Anti-SARS-CoV-2 Viral Activity of Sweet Potato Trypsin Inhibitor via Downregulation of TMPRSS2 Activity and ACE2 Expression In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.

Prebound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations.

The serine protease trypsin forms a tightly bound inhibitor complex with the bovine pancreatic trypsin inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to α-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores the inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown, and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before, and unrestrained umbrella sampling simulations of these complexes suggest additional energetic minima. Here, we use random acceleration molecular dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the prebound state. The prebound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the BPTI variants, and changes in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between the fully bound state and prebound state and also lowers the energy minimum of the prebound state. While the effect of fluorination is in general difficult to quantify, here, it is in part caused by favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the prebound state offers insights into the inhibitory mechanism of BPTI and might add valuable information for the design of serine protease inhibitors.

Antifungal activity of a trypsin inhibitor from Salvia hispanica L. (chia) seeds against fluconazole-resistant strains of Candida spp. and evaluation of its toxicity in vitro.

The incidence of Candida species resistant to traditional antifungal drugs is increasing globally. This issue significantly impacts patients' lives and increases healthcare expenses, confirming the need to develop novel therapeutic strategies. Recently, a thermostable trypsin inhibitor named ShTI (11.558 kDa), which has antibacterial effects on Staphylococcus aureus, was isolated from Salvia hispanica L. (chia) seeds. This study aimed to assess the antifungal effect of ShTI against Candida species and its synergism with fluconazole and to evaluate its mode of action. Preliminary toxicological studies on mouse fibroblasts were also performed. ShTI exhibited antifungal effects against C. parapsilosis (ATCC® 22,019), C. krusei (ATCC® 6258), and six clinical fluconazole-resistant strains of C. albicans (2), C. parapsilosis (2), and C. tropicalis (2). The minimum inhibitory concentration (MIC) values were 4.1 µM (inhibiting 50% of the isolates) and 8.2 µM (inhibiting 100% of the isolates). Additionally, when combined with fluconazole, ShTI had a synergistic effect on C. albicans, altering the morphological structure of the yeast. The mode of action of ShTI against C. krusei (ATCC® 6258) and C. albicans involves cell membrane permeabilization, the overproduction of reactive oxygen species, the formation of pseudohyphae, pore formation, and consequently, cell death. In addition, ShTI (8.65 and 17.3 µM) had noncytotoxic and nongenotoxic effects on L929 mouse fibroblasts. These findings suggest that ShTI could be a promising antimicrobial candidate, but further research is necessary to advance its application as a novel antifungal agent.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

MDR49 coding for both P-glycoprotein and TMOF transporter functions in ivermectin resistance, trypsin activity inhibition, and fertility in the yellow fever mosquito, Aedes aegypti.

This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.

The Preventive Effect of Urinary Trypsin Inhibitor on Postoperative Cognitive Dysfunction, on the Aspect of Behavior, Evaluated by Y-Maze Test, via Modulation of Microglial Activity.

This experimental study was designed to evaluate the effect of ulinastatin, a urinary trypsin inhibitor, on postoperative cognitive dysfunction (POCD) in rats under general anesthesia with isoflurane, on the aspect of behavior, as evaluated using a Y-maze test and focusing on microglial activity. Ulinastatin (50,000 U/mL) and normal saline (1 mL) were randomly (1:1) administered intraperitoneally to the ulinastatin and control groups, respectively, before general anesthesia. Anesthesia with isoflurane 1.5 volume% was maintained for 2 h. The Y-maze test was used to evaluate cognitive function. Neuronal damage using caspase-1 expression, the degree of inflammation through cytokine detection, and microglial activation with differentiation of the phenotypic expression were evaluated. Twelve rats were enrolled in the study and evenly allocated into the two groups, with no dropouts from the study. The Y-maze test showed similar results in the two groups before general anesthesia (63 ± 12% in the control group vs. 64 ± 12% in the ulinastatin group, p = 0.81). However, a significant difference was observed between the two groups after general anesthesia (17 ± 24% in the control group vs. 60 ± 12% in the ulinastatin group, p = 0.006). The ulinastatin group showed significantly lower expression of caspase-1. Pro-inflammatory cytokine levels were significantly lower in the ulinastatin group than in the control group. The ulinastatin group had a significantly lower microglial activation (41.74 ± 10.56% in the control group vs. 4.77 ± 0.56% in the ulinastatin, p < 0.001), with a significantly lower activation of M1 phenotypes (52.19 ± 7.83% in the control group vs. 5.58 ± 0.76% in the ulinastatin group, p < 0.001). Administering ulinastatin before general anesthesia prevented neuronal damage and cognitive decline after general anesthesia, in terms of the aspect of behavior, as evaluated by the Y-maze test. The protective effect of ulinastatin was associated with the inhibition of microglial activation, especially the M1 phenotype.

Correlation of Experimental and Calculated Inhibition Constants of Protease Inhibitor Complexes.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

DbGTi: Thermostable trypsin inhibitor from Dioscorea bulbifera L. ground tubers: assessment of antioxidant and antibacterial properties and cytotoxicity evaluation using zebrafish model.

