Three decades targeting falcipains to develop antiplasmodial agents: what have we learned and what can be done next?

Malaria is a devastating infectious disease that affects large swathes of human populations across the planet's tropical regions. It is caused by parasites of the genus Plasmodium, with Plasmodium falciparum being responsible for the most lethal form of the disease. During the intraerythrocytic stage in the human hosts, malaria parasites multiply and degrade hemoglobin (Hb) using a battery of proteases, which include two cysteine proteases, falcipains 2 and 3 (FP-2 and FP-3). Due to their role as major hemoglobinases, FP-2 and FP-3 have been targeted in studies aiming to discover new antimalarials and numerous inhibitors with activity against these enzymes, and parasites in culture have been identified. Nonetheless, cross-inhibition of human cysteine cathepsins remains a serious hurdle to overcome for these compounds to be used clinically. In this article, we have reviewed key functional and structural properties of FP-2/3 and described different compound series reported as inhibitors of these proteases during decades of active research in the field. Special attention is also paid to the wide range of computer-aided drug design (CADD) techniques successfully applied to discover new active compounds. Finally, we provide guidelines that, in our understanding, will help advance the rational discovery of new FP-2/3 inhibitors.

Design, synthesis and biological evaluation of novel thiosemicarbazones as cruzipain inhibitors.

Based on the activity of 23 TSCs on CZ taken from the literature, we have developed a QSAR model for predicting the activity of TSCs. New TSCs were designed and then tested against CZP, resulting in inhibitors with IC50 values in the nanomolar range. The modelling of the corresponding TSC-CZ complexes by molecular docking and QM/QM ONIOM refinement indicates a binding mode compatible with what was expected for active TSCs, according to a geometry-based theoretical model previously developed by our research group. Kinetic experiments on CZP suggest that the new TSCs act by a mechanism that involves the formation of a reversible covalent adduct with slow association and dissociation kinetics. These results demonstrate the strong inhibitory effect of the new TSCs and the benefit of the combined use of QSAR and molecular modelling techniques in the design of new and potent CZ/CZP inhibitors.

The Trypanosoma cruzi Antigen and Epitope Atlas: antibody specificities in Chagas disease patients across the Americas.

During an infection the immune system produces pathogen-specific antibodies. These antibody repertoires become specific to the history of infections and represent a rich source of diagnostic markers. However, the specificities of these antibodies are mostly unknown. Here, using high-density peptide arrays we examined the human antibody repertoires of Chagas disease patients. Chagas disease is a neglected disease caused by Trypanosoma cruzi, a protozoan parasite that evades immune mediated elimination and mounts long-lasting chronic infections. We describe a proteome-wide search for antigens, characterised their linear epitopes, and show their reactivity on 71 individuals from diverse human populations. Using single-residue mutagenesis we revealed the core functional residues for 232 of these epitopes. Finally, we show the diagnostic performance of identified antigens on challenging samples. These datasets enable the study of the Chagas antibody repertoire at an unprecedented depth and granularity, while also providing a rich source of serological biomarkers.

Enzyme Inhibitors from Gorgonians and Soft Corals.

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

Thiosemicarbazone derivatives: Evaluation as cruzipain inhibitors and molecular modeling study of complexes with cruzain.

The development of cruzipain inhibitors represents one of the most attractive challenges in the search for drugs for the treatment of Chagas disease. A recombinant form of this enzyme, cruzain, has been crystallized with numerous inhibitors, excluding thiosemicarbazones. These compounds have been established as potent inhibitors of cruzain, although there is very little data in the literature of thiosemicarbazones tested on cruzipain. In this work, we present the results of the evaluation of eleven thiosemicarbazones on cruzipain, isolated from T. cruzi epimastigotes, six of them previously evaluated on cruzain. For these latter, we studied through computational methods, the mode of interaction with the active site of cruzain and the contribution of geometric parameters to the possible mechanism of action involved in the observed inhibition. Finally, from some geometric parameters analyzed on modeled TSC-cruzain complexes, a semi-quantitative relationship was established that could explain the inhibitory activity of thiosemicarbazones on cruzipain, the enzyme actually present in the parasite.

