Chem. –A Eur. J. 2001, 7, 4296–4310. [CrossRef] 125. Alonso, I.P.; Sanchez, Y.M.A.; Ruiz, G.A.; González, M.L.R.; Hernández-Zanuy, A. Identificación De Actividad Inhibidora De Aminopeptidasa N Aislada De Riñón Porcino, En Invertebrados Marinos De La Plataforma Insular De La Habana/Identification of Porcine Kidney Aminopeptidase N Inhibitory Activity, in Marine Invertebrates from Havana Coastline. Rev. Cuba. Cienc. Biológicas 2016, 5, 15. 126. Alonso, I.P.; Pedroso, A.; Sánchez, Y.M.A.; Valdés-Tresanco, M.E.; Méndez, L.R.; Fortun, S. La Aminopeptidasa a De Mamíferos: Características Bioquímicas, Funciones Fisiológicas Y Su Implicación En Procesos Fisiopatológicos En Humanos/Aminopeptidase a from Mammals: Biochemical Characteristics, Physiological Roles and Implication in Physiopathological Processes in Humans. Rev. Cuba. Cienc. Biológicas 2018, 6, 20. 127. Göhring, B.; Holzhausen, H.; Meye, A.; Heynemann, H.; Rebmann, U.; Langner, J.; Riemann, D. Endopeptidase 24.11/Cd10 Is Down-Regulated in Renal Cell Cancer. Int. J. Mol. Med. 1998, 2, 409–423. [CrossRef] [PubMed] 128. Tonna, S.; Dandapani, S.V.; Uscinski, A.; Appel, G.B.; Schlöndorff, J.S.; Zhang, K.; Denker, B.M.; Pollak, M.R. Functional Genetic Variation in Aminopeptidase a (Enpep): Lack of Clear Association with Focal and Segmental Glomerulosclerosis (Fsgs). Gene 2008, 410, 44–52. [CrossRef] 129. del Carmen Puertas, M.; Martínez-Martos, J.M.; Cobo, M.; Lorite, P.; Sandalio, R.M.; Palomeque, T.; Torres, M.I.; Carrera-González, M.P.; Mayas, M.D.; Ramírez-Expósito, M.J. Plasma Renin–Angiotensin System-Regulating Aminopeptidase Activities Are Modified in Early Stage Alzheimer’s Disease and Show Gender Differences but Are Not Related to Apolipoprotein E Genotype. Exp. Gerontol. 2013, 48, 557–564. [CrossRef] [PubMed] 130. Blanco, L.; Sanz, B.; Perez, I.; Sánchez, C.E.; Cándenas, M.L.; Pinto, F.M.; Gil, J.; Casis, L.; López, J.I.; Larrinaga, G. Altered Glutamyl-Aminopeptidase Activity and Expression in Renal Neoplasms. BMC Cancer 2014, 14, 386. [CrossRef] 131. Bodineau, L.; Frugiere, A.; Marc, Y.; Inguimbert, N.; Fassot, C.; Balavoine, F.; Roques, B.; Llorens-Cortes, C. Orally Active Aminopeptidase a Inhibitors Reduce Blood Pressure: A New Strategy for Treating Hypertension. Hypertension 2008, 51, 1318–1325. [CrossRef] [PubMed] 132. Matsui, M.; Fowler, J.H.; Walling, L.L. Leucine Aminopeptidases: Diversity in Structure and Function. Biol. Chem. 2006, 387, 1535–1544. [CrossRef] [PubMed] 133. Scranton, M.A.; Yee, A.; Park, S.-Y.; Walling, L.L. Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-Induced Damage. J. Biol. Chem. 2012, 287, 18408–18417. [CrossRef] 134. Chao, W.S.; Gu, Y.-Q.; Pautot, V.; Bray, E.A.; Walling, L.L. Leucine Aminopeptidase Rnas, Proteins, and Activities Increase in Response to Water Deficit, Salinity, and the Wound Signals Systemin, Methyl Jasmonate, and Abscisic Acid. Plant Physiol. 1999, 120, 979–992. [CrossRef] 135. Alén, C.; Sherratt, D.J.; Colloms, S. Direct Interaction of Aminopeptidase a with Recombination Site DNA in Xer Site-Specific Recombination. EMBO J. 1997, 16, 5188–5197. [CrossRef] 136. Behari, J.; Stagon, L.; Calderwood, S.B. Pepa, a Gene Mediating Ph Regulation of Virulence Genes in Vibrio cholerae. J. Bacteriol. 2001, 183, 178–188. [CrossRef] 137. Aly, A.S.; Vaughan, A.M.; Kappe, S.H. Malaria Parasite Development in the Mosquito and Infection of the Mammalian Host. Annu. Rev. Microbiol. 