Oxidative stress disorders and diseases caused by drug-resistant bacteria have emerged as significant public health concerns. Plant-based medications like protease inhibitors are growing despite adverse effects therapies. Consecutively, in this study, trypsin inhibitors from Dioscorea bulbifera L. (DbGTi trypsin inhibitor) ground tubers were isolated, purified, characterized, and evaluated for their potential cytotoxicity, antibacterial, and antioxidant activities. DbGTi protein was purified by Q-Sepharose matrix, followed by trypsin inhibitory activity. The molecular weight of the DbGTi protein was found to be approximately 31 kDa by SDS-PAGE electrophoresis. The secondary structure analysis by circular dichroism (CD) spectroscopy revealed that the DbGTi protein predominantly comprises ß sheets followed by α helix. DbGTi protein showed competitive type of inhibition with Vmax = 2.1372 × 10-1 µM/min, Km = 1.1805 × 102 µM, & Ki = 8.4 × 10-9 M and was stable up to 70 °C. DbGTi protein exhibited 58 % similarity with Dioscorin protein isolated from Dioscorea alata L. as revealed by LC-MS/MS analysis. DbGTi protein showed a non-toxic effect, analyzed by MTT, Haemolytic assay and in vivo studies on zebrafish model. DbGTi protein significantly inhibited K. pneumoniae and has excellent antioxidant properties, confirmed by various antioxidant assays. The results of anti-microbial, cytotoxicity and antioxidant assays demonstrate its bioactive potential and non-toxic nature.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

A soybean trypsin inhibitor reduces the resistance to transgenic maize in a population of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells.

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Exploring Photoswitchable Binding Interactions with Small-Molecule- and Peptide-Based Inhibitors of Trypsin.

The ability to photochemically activate a drug, both when and where needed, requires optimisation of the difference in biological activity between each isomeric state. As a step to this goal, we report small-molecule- and peptide-based inhibitors of the same protease-trypsin-to better understand how photoswitchable drugs interact with their biological target. The best peptidic inhibitor displayed a more than fivefold difference in inhibitory activity between isomeric states, whereas the best small-molecule inhibitor only showed a 3.4-fold difference. Docking and molecular modelling suggest this result is due to a large change in 3D structure in the key binding residues of the peptidic inhibitor upon isomerisation; this is not observed for the small-molecule inhibitor. Hence, we demonstrate that significant structural changes in critical binding motifs upon irradiation are essential for maximising the difference in biological activity between isomeric states. This is an important consideration in the design of future photoswitchable drugs for clinical applications.

Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis.

OBJECTIVE: Wheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in experimental autoimmune encephalitis (EAE), a model of multiple sclerosis (MS). DESIGN: EAE was induced in C57BL/6J mice on standardised dietary regimes with defined content of gluten/ATI. Mice received a gluten and ATI-free diet with defined carbohydrate and protein (casein/zein) content, supplemented with: (a) 25% of gluten and 0.75% ATI; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified ATI. The effect of dietary ATI on clinical EAE severity, on intestinal, mesenteric lymph node, splenic and central nervous system (CNS) subsets of myeloid cells and lymphocytes was analysed. Activation of peripheral blood mononuclear cells from patients with MS and healthy controls was compared. RESULTS: Dietary ATI dose-dependently caused significantly higher EAE clinical scores compared with mice on other dietary regimes, including on gluten alone. This was mediated by increased numbers and activation of pro-inflammatory intestinal, lymph node, splenic and CNS myeloid cells and of CNS-infiltrating encephalitogenic T-lymphocytes. Expectedly, ATI activated peripheral blood monocytes from both patients with MS and healthy controls. CONCLUSIONS: Dietary wheat ATI activate murine and human myeloid cells. The amount of ATI present in an average human wheat-based diet caused mild intestinal inflammation, which was propagated to extraintestinal sites, leading to exacerbation of CNS inflammation and worsening of clinical symptoms in EAE. These results support the importance of the gut-brain axis in inflammatory CNS disease.

Development of a rapid process for purification of Bowman-Birk and Kunitz inhibitors from legume seeds, and evaluation of their biophysical, insecticidal, and antimicrobial properties.

Bowman-Birk inhibitor (BBI ~10 kDa) and Kunitz inhibitor (KI ~20 kDa) are serine protease/proteinase inhibitor(s) [PI(s)] ubiquitously found in several Leguminous plant species with insecticidal and therapeutic properties. Due to narrow molecular mass differences, the separation of these inhibitors from a single seed variety is tedious. The present study is aimed to develop a rapid protocol (<24 h) for purifying BBI and KI from legume seeds using mild trichloroacetic acid (TCA) extraction followed by trypsin-affinity chromatography. The mature seeds of Vigna radiata and Cajanus platycarpus are used as a model to purify BBI and KI using this protocol. The BBI and KI purified from the seeds of V. radiata are labeled as VrBBI & VrKI, and C. platycarpus are labeled as CpBBI & CpKI, respectively. These PIs are confirmed by immunodetection and MALDI-TOF studies and further characterized for their structural (CD & fluorescence spectroscopy) and functional properties (temperature & DTT stability). BBI(s) purified using the above process are effective in the management of castor semi-looper 'Achaea janata', while KI(s) are effective in the management of pod borer 'Helicoverpa armigera'. Besides, both BBI(s) and KI(s) have significant potential in controlling the growth of methicillin-sensitive 'Staphylococcus aureus', a gram-positive pathogenic bacterium.