Tetracycline Derivatives Inhibit Plasmodial Cysteine Protease Falcipain-2 through Binding to a Distal Allosteric Site.

Allosteric inhibitors regulate enzyme activity from remote and usually specific pockets. As they promise an avenue for less toxic and safer drugs, the identification and characterization of allosteric inhibitors has gained great academic and biomedical interest in recent years. Research on falcipain-2 (FP-2), the major papain-like cysteine hemoglobinase of Plasmodium falciparum, might benefit from this strategy to overcome the low selectivity against human cathepsins shown by active site-directed inhibitors. Encouraged by our previous finding that methacycline inhibits FP-2 noncompetitively, here we assessed other five tetracycline derivatives against this target and characterized their inhibition mechanism. As previously shown for methacycline, tetracycline derivatives inhibited FP-2 in a noncompetitive fashion, with Ki values ranging from 121 to 190 µM. A possible binding to the S' side of the FP-2 active site, similar to that described by X-ray crystallography (PDB: 6SSZ) for the noncompetitive inhibitor E-chalcone 48 (EC48), was experimentally discarded by kinetic analysis using a large peptidyl substrate spanning the whole active site. By combining lengthy molecular dynamics (MD) simulations that allowed methacycline to diffuse from solution to different FP-2 surface regions and free energy calculations, we predicted the most likely binding mode of the ligand. Of note, the proposed binding pose explains the low differences in Ki values observed for the tested tetracycline derivatives and the calculated binding free energies match the experimental values. Overall, this study has implications for the design of novel allosteric inhibitors against FP-2 and sets the basis for further optimization of the tetracycline scaffold to produce more potent and selective inhibitors.

In silico identification of noncompetitive inhibitors targeting an uncharacterized allosteric site of falcipain-2.

Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.

Screening and Identification of Metacaspase Inhibitors: Evaluation of Inhibition Mechanism and Trypanocidal Activity.

A common strategy to identify new antiparasitic agents is the targeting of proteases, due to their essential contributions to parasite growth and development. Metacaspases (MCAs) are cysteine proteases present in fungi, protozoa, and plants. These enzymes, which are associated with crucial cellular events in trypanosomes, are absent in the human host, thus arising as attractive drug targets. To find new MCA inhibitors with trypanocidal activity, we adapted a continuous fluorescence enzymatic assay to a medium-throughput format and carried out screening of different compound collections, followed by the construction of dose-response curves for the most promising hits. We used MCA5 from Trypanosoma brucei (TbMCA5) as a model for the identification of inhibitors from the GlaxoSmithKline HAT and CHAGAS chemical boxes. We also assessed a third collection of nine compounds from the Maybridge database that had been identified by virtual screening as potential inhibitors of the cysteine peptidase falcipain-2 (clan CA) from Plasmodium falciparum Compound HTS01959 (from the Maybridge collection) was the most potent inhibitor, with a 50% inhibitory concentration (IC50) of 14.39 µM; it also inhibited other MCAs from T. brucei and Trypanosoma cruzi (TbMCA2, 4.14 µM; TbMCA3, 5.04 µM; TcMCA5, 151 µM). HTS01959 behaved as a reversible, slow-binding, and noncompetitive inhibitor of TbMCA2, with a mechanism of action that included redox components. Importantly, HTS01959 displayed trypanocidal activity against bloodstream forms of T. brucei and trypomastigote forms of T. cruzi, without cytotoxic effects on Vero cells. Thus, HTS01959 is a promising starting point to develop more specific and potent chemical structures to target MCAs.

In silico Guided Drug Repurposing: Discovery of New Competitive and Non-competitive Inhibitors of Falcipain-2.

