Effects of plant protease inhibitors (Pep-3-EcTI, Pep-BbKI, and Pep-BrTI) versus corticosteroids on inflammation, remodeling, and oxidative stress in an asthma-COPD (ACO) model.

The peptide derived from E. contortisiliquum trypsin inhibitor (Pep-3-EcTI), peptide derived from kallikrein inhibitor isolated from B. bauhinioides (Pep-BbKI), and B. rufa peptide modified from B. bauhinioides (Pep-BrTI) peptides exhibit anti-inflammatory and antioxidant activities, suggesting their potential for treating asthma-chronic obstructive pulmonary disease (COPD) overlap (ACO). We compared the effects of these peptides with dexamethasone (DX) treatment in an ACO model. In this study, 11 groups of male BALB/c mice were pre-treated under different conditions, including sensitization with intraperitoneal injection and inhalation of ovalbumin (OVA), intratracheal instillation of porcine pancreatic elastase (ELA), sensitization with intraperitoneal injection, and various combinations of peptide treatments with Pep-3-EcTI, Pep-BbKI, Pep-BrTI, dexamethasone, and non-treated controls (SAL-saline). Respiratory system resistance, airway resistance, lung tissue resistance, exhaled nitric oxide, linear mean intercept, immune cell counts in the bronchoalveolar lavage fluid, cytokine expression, extracellular matrix remodeling, and oxidative stress in the airways and alveolar septa were evaluated on day 28. Results showed increased respiratory parameters, inflammatory markers, and tissue remodeling in the ACO group compared to controls. Treatment with the peptides or DX attenuated or reversed these responses, with the peptides showing effectiveness in controlling hyperresponsiveness, inflammation, remodeling, and oxidative stress markers. These peptides demonstrated an efficacy comparable to that of corticosteroids in the ACO model. However, this study highlights the need for further research to assess their safety, mechanisms of action, and potential translation to clinical studies before considering these peptides for human use.

Investigating the Effects of a New Peptide, Derived from the Enterolobium contortisiliquum Proteinase Inhibitor (EcTI), on Inflammation, Remodeling, and Oxidative Stress in an Experimental Mouse Model of Asthma-Chronic Obstructive Pulmonary Disease Overlap (ACO).

The synthesized peptide derived from Enterolobium contortisiliquum (pep3-EcTI) has been associated with potent anti-inflammatory and antioxidant effects, and it may be a potential new treatment for asthma-COPD overlap-ACO). Purpose: To investigate the primary sequence effects of pep3-EcTI in an experimental ACO. BALB/c mice were divided into eight groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep3-EcTI (treated with inhibitor), ACO-DX (treated with dexamethasone), ACO-DX-pep3-EcTI (treated with dexamethasone and inhibitor), and SAL-pep3-EcTI (saline group treated with inhibitor). We evaluated the hyperresponsiveness to methacholine, exhaled nitric oxide, bronchoalveolar lavage fluid (BALF), mean linear intercept (Lm), inflammatory markers, tumor necrosis factor (TNF-α), interferon (IFN)), matrix metalloproteinases (MMPs), growth factor (TGF-ß), collagen fibers, the oxidative stress marker inducible nitric oxide synthase (iNOS), transcription factors, and the signaling pathway NF-κB in the airways (AW) and alveolar septa (AS). Statistical analysis was conducted using one-way ANOVA and t-tests, significant when p < 0.05. ACO caused alterations in the airways and alveolar septa. Compared with SAL, ACO-pep3-EcTI reversed the changes in the percentage of resistance of the respiratory system (%Rrs), the elastance of the respiratory system (%Ers), tissue resistance (%Gtis), tissue elastance (%Htis), airway resistance (%Raw), Lm, exhaled nitric oxide (ENO), lymphocytes, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, transforming growth factor (TGF)-ß, collagen fibers, and iNOS. ACO-DX reversed the changes in %Rrs, %Ers, %Gtis, %Htis, %Raw, total cells, eosinophils, neutrophils, lymphocytes, macrophages, IL-1ß, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, TGF-ß, collagen fibers, and iNOS. ACO-DX-pep3-EcTI reversed the changes, as was also observed for the pep3-EcTI and the ACO-DX-pep3-EcTI. Significance: The pep3-EcTI was revealed to be a promising strategy for the treatment of ACO, asthma, and COPD.

Effects of a Peptide Derived from the Primary Sequence of a Kallikrein Inhibitor Isolated from Bauhinia bauhinioides (pep-BbKI) in an Asthma-COPD Overlap (ACO) Model.

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-ß, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO (p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1ß(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-ß (AS), collagen fibers (AW), iNOS, and eNO (p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons (p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Impairment of SK-MEL-28 Development-A Human Melanoma Cell Line-By the Crataeva tapia Bark Lectin and Its Sequence-Derived Peptides.

Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstrated deleterious effects on prostate and glioblastoma tumor cells. However, neither rBbKIm nor its derived peptides affected the viability of SK-MEL-28, a melanoma cell line, while CrataBL decreased viability by over 60%. Two peptides, Pep. 26 (Ac-Q-N-S-S-L-K-V-V-P-L-NH2) and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2), were also tested. Pep. 27 suppressed cell migration and induced apoptosis when combined with vemurafenib, while Pep. 26 inhibited cell migration and reduced nitric oxide and the number of viable cells. Vemurafenib, a chemotherapy drug used to treat melanoma, was found to decrease the release of interleukin 8 and PDGF-AB/BB cytokines and potentiated the effects of proteins and peptides in reducing these cytokines. These findings suggest that protease inhibitors may be effective in blocking melanoma cells and highlight the potential of CrataBL and its derived peptides.

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time.

BACKGROUND: (p-BthTX-I)2 K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required. OBJECTIVES: To determine whether (p-BthTX-I)2 K affects the hemostatic system. METHODS: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I)2 K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg- 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I)2 K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline administration. RESULTS: (p-BthTX-I)2 K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I)2 K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I)2 K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I)2 K did not prolong the bleeding time in the mouse model of arterial thrombosis. CONCLUSION: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I)2 K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.

Modulation of STAT-1, STAT-3, and STAT-6 activities in THP-1 derived macrophages infected with two Trypanosoma cruzi strains.

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi, co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1ß) and/or increasing IL-4, IL-10, and TGF-ß. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-ß, and/or promotion of IFN-γ and IL-1ß release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells.

Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

Plant Kunitz Inhibitors and Their Interaction with Proteases: Current and Potential Pharmacological Targets.

The action of proteases can be controlled by several mechanisms, including regulation through gene expression; post-translational modifications, such as glycosylation; zymogen activation; targeting specific compartments, such as lysosomes and mitochondria; and blocking proteolysis using endogenous inhibitors. Protease inhibitors are important molecules to be explored for the control of proteolytic processes in organisms because of their ability to act on several proteases. In this context, plants synthesize numerous proteins that contribute to protection against attacks by microorganisms (fungi and bacteria) and/or invertebrates (insects and nematodes) through the inhibition of proteases in these organisms. These proteins are widely distributed in the plant kingdom, and are present in higher concentrations in legume seeds (compared to other organs and other botanical families), motivating studies on their inhibitory effects in various organisms, including humans. In most cases, the biological roles of these proteins have been assigned based mostly on their in vitro action, as is the case with enzyme inhibitors. This review highlights the structural evolution, function, and wide variety of effects of plant Kunitz protease inhibitors, and their potential for pharmaceutical application based on their interactions with different proteases.

A Bifunctional Molecule with Lectin and Protease Inhibitor Activities Isolated from Crataeva tapia Bark Significantly Affects Cocultures of Mesenchymal Stem Cells and Glioblastoma Cells.