2009, 63, 195–221. [CrossRef] [PubMed] 138. Goldberg, D.E. Complex Nature of Malaria Parasite Hemoglobin Degradation. Proc. Natl. Acad. Sci. USA 2013, 110, 5283–5284. [CrossRef] [PubMed] 139. Skinner-Adams, T.S.; Stack, C.M.; Trenholme, K.R.; Brown, C.L.; Grembecka, J.; Lowther, J.; Mucha, A.; Drag, M.; Kafarski, P.; McGowan, S.; et al. Plasmodium falciparum Neutral Aminopeptidases: New Targets for Anti-Malarials. Trends Biochem. Sci. 2010, 35, 53–61. [CrossRef] [PubMed] 140. Calic, P.P.; Vinh, N.B.; Webb, C.T.; Malcolm, T.R.; Ngo, A.; Lowes, K.; Drinkwater, N.; McGowan, S.; Scammells, P.J. Structure- Based Development of Potent Plasmodium Falciparum M1 and M17 Aminopeptidase Selective and Dual Inhibitors Via S1’-Region Optimisation. Eur. J. Med. Chem. 2023, 248, 115051. [CrossRef] [PubMed] 141. Drinkwater, N.; Vinh, N.B.; Mistry, S.N.; Bamert, R.S.; Ruggeri, C.; Holleran, J.P.; Loganathan, S.; Paiardini, A.; Charman, S.A.; Powell, A.K.; et al. Potent Dual Inhibitors of Plasmodium falciparum M1 and M17 Aminopeptidases through Optimization of S1 Pocket Interactions. Eur. J. Med. Chem. 2016, 110, 43–64. [CrossRef] 142. Bauvois, B.; Dauzonne, D. Aminopeptidase-N/Cd13 (Ec 3.4. 11.2) Inhibitors: Chemistry, Biological Evaluations, and Therapeutic Prospects. Med. Res. Rev. 2006, 26, 88–130. [CrossRef] 143. Kancharla, P.; Li, Y.; Yeluguri, M.; Dodean, R.A.; Reynolds, K.A.; Kelly, J.X. Total Synthesis and Antimalarial Activity of 2-(P- Hydroxybenzyl)-Prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine Myp1 Isolated from Marine Bacteria. J. Med. Chem. 2021, 64, 8739–8754. [CrossRef] 144. Nweze, J.A.; Mbaoji, F.N.; Li, Y.-M.; Yang, L.-Y.; Huang, S.-S.; Chigor, V.N.; Eze, E.A.; Pan, L.-X.; Zhang, T.; Yang, D.-F. Potentials of Marine Natural Products against Malaria, Leishmaniasis, and Trypanosomiasis Parasites: A Review of Recent Articles. Infect. Dis. Poverty 2021, 10, 1–19. [CrossRef] 145. Zayed, A.; Negm, W.; Kabbash, A.; Ezzat, S.M. Marine-Derived Metabolites as Antimalarial Candidates Targeting Various Life Stages. J. Adv. Med. Pharm. Res. 2022, 3, 12–18. [CrossRef] Mar. Drugs 2023, 21, 279 37 of 37 146. Singh, H.; Parida, A.; Debbarma, K.; Ray, D.P.; Banerjee, P. Common Marine Organisms: A Novel Source of Medicinal Compounds. Int. J. Bioresour. Sci. 2020, 7, 39–49. [CrossRef] 147. Zhang, M.; Tian, Z.; Wang, J.; Tian, X.; Wang, C.; Cui, J.; Huo, X.; Feng, L.; Yu, Z.; Ma, X. Visual Analysis and Inhibitor Screening of Leucine Aminopeptidase, a Key Virulence Factor for Pathogenic Bacteria-Associated Infection. ACS Sens. 2021, 6, 3604–3610. [CrossRef] [PubMed] 148. Fang, C.; Zhang, J.; Yang, H.; Peng, L.; Wang, K.; Wang, Y.; Zhao, X.; Liu, H.; Dou, C.; Shi, L.; et al. Leucine Aminopeptidase 3 Promotes Migration and Invasion of Breast Cancer Cells through Upregulation of Fascin and Matrix Metalloproteinases-2/9 Expression. J. Cell. Biochem. 2019, 120, 3611–3620. [CrossRef] [PubMed] 149. Yang, H.; Dai, G.; Wang, S.; Zhao, Y.; Wang, X.; Zhao, X.; Zhang, H.; Wei, L.; Zhang, L.; Guo, S.; et al. Inhibition of the Proliferation, Migration, and Invasion of Human Breast Cancer Cells by Leucine Aminopeptidase 3 Inhibitors Derived from Natural Marine Products. Anti-Cancer Drugs 2020, 31, 60–66. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.