Malaria is among the leading causes of death worldwide. The emergence of Plasmodium falciparum resistant strains with reduced sensitivity to the first line combination therapy and suboptimal responses to insecticides used for Anopheles vector management have led to renewed interest in novel therapeutic options. Here, we report the development and validation of an ensemble of ligand-based computational models capable of identifying falcipain-2 inhibitors, and their subsequent application in the virtual screening of DrugBank and Sweetlead libraries. Among four hits submitted to enzymatic assays, two (odanacatib, an abandoned investigational treatment for osteoporosis and bone metastasis, and the antibiotic methacycline) confirmed inhibitory effects on falcipain-2, with Ki of 98.2 nM and 84.4 µM. Interestingly, Methacycline proved to be a non-competitive inhibitor (α = 1.42) of falcipain-2. The effects of both hits on falcipain-2 hemoglobinase activity and on the development of P. falciparum were also studied.

Potent and selective inhibitors for M32 metallocarboxypeptidases identified from high-throughput screening of anti-kinetoplastid chemical boxes.

Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas disease, respectively. These enzymes are absent in humans and several functions have been proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been reported so far for these enzymes. Here, we present the identification of a set of inhibitors for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two collections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzymatic assays to a medium-throughput format and carried out the screening of both collections, followed by the construction of dose-response curves for the most promising hits. As a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE), a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor reported for this family of MCPs and considering its potency and specificity, TCMDC-143620 seems to be a promissory starting point to develop more specific and potent chemical tools targeting M32 MCPs from trypanosomatid parasites.

Target-based Screening of the Chagas Box: Setting Up Enzymatic Assays to Discover Specific Inhibitors Across Bioactive Compounds.

Chagas disease is a neglected tropical illness caused by the protozoan parasite Trypanosoma cruzi. The disease is endemic in Latin America with about 6 million people infected and many more being at risk. Only two drugs are available for treatment, Nifurtimox and Benznidazole, but they have a number of side effects and are not effective in all cases. This makes urgently necessary the development of new drugs, more efficient, less toxic and affordable to the poor people, who are most of the infected population. In this review we will summarize the current strategies used for drug discovery considering drug repositioning, phenotyping screenings and target-based approaches. In addition, we will describe in detail the considerations for setting up robust enzymatic assays aimed at identifying and validating small molecule inhibitors in high throughput screenings.

Identification of (4-(9H-fluoren-9-yl) piperazin-1-yl) methanone derivatives as falcipain 2 inhibitors active against Plasmodium falciparum cultures.

BACKGROUND: Falcipain 2 (FP-2) is the hemoglobin-degrading cysteine protease of Plasmodium falciparum most extensively targeted to develop novel antimalarials. However, no commercial antimalarial drugs based on FP-2 inhibition are available yet due to the low selectivity of most FP-2 inhibitors against the human cysteine proteases. METHODS: A structure-based virtual screening (SVBS) using Maybridge HitFinder™ compound database was conducted to identify potential FP-2 inhibitors. In vitro enzymatic and cell-growth inhibition assays were performed for the top-scoring compounds. Docking, molecular dynamics (MD) simulations and free energy calculations were employed to study the interaction of the best hits with FP-2 and other related enzymes. RESULTS AND CONCLUSIONS: Two hits based on 4-(9H-fluoren-9-yl) piperazin-1-yl) methanone scaffold, HTS07940 and HTS08262, were identified as inhibitors of FP-2 (half-maximal inhibitory concentration (IC50) = 64 µM and 14.7 µM, respectively) without a detectable inhibition against the human off-target cathepsin K (hCatK). HTS07940 and HTS08262 inhibited the growth of the multidrug-resistant P. falciparum strain FCR3 in culture (half-maximal inhibitory concentrations (IC50) = 2.91 µM and 34 µM, respectively) and exhibited only moderate cytotoxicity against HeLa cells (Half-maximal cytotoxic concentration (CC50) = 133 µM and 350 µM, respectively). Free energy calculations reproduced the experimental affinities of the hits for FP-2 and explained the selectivity with respect to hCatK. GENERAL SIGNIFICANCE: To the best of our knowledge, HTS07940 stands among the most selective FP-2 inhibitors identified by SBVS reported so far, displaying moderate antiplasmodial activity and low cytotoxicity against human cells. Hence, this compound constitutes a promising lead for the design of more potent and selective FP-2 inhibitors.

