BACKGROUND: Human African Trypanosomiasis (HAT), also known as sleeping sickness, and Chagas disease are neglected tropical diseases caused by Trypanosoma brucei and Trypanosoma cruzi, respectively. These diseases present significant challenges in treatment due to the toxicity, low efficacy, and drug-resistant strains associated with current therapies.
BACKGROUND: Cysteine proteases play vital roles in the life cycles of these parasites, making them potential targets for therapeutic intervention. Natural inhibitors sourced from plants, marine organisms, and microorganisms show promise for developing novel therapies.
METHODS: This review surveys the potential of natural inhibitors as therapeutic agents against HAT and Chagas disease. It compiles PubMed and PubChem information from various studies to provide an overview of their activities and characteristics, including their ability to inhibit cysteine proteases, modulate the host immune response, and interfere with other parasite proteins.
RESULTS: Several natural inhibitors, such as berberine, curcumin, and tannins, have been identified and characterized. These inhibitors have demonstrated encouraging outcomes in both in vitro and in vivo experiments, indicating their potential as therapeutic agents for HAT and Chagas disease.
CONCLUSIONS: Natural inhibitors of cysteine proteases offer a promising avenue for developing novel therapies against HAT and Chagas disease. Further research is needed to identify additional natural inhibitors and optimize their efficacy and safety for human use. The significance of this study lies in its potential to contribute to the discovery of effective, safe, and affordable treatments for these neglected tropical diseases.

The malaria parasite Plasmodium expresses four related papain-family cysteine proteases. Targeting these different cysteine proteases can elucidate their roles and potential as therapeutic targets, thereby expanding the pool of antimalarial targets. During gametogenesis, cysteine proteases like SERA-5, SERA-3, DPAP-1, DPAP-2, DPAP- 3, and Falcipain-1 are required for parasitophorous vacuole membrane (PVM) rupture. In the liver stage, cysteine proteases such as Falcipain-1 and SERA-3, SERA-4, SERA-5, and SERA-6 are essential. Additionally, cysteine proteases like DPAP-3, Falcipain- 1, Falcipain-2, Falcipain-3, and SERA-5, SERA-6 play crucial roles in merozoite invasion into red blood cells (RBCs), hemoglobin degradation, and merozoite release from RBCs. This review summarizes the available literature describing the key roles of various cysteine proteases in the life cycle of the malaria parasite and their potential as targets for antimalarial therapy. Understanding these proteases could aid in developing novel antimalarial treatments and overcoming drug resistance.

Substituted 5-hydroxy γ-pyrones have shown promise as covalent inhibitor leads against cysteine proteases and transcription factors, but their hydrolytic instability has hindered optimization efforts. Previous mechanistic proposals have suggested that these molecules function as Michael acceptor prodrugs, releasing a leaving group to generate an o-quinone methide-like structure. Addition to this electrophile of either water or an active site cysteine was purported to lead to inhibitor hydrolysis or enzyme inhibition, respectively. Through the use of kinetic nuclear magnetic resonance experiments, Hammett analysis, kinetic isotope effect studies, and density functional theory calculations, our findings suggest that enzyme inhibition and hydrolysis proceed by distinct pathways and are differentially influenced by substituent electronics. This mechanistic revision helps enable a more rational optimization for this class of promising compounds.

Legionella pneumophila grows within membrane-bound vacuoles in phylogenetically diverse hosts. Intracellular growth requires the function of the Icm/Dot type-IVb secretion system, which translocates more than 300 proteins into host cells. A screen was performed to identify L. pneumophila proteins that stimulate mitogen-activated protein kinase (MAPK) activation, using Icm/Dot translocated proteins ectopically expressed in mammalian cells. In parallel, a second screen was performed to identify L. pneumophila proteins expressed in yeast that cause growth inhibition in MAPK pathway-stimulatory high-osmolarity medium. LegA7 was shared in both screens, a protein predicted to be a member of the bacterial cysteine protease family that has five carboxyl-terminal ankyrin repeats. Three conserved residues in the predicted catalytic triad of LegA7 were mutated. These mutations abolished the ability of LegA7 to inhibit yeast growth. To identify other residues important for LegA7 function, a generalizable selection strategy in yeast was devised to isolate mutants that have lost function and no longer cause growth inhibition on a high-osmolarity medium. Mutations were isolated in the two carboxyl-terminal ankyrin repeats, as well as an inter-domain region located between the cysteine protease domain and the ankyrin repeats. These mutations were predicted by AlphaFold modeling to localize to the face opposite from the catalytic site, arguing that they interfere with the positive regulation of the catalytic activity. Based on our data, we present a model in which LegA7 harbors a cysteine protease domain with an inter-domain and two carboxyl-terminal ankyrin repeat regions that modulate the function of the catalytic domain.
OBJECTIVE: Legionella pneumophila grows in a membrane-bound compartment in macrophages during disease. Construction of the compartment requires a dedicated secretion system that translocates virulence proteins into host cells. One of these proteins, LegA7, is shown to activate a stress response pathway in host cells called the mitogen-activated protein kinase (MAPK) pathway. The effects on the mammalian MAPK pathway were reconstructed in yeast, allowing the development of a strategy to identify the role of individual domains of LegA7. A domain similar to cysteine proteases is demonstrated to be critical for impinging on the MAPK pathway, and the catalytic activity of this domain is required for targeting this path. In addition, a conserved series of repeats, called ankyrin repeats, controls this activity. Data are provided that argue the interaction of the ankyrin repeats with unknown targets probably results in activation of the cysteine protease domain.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and β-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded β-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the β-flap inducing the active conformation of catalytic residues. The β-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δβ-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the β-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the β-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.

In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.

In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a ∼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances.