Search Results (210)

Chagas disease, caused by Trypanosoma cruzi, poses a significant public health challenge due to its widespread prevalence, limited therapeutic options, and adverse effects associated with available medications. In this study, we developed 13 novel pyrazole-imidazoline derivatives, inspired by a previously identified cysteine protease inhibitor, and evaluated their antiparasitic activity. Our in silico analyses predicted favorable physicochemical profiles and promising oral bioavailability for these derivatives. Upon phenotypic screening, we observed that these new derivatives exhibited low cytotoxicity (CC₅₀ > 100 µM) and marked efficacy against intracellular amastigotes. Derivative 1k showed high activity (IC₅₀ = 3.3 ± 0.2 µM), selectivity (SI = 73.9), and potency (pIC₅₀ = 5.4). In a 3D cardiac microtissue model, 1k significantly reduced parasite load, matching the efficacy of benznidazole (Bz) even at lower concentrations. Both 1k and Bz effectively prevented parasite recrudescence; however, neither resulted in parasite sterility under the experimental conditions employed. The combination of 1k–Bz yielded an additive interaction, highlighting its potential for in vivo combination therapy. While structural changes abolished cysteine protease inhibition, incorporating a CF₃ substituent at the para position and excluding the amino group enhanced antiparasitic activity. These findings reinforce the promise of the pyrazole-imidazoline scaffold and support further structural optimizations to develop innovative candidates for treating Chagas disease. Full article

Background/Objectives: Gingipains produced by P. gingivalis have been shown to be directly related to periodontal tissue degradation and are significant molecular targets in therapy of periodontitis. Blocking the activity of these enzymes should reduce survival of this pathogen and mitigate the effects of inflammation in periodontitis. Therefore, gingipains inhibitors and specific antibodies could be recommended in the treatment of periodontitis. Cysteine peptidase inhibitors can be obtained by chemical synthesis, or isolated from natural raw materials. This research has the following aims: 1. to analyze in vitro the inhibition of cysteine protease activity in the gingival crevicular fluid (GCF) and 2. to compare the toxicity of natural raw inhibitors (obtained from Fallopia japonica plant and egg white) with chlorhexidine (CHX) using an MTS viability test. Methods: Samples of GCF were collected from healthy (N = 17) individuals and (N = 65) periodontal patients. Cysteine peptidase activity was inhibited by adding a solution of cystatin from egg white (with 20% glycerol), or cystatin from knotweed, or low molecular weight inhibitors (MW < 3 kDa) from egg white and knotweed against Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride. Results: There was a statistically significant difference between the inhibition means of cysteine protease activity for the five groups (p < 0.001). Means for the four groups of patients with periodontitis were not statistically significant different from each other (p = 0.320). The inhibition rates were higher in periodontitis patients. The toxicity of knotweed cystatin inhibitor was several times lower than the toxicity of E-64d, and of CHX. Conclusion: Cysteine protease inhibitors isolated from egg or plants were non-toxic, effectively inhibited the activity of cysteine proteases in GCF, and may be a promising alternative to more toxic standard antimicrobials (CHX) in preventing periodontal tissue breakdown. Full article

Norovirus, a major cause of acute gastroenteritis, possesses a single-stranded positive-sense RNA genome. The viral 3C-like cysteine protease (3CL^pro) plays a critical role in processing the viral polyprotein into mature non-structural proteins, a step essential for viral replication. Targeting 3CL^pro has emerged as a promising strategy for developing small-molecule inhibitors against Norovirus. In this study, we employed a combination of virtual screening and the FlipGFP assay to identify potential inhibitors targeting the 3CL^pro of Norovirus genotype GII.4. A library of approximately 58,800 compounds was screened using AutoDock Vina tool, yielding 20 candidate compounds based on their Max Affinity scores. These compounds were subsequently evaluated using a cell-based FlipGFP assay. Among them, eight compounds demonstrated significant inhibitory activity against 3CL^pro, with Gedatolisib showing the most potent effect (IC₅₀ = 0.06 ± 0.01 μM). Molecular docking and molecular dynamics simulations were conducted to explore the binding mechanisms and structural stability of the inhibitor–3CL^pro complexes. Our findings provide valuable insights into the development of antiviral drugs targeting Norovirus 3CL^pro, offering potential therapeutic strategies to combat Norovirus infections. Full article

Background/Objectives: Nifuroxazide (Nfz) is a drug that has been used as a scaffold for designing antimicrobial and antiparasitic agents. This study aimed to synthesize and evaluate in vitro of Nfz and twenty-five 4-hydroxybenzhydrazone derivatives as potential anti-Trypanosoma cruzi, anti-Leishmania mexicana, and anti-Mycobacterium tuberculosis agents. Methods: The compounds were synthesized by condensing 4-hydroxybenzhydrazide with appropriate aldehydes in acidic conditions and structurally confirmed by spectroscopic techniques. All compounds were evaluated in vitro against T. cruzi strains (NINOA and A1), L. mexicana (M379 and FCQEPS strains), and M. tuberculosis (H37Rv strain), followed by enzymatic assays against T. cruzi cysteine proteases. Results: Compound Nfz-24 (IC₅₀ = 6.8 μM) had better trypanocidal activity than the reference drugs benznidazole (IC₅₀ > 30 μM) and nifurtimox (IC₅₀ > 7 μM) against the NINOA strain, and Nfz-8 (IC₅₀ = 7.2 μM) was the compound most active against the A1 strain with a high inhibition of T. cruzi cysteine proteases (IC₅₀ = 4.6 μM) and low cytotoxic effects (CC₅₀ >100 μM). On the other hand, compound Nfz-5 (IC₅₀ = 5.2 μM) had a 25-fold better leishmanicidal effect than glucantime (IC₅₀ > 125 μM) against the L. mexicana M379 strain, and compound Nfz-13 had the best leishmanicidal effects (IC₅₀ = 10.2 μM) against the FCQEPS strain. Finally, Nfz, Nfz-1, and Nfz-2 had minimum inhibitory concentration (MIC) values of 12.3, 5.1, and 18.8 μg/mL against M. tuberculosis, respectively. Conclusions: In summary, these results suggest that the compounds Nfz-1, Nfz-2, Nfz-5, Nfz-8, Nfz-10, Nfz-15, Nfz-24, and Nfz-25 are candidates for further studies to develop new and more potent anti-T. cruzi, anti-leishmaniasis, and anti-M. tuberculosis agents. Full article

Autolysis in sea cucumber has long been a threat to raw material storage and product processing. The involvement of endogenous cysteine protease in sea cucumber autolysis has been proved extendedly. However, as an essential part of the mechanism of autolysis, the role of its endogenous inhibitor has seldom been reported. To investigate the role of cysteine protease inhibitors in the early stage of hypoxic exposure-induced autolysis, a novel cystatin gene (SjCyt) belonging to the subfamily of cystatin C was cloned from Apostichopus japonicus by homology cloning and rapid amplification of cDNA ends. The affinity of SjCyt to cysteine protease (cathepsin L and cathepsin B) was investigated by molecular dynamics simulations. Pertinent metrics, including the root mean square deviation, radius of gyration, Gibbs free energy, binding free energy, and bond-forming frequency, showed that the conformation of SjCyt–SjCL was more stable and confirmed a stronger interaction of SjCyt with cathepsin L than with cathepsin B. Thus, cathepsin L (SjCL) was selected to further study its co-expression with SjCyt over a period of 9 h at an early stage of hypoxic exposure. Quantitative RT-qPCR revealed a ubiquitous transcriptional profile of SjCyt and SjCL in all the tested tissues, with the highest abundance in the dorsal epidermis, tube feet, and coelomocytes. Temporal transcription of them showed an overall up-regulated co-expression in the dorsal epidermis and tube feet. However, up-regulated SjCyt and down-regulated SjCL were observed at the protein level. Further immunofluorescence double labeling also found increased staining of SjCyt and SjCyt–SjCL complexes and decreased SjCL. Additionally, recombinant SjCyt was prepared and demonstrated an evident autolysis-inhibiting effect. The results of this study indicated that the anti-autolytic regulation of SjCyt functions at the very early stage of hypoxic exposure, exerting effects at both the transcriptional and translational levels. The above finding offers new insights into the mechanisms of sea cucumber autolysis. Full article

The SARS-CoV-2 proteases M^pro and PL^pro are critical targets for antiviral drug development for the treatment of COVID-19. The 1,2,4-thiadiazole functional group is an inhibitor of cysteine proteases, such as papain and cathepsins. This chemical moiety is also present in ceftaroline fosamil (CF), an FDA-approved fifth-generation cephalosporin antibiotic. This study investigates the interactions between CF, its primary metabolites (M1 is dephosphorylated CF and M2 is an opened β-lactam ring) and derivatives (protonated M1H and M2H), and its open 1,2,4-thiadiazole rings derivatives (open-M1H and open-M2H) with SARS-CoV-2 proteases and evaluates CF’s effects on in vitro viral replication. In silico analyses (molecular docking and molecular dynamics (MD) simulations) demonstrated that CF and its metabolites are potential inhibitors of PL^pro and M^pro. Docking analysis indicated that the majority of the ligands were more stable with M^pro than PL^pro; however, in vitro biochemical analysis indicated PL^pro as the preferred target for CF. CF inhibited viral replication in the human Calu-3 cell model at submicromolar concentrations when added to cell culture medium at 12 h. Our results suggest that CF should be evaluated as a potential repurposing agent for COVID-19, considering not only viral proteases but also other viral targets and relevant cellular pathways. Additionally, the reactivity of sulfur in the 1,2,4-thiadiazole moiety warrants further exploration for the development of viral protease inhibitors. Full article