Author Correction: Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

DNA-damage inducible protein 1 is a conserved metacaspase substrate that is cleaved and further destabilized in yeast under specific metabolic conditions.

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death in plants and in progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. Here, we report that the DNA-damage inducible protein 1 (Ddi1) represents a conserved protein substrate for metacaspases belonging to divergent unicellular eukaryotes (trypanosomes and yeasts). We show that although the recognized cleavage sequence is not identical among the different model organisms tested, in all of them the proteolysis consequence is the removal of the ubiquitin-associated domain (UBA) present in the protein. We also demonstrate that Ddi1 cleavage is tightly regulated in vivo as it only takes place in yeast when calcium increases but under specific metabolic conditions. Finally, we show that metacaspase-mediated Ddi1 cleavage reduces the stability of this protein which can certainly impact on the many functions ascribed for it, including shuttle to the proteasome, cell cycle control, late secretory pathway regulation, among others.

Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes.

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.

Intensity fading MALDI-TOF mass spectrometry and functional proteomics assignments to identify protease inhibitors in marine invertebrates.

Proteases and their inhibitors have become molecules of increasing fundamental and applicative value. Here we report an integrated strategy to identify and analyze such inhibitors from Caribbean marine invertebrates extracts by a fast and sensitive functional proteomics-like approach. The strategy works in three steps: i) multiplexed enzymatic inhibition kinetic assays, ii) Intensity Fading MALDI-TOF MS to establish a link between inhibitory molecules and the related MALDI signal(s) detected in the extract(s), and iii) ISD-CID-T3 MS fragmentation on the parent MALDI signals selected in the previous step, enabling the partial or total top-down sequencing of the molecules. The present study has allowed validation of the whole approach, identification of a substantial number of novel protein protease inhibitors, as well as full or partial sequencing of reference molecular species and of many unknown ones, respectively. Such inhibitors correspond to six protease subfamilies (metallocarboxypeptidases-A and -B, pepsin, papain, trypsin and subtilisin), are small (1-10KDa) disulfide-rich proteins, and have been found at diverse frequencies among the invertebrates (13 to 41%). The overall procedure could be tailored to other enzyme-inhibitor and protein interacting systems, analyzing samples at medium-throughput level and leading to the functional and structural characterization of proteinaceous ligands from complex biological extracts. SIGNIFICANCE: Invertebrate animals, and marine ones among, display a remarkable diversity of species and contained biomolecules. Many of their proteins-peptides have high biological, biotechnological and biomedical potential interest but, because of the lack of sequenced genomes behind, their structural and functional characterization constitutes a great challenge. Here, looking at the small, disulfide-rich, proteinaceous inhibitors of proteases found in them, it is shown that such problem can be significatively facilitated by integrative multiplexed enzymatic assays, affinity-based Intensity-Fading (IF-) MALDI-TOF mass spectrometry (MS), and on-line MS fragmentation, in a fast and easy approach.

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.

Natural products from marine origin constitute a very promising and underexplored source of interesting compounds for modern biotechnological and pharmaceutical industries. However, their evaluation is quite challenging and requires specifically designed assays to reliably identify the compounds of interest in a highly heterogeneous and interfering context. In the present study, we describe a general strategy for the confident identification of tight-binding protease inhibitors in the aqueous extracts of 62 Cuban marine invertebrates, using Plasmodium falciparum hemoglobinases Plasmepsin II and Falcipain 2 as model enzymes. To this end, we first developed a screening strategy that combined enzymatic with interaction-based assays and then validated screening conditions using five reference extracts. Interferences were evaluated and minimized. The results from the massive screening of such extracts, the validation of several hits by a variety of interaction-based assays and the purification and functional characterization of PhPI, a multifunctional and reversible tight-binding inhibitor for Plasmepsin II and Falcipain 2 from the gorgonian Plexaura homomalla, are presented.

Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes.

Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2 µmol aldehyde/cm2). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH2, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV-GCM showed the highest maximal immobilized protein load (2.1 µg/cm2), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.