Currently available drugs for treatment of glioblastoma, the most aggressive brain tumor, remain inefficient, thus a plethora of natural compounds have already been shown to have antimalignant effects. However, these have not been tested for their impact on tumor cells in their microenvironment-simulated cell models, e.g., mesenchymal stem cells in coculture with glioblastoma cell U87 (GB). Mesenchymal stem cells (MSC) chemotactically infiltrate the glioblastoma microenvironment. Our previous studies have shown that bone-marrow derived MSCs impair U87 growth and invasion via paracrine and cell-cell contact-mediated cross-talk. Here, we report on a plant-derived protein, obtained from Crataeva tapia tree Bark Lectin (CrataBL), having protease inhibitory/lectin activities, and demonstrate its effects on glioblastoma cells U87 alone and their cocultures with MSCs. CrataBL inhibited U87 cell invasion and adhesion. Using a simplified model of the stromal microenvironment, i.e., GB/MSC direct cocultures, we demonstrated that CrataBL, when added in increased concentrations, caused cell cycle arrest and decreased cocultured cells' viability and proliferation, but not invasion. The cocultured cells' phenotypes were affected by CrataBL via a variety of secreted immunomodulatory cytokines, i.e., G-CSF, GM-CSF, IL-6, IL-8, and VEGF. We hypothesize that CrataBL plays a role by boosting the modulatory effects of MSCs on these glioblastoma cell lines and thus the effects of this and other natural lectins and/or inhibitors would certainly be different in the tumor microenvironment compared to tumor cells alone. We have provided clear evidence that it makes much more sense testing these potential therapeutic adjuvants in cocultures, mimicking heterogeneous tumor-stroma interactions with cancer cells in vivo. As such, CrataBL is suggested as a new candidate to approach adjuvant treatment of this deadly tumor.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

A plant proteinase inhibitor from Enterolobium contortisiliquum attenuates airway hyperresponsiveness, inflammation and remodeling in a mouse model of asthma.

INTRODUCTION: Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for asthma. PURPOSE: The aim of the present study was to evaluate the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on pulmonary mechanical function, eosinophilic recruitment, inflammatory cytokines, remodeling and oxidative stress in an experimental model of chronic allergic pulmonary inflammation. METHODS: BALB/c mice were divided into 4 groups: C (saline i.p and inhalations with saline), OVA (ovalbumin i.p and inhalations with ovalbumin); C+EC (saline i.p, inhalations with s aline and treatment with EcTI); OVA+EC (ovalbumin i.p, inhalations with ovalbumin and treatment with EcTI). On day 29, we performed the following tests: resistance (Rrs) and elastance (Ers) of the respiratory system; (b) quantify eosinophils, 8-ISO-PGF2α, collagen and elastic fiber volume fractions; (c) IFN-γ, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-ß, iNOS and p65-NFκB-positive cells in the airway and alveolar walls. RESULTS: In OVA+EC group, there was an attenuation of the Rrs and Ers, reduction of eosinophils, IL-4, IL-5, IL-13, IFN-γ, iNOS and p65-NFκB-positive cells compared to OVA group. The 8-ISO-PGF2α, elastic and collagen fibers volume fractions as well as the positive cells for MMP-9, TIMP-1 and TGF-ß positive cells were decreased in OVA+EC compared to the OVA group. CONCLUSION: EcTI attenuates bronchial hyperresponsiveness, inflammation, remodeling and oxidative stress activation in this experimental mouse model of asthma.

Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer?

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the ß1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line.

Theoretical insight into the mechanism for the inhibition of the cysteine protease cathepsin B by 1,2,4-thiadiazole derivatives.

Several cellular disorders have been related to the overexpression of the cysteine protease cathepsin B (CatB), such as rheumatic arthritis, muscular dystrophy, osteoporosis, Alzheimer's disease, and tumor metastasis. Therefore, inhibiting CatB may be a way to control unregulated cellular functions and prevent tissue malformations. The inhibitory action of 1,2,4-thiadiazole (TDZ) derivatives has been associated in the literature with their ability to form disulfide bridges with the catalytic cysteine of CatB. In this work, we present molecular modeling and docking studies of a series of eight 1,2,4-thiadiazole compounds. Substitutions at two positions (3 and 5) on the 1,2,4-thiadiazole ring were analyzed, and the docking scores were correlated to experimental data. A correlation was found with the sequence of scores of four related compounds with different substituents at position 5. No correlation was observed for changes at position 3. In addition, quantum chemistry calculations were performed on smaller molecular models to study the mechanism of inhibition of TDZ at the active site of CatB. All possible protonation states of the ligand and the active site residues were assessed. The tautomeric form in which the proton is located on N2 was identified as the species that has the structural and energetic characteristics that would allow the ring opening of 1,2,4-thiadiazole.