Proteases play a pivotal role in cancer progression, facilitating processes such as extracellular matrix degradation, angiogenesis, and metastasis. Consequently, protease inhibitors have emerged as promising therapeutic agents in oncology. This review provides a comprehensive overview of the mechanisms by which protease inhibitors modulate cancer biology, categorizing inhibitors by their target protease classes, including matrix metalloproteinases, cysteine proteases, and serine proteases. We discuss the therapeutic potential of both synthetic and natural protease inhibitors, highlighting their applications in preclinical and clinical settings. Furthermore, challenges such as specificity, toxicity, and resistance mechanisms are addressed, alongside strategies to overcome these limitations through innovative drug designs and combination therapies. The future of protease inhibitors in cancer treatment lies in precision medicine, leveraging proteomic profiling to tailor therapies to individual tumors. This review underscores the importance of ongoing research and the development of novel approaches to harness protease inhibitors effectively for cancer management. Full article

The discovery of effective cysteine protease inhibitors with crude protein kiwi extracts (CPKEs) has created novel challenges and prospects for pharmaceutical development. Despite extensive research on CPKEs, limited research has been conducted on treating atopic dermatitis (AD). Therefore, the objective of this work was to investigate the anti-inflammatory effects of CPKEs on TNF-α activation in a HaCaT cell model and in a DNCB (1-chloro-2, 4-dinitrochlorobenzene)-induced atopic dermatitis animal model. The molecular weight of the CPKE was determined using SDS-PAGE under non-reducing (17 kDa and 22 kDa) and reducing conditions (25 kDa, 22 kDa, and 15 kDa), whereas gelatin zymography was performed to examine the CPKE’s inhibitory impact on cysteine protease (actinidin and papain) activity. Moreover, the CPKE remains stable at 60 °C, with pH levels varying from 4 to 11, as determined by the azocasein assay. CPKE treatment decreased the phosphorylation of mitogen-activated protein kinase (MAPK) and Akt, along with the activation of nuclear factor-kappa B (NF-κB)-p65 in tumor necrosis factor-α (TNF-α)-stimulated HaCaT cells. Five-week-old BALB/c mice were treated with DNCB to act as an AD-like animal model. The topical application of CPKE to DNCB-treated mice for three weeks substantially decreased clinical dermatitis severity and epidermal thickness and reduced eosinophil infiltration and mast cells into ear and skin tissues. These findings imply that CPKE derived from kiwifruit might be a promising therapy option for inflammatory skin diseases such as AD. Full article

Wheat (Triticum aestivum L.) is a primary crop globally. Among the numerous pathogens affecting wheat production, Puccinia striiformis f. sp. tritici (Pst) is a significant biotic stress agent and poses a major threat to world food security by causing stripe rust or yellow rust disease. Understanding the molecular basis of plant–pathogen interactions is crucial for developing new means of disease management. It is well established that the effector proteins play a pivotal role in pathogenesis. Therefore, studying effector proteins has become an important area of research in plant biology. Our previous work identified differentially expressed candidate secretory effector proteins of stripe rust based on transcriptome sequencing data from susceptible wheat (Avocet S) and resistant wheat (Avocet YR10) infected with Pst. Among the secreted effector proteins, PSTG_14090 contained an ancient double-psi beta-barrel (DPBB) fold, which is conserved in the rare lipoprotein A (RlpA) superfamily. This study investigated the role of PSTG_14090 in plant immune responses, which encodes a protein, here referred to as Pst-DPBB, having 131 amino acids with a predicted signal peptide (SP) of 19 amino acids at the N-terminal end, and the DNA sequence of this effector is highly conserved among different stripe rust races. qRT-PCR analysis indicated that expression levels are upregulated during the early stages of infection. Subcellular localization studies in Nicotiana benthamiana leaves and wheat protoplasts revealed that it is distributed in the cytoplasm, nucleus, and apoplast. We demonstrated that Pst-DPBB negatively regulates the immune response by functioning in various compartments of the plant cells. Based on Co-IP and structural predictions and putative interaction analyses by AlphaFold 3, we propose the probable biological function(s). Pst-DPBB behaves as a papain inhibitor of wheat cysteine protease; Pst-DPBB has high structural homology to kiwellin, which is known to interact with chorismate mutase, suggesting that Pst-DPBB inhibits the native function of the host chorismate mutase involved in salicylic acid synthesis. The DPBB fold is also known to interact with DNA and RNA, which may suggest its possible role in regulating the host gene expression. Full article

The processes before heating in surimi gel production, such as grinding and molding, are performed below 10 °C to prevent unintentional protein gelation (suwari or setting) catalyzed by endogenous transglutaminase. Adding transglutaminase and protease inhibitors contributes to obtaining surimi gel with superior breaking properties after suwari suppression. However, the impact of suwari suppression and enzyme inhibitors on the water holding capacity (WHC) of surimi gel remains unclear. This study investigated changes in the WHC of surimi gel during long-term suwari suppression by adding enzyme inhibitors at 25 °C and clarified its mechanism. The expressible drip of samples with EGTA, a transglutaminase inhibitor, increased with heating time at 25 °C to 12.3% owing to proteolysis. The use of N-ethylmaleimide and E-64, cysteine protease inhibitors, further increased the expressible drip of samples (18.9–19.4%) because of protein conformational changes which were observed by DSC analysis and salt solubility measurement. However, this increase was inhibited by a modified grinding method where NaCl was mixed with surimi before adding enzyme inhibitors. This modified method prevented the reduction in protein salt solubility due to protein conformational changes, thereby maintaining WHC in surimi gel with suwari suppression. Full article

This study explores the extraction and characterization of proteolytic enzymes from brewer’s spent grain (BSG) and their potential as sustainable coagulants in the dairy industry. BSG samples from various beer types (Blonde Ale, IPA, Kölsch, Honey, and Porter) were obtained from two artisanal breweries in Mar del Plata, Argentina. Optimization of caseinolytic activity (CA) and protein extraction was conducted using a Plackett–Burman design, followed by a Box–Behnken design. Optimal protein concentration was achieved at intermediate pH and high temperature, while CA peaked at pH 8.0. The specific caseinolytic activity (SCA) varied among the extracts, with BSG3 showing the highest activity (99.6 U mg⁻¹) and BSG1 the lowest (60.4 U mg⁻¹). Protease inhibitor assays suggested the presence of aspartic, serine, metallo, and cysteine proteases. BSG3 and BSG4 showed the highest hydrolysis rates for α-casein (70% and 78%). For κ-casein, BSG1, BSG2, and BSG3 demonstrated moderate activity (56.5%, 49%, and 55.8), while BSG4 and BSG5 exhibited the lowest activity. Additionally, the milk-clotting activity (MCA) of BSG extracts was comparable to plant-based coagulants like Cynara cardunculus and Ficus carica. These findings highlight the potential of BSG-derived proteases as alternative coagulants for cheese production, offering a sustainable link between the brewing and dairy industries. Full article

Endometriosis is a common chronic disorder characterized by the growth of endometrium-like tissue outside the uterine cavity. The disease is associated with chronic inflammation and pelvic pain and may have an impact on the patient’s fertility. The causative factors and pathophysiology of the disease are still poorly recognized. The dysregulation of the immune system, aberrant tissue remodeling, and angiogenesis contribute to the disease progression. In endometriosis patients, the proteins regulating the breakdown and reorganization of the connective tissue, e.g., collagenases, and other proteases, as well as their inhibitors, show an incorrect pattern of expression. Here, we report that the expression of reversion-inducing cysteine-rich protein with Kazal motifs (RECK), one of the inhibitors of connective tissue proteases, is elevated in endometrioma cysts as compared to normal endometrium from unaffected women. We also demonstrate a reduced level of miR200b in endometriotic tissue that correlates with RECK mRNA levels. Furthermore, we employ the 12Z cell line, derived from a peritoneal endometriotic lesion, and the Ishikawa cell line, originating from endometrial adenocarcinoma to identify RECK as a direct target of miR200b. The described effect of miR200b on RECK, together with the aberrant expression of both genes in endometrioma, may help to understand the role played by the tissue remodeling system in the pathogenesis of endometriosis. Full article

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC₅₀ values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC₅₀ in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves. Full article

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system. Full article

SARS-CoV-2 is a spherical, positive-sense, single-stranded RNA virus with a large genome, responsible for encoding both structural proteins, vital for the viral particle’s architecture, and non-structural proteins, critical for the virus’s replication cycle. Among the non-structural proteins, two cysteine proteases emerge as promising molecular targets for the design of new antiviral compounds. The main protease (M^pro) is a homodimeric enzyme that plays a pivotal role in the formation of the viral replication–transcription complex, associated with the papain-like protease (PL^pro), a cysteine protease that modulates host immune signaling by reversing post-translational modifications of ubiquitin and interferon-stimulated gene 15 (ISG15) in host cells. Due to the importance of these molecular targets for the design and development of novel anti-SARS-CoV-2 drugs, the purpose of this review is to address aspects related to the structure, mechanism of action and strategies for the design of inhibitors capable of targeting the M^pro and PL^pro. Examples of covalent and non-covalent inhibitors that are currently being evaluated in preclinical and clinical studies or already approved for therapy will be also discussed to show the advances in medicinal chemistry in the search for new molecules to treat COVID-19. Full article