Diseño asistido por computadora de la inmovilización covalente de bromelina y papaína / Computer-aided design of bromelain and papain covalent immobilization

Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The aim of this work was to optimize the covalent immobilization of bromelain and papain via rational design of immobilized derivatives strategy (RDID) and RDID1.0 program. Were determined the maximum protein quantity to immobilize, the optimum immobilization pH (in terms of functional activity retention), was predicted the most probable configuration of the immobilized derivative and the probabilities of multipoint covalent attachment. As support material was used Glyoxyl-Sepharose CL 4B. The accuracy of RDID1.0 program´s prediction was demonstrated comparing with experimental results. Bromelain and papain immobilized derivatives showed desired characteristics for industrial biocatalysis, such as: elevate pH stability retaining 95% and 100% residual activity at pH 7.0 and 8.0, for bromelain and papain, respectively; high thermal stability at 30 °C retaining 90% residual activity for both immobilized enzymes; a catalytic configuration bonded by immobilization at optimal pH; and the ligand load achieve ensure the minimization of diffusional restrictions.

Las enzimas inmovilizadas han sido ampliamente utilizadas en las industrias Alimentaria, Agroquímica, Farmacéutica y Biotecnológica. La inmovilización de proteínas es, probablemente, la tecnología más empleada para elevar la estabilidad operacional de estas moléculas. La bromelina (Ananas comosus) y la papaína (Carica papaya) son cisteín proteasas extensamente usadas como biocatalizadores inmovilizados con disímiles aplicaciones en la terapéutica, resolución de mezclas racémicas, cromatografía de afinidad, entre otros escenarios industriales. El objetivo del presente trabajo fue optimizar la inmovilización covalente de las enzimas bromelina y papaína a través de la estrategia de diseño racional de derivados inmovilizados (RDID) y el programa RDID1.0. Se predijo la cantidad máxima de proteína a inmovilizar, el pH óptimo de inmovilización (en términos de retención de la actividad funcional), la configuración más probable del derivado inmovilizado y la probabilidad de enlazamiento covalente multipuntual. Como soporte de inmovilización de empleó Glioxil-Sepharose CL 4B. La precisión de las predicciones llevadas a cabo con el programa RDID1.0 fue validada comparando con los resultados experimentales obtenidos. Los derivados inmovilizados de bromelina y papaína mostraron características deseadas para la biocatálisis a nivel industrial, tales como: elevada estabilidad al pH reteniendo el 95% y 100% de actividad residual a pH 7.0 y 8.0, para la bromelina y la papaína, respectivamente; una elevada estabilidad térmica con la retención del 90% de actividad residual a 30 °C para ambas enzimas; al pH de inmovilización óptimo la configuración obtenida es catalíticamente competente; y la carga de ligando alcanzada asegura la disminución de las restricciones difusionales.

Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla.

Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum. The resultant fraction was chromatographically enriched in tight-binding inhibitors active against Papain-like cysteine peptidases (92%) and Pepsin-like aspartyl peptidases (8%). Globally, the inhibitors present in the enriched fraction showed no competition with substrates and apparent Ki values of 1.99 and 4.81nM for Falcipain 2 and Cruzipain, the major cysteine peptidases from P. falciparum and T. cruzi, respectively. The inhibitor-enriched fraction showed promising antiparasitic activity in cultures. It reduced the growth of the chloroquine-resistant P. falciparum strain Dd2 (IC50=0.46µM) and promoted the apparent accumulation of trophozoites, both consistent with a blockade in the hemoglobin degradation pathway. At sub-micromolar concentrations, the inhibitor-enriched fraction reduced the infection of VERO cells by T. cruzi (CL Brener clone) trypomastigotes and interfered with intracellular differentiation and/or replication of the parasites. This study provides new scientific evidence that confirms P. homomalla as an excellent source of tight-biding protease inhibitors for different proteases with biomedical relevance, and suggests that either the individual inhibitors or the enriched fraction itself could be valuable as antiparasitic compounds.