Ultrasound-enhanced interfacial adsorption and inactivation of soy trypsin inhibitors.

In this study, liquid-liquid interfacial protein adsorption was proposed as a means of inactivating soy trypsin inhibitors (TIs, including Kunitz (KTI) and Bowman-Birk inhibitor (BBI)). Hexane-water was first selected as a model system to compare three emulsification methods (hand shaking, rotor-stator and ultrasound mixing). Ultrasound could generate the smallest and least polydisperse emulsion droplets, resulting in highest interfacial adsorption amount of KTI and BBI as well as the highest inactivation percentage of TIs (p < 0.05). Therefore, ultrasound was selected to further explore the effect of the non-aqueous phase on interfacial adsorption and inactivation kinetics of TIs in a food emulsion system containing vegetable oil (VTO). The adsorption amounts of KTI and BBI in the VTO-aqueous emulsion increased by âˆ¼ 25 % compared to the hexane-aqueous emulsion. In addition, the adsorption amounts of KTI and BBI were rapidly increased as a function of sonication time, especially for the hexane-aqueous emulsion system. This result suggests that such inactivation of TIs could be implemented in continuous systems for large-scale processing. Finally, the pathways of interface-induced inactivation of BBI and KTI were investigated based on separate experiments on individual BBI and KTI systems. The results showed that the interface adsorption caused the changes in the secondary and tertiary structure of KTI that led to its activitation. However, BBI was quite stable at the liquid-liquid interface without significant conformational change. Overall, ultrasound-assisted interfacial adsorption can be considered a rapid and highly efficient method to inactivate KTI.

An innovative insecticidal approach based on plant protease inhibitor and Bt protoxins inhibits trypsin-like activity in zebrafish.

The Leucaena leucocephala trypsin inhibitor (LTI) + Bacillus thuringiensis (Bt) protoxins mix has been proposed as a novel larvicide agent in order to control the vector mosquito of dengue virus, Aedes aegypti, in their aquatic breeding sites. However, use of this insecticide formulation has raised concerns about its impacts on aquatic biota. In this context, this work aimed to assess the effects of LTI and Bt protoxins, separately or in combination, in zebrafish, in regard to the evaluation of toxicity at early life stages and to the presence of LTI inhibitory effects on intestinal proteases of this fish. Results showed that LTI and Bt concentrations (250 mg/L, and 0.13 mg/L, respectively), and LTI + Bt mix (250 mg/L + 0.13 mg/L) - 10 times superior to those with insecticidal action - did not cause death nor did it induce morphological changes during embryonic and larval development (3 to 144 h post-fertilization) of zebrafish. Molecular docking analyses highlighted a possible interaction between LTI and zebrafish trypsin, especially through hydrophobic interactions. In concentrations near to those with larvicidal action, LTI (0.1 mg/mL) was able to inhibit in vitro intestinal extracts of trypsin in female and male fish by 83 % and 85 %, respectively, while LTI + Bt mix promoted trypsin inhibition of 69 % in female and 65 % in male ones. These data show that the larvicidal mix can potentially promote deleterious effects to nutrition and survival in non-target aquatic organisms, especially those with trypsin-like dependent protein digestion.

Spiders' digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family.

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Prospecting in silico antibacterial activity of a peptide from trypsin inhibitor isolated from tamarind seed.

Bacterial infections have become a global concern, stimulating the growing demand for natural and biologically safe therapeutic agents with antibacterial action. This study was evaluated the genotoxicity of the trypsin inhibitor isolated from tamarind seeds (TTI) and the antibacterial effect of TTI theoric model, number 56, and conformation number 287 (TTIp 56/287) and derived peptides in silico. TTI (0.3 and 0.6 mg.mL-1) did not cause genotoxicity in cells (p > 0.05). In silico, a greater interaction of TTIp 56/287 with the Gram-positive membrane (GP) was observed, with an interaction potential energy (IPE) of -1094.97 kcal.mol-1. In the TTIp 56/287-GP interaction, the Arginine, Threonine (Thr), and Lysine residues presented lower IPE. In molecular dynamics (MD), Peptidotrychyme59 (TVSQTPIDIPIGLPVR) showed an IPE of -518.08 kcal.mol-1 with the membrane of GP bacteria, and the Thr and Arginine residues showed the greater IPE. The results highlight new perspectives on TTI and its derived peptides antibacterial activity.