Chymotrypsin Inhibitor Research Articles

Soybean Trypsin Inhibitors: Selective Inactivation at Hydrolysis of Soybean Proteins by Some Enzymatic Complexes

Inactivation of the Kunitz and Bowman-Birk soybean trypsin inhibitors at hydrolysis of soybean proteins by enzymatic complexes was studied. These complexes are derived from king crab hepatopancreas, cod fish pyloric caeca, and a commercial enzymatic complex of protosubtilin. Analysis of the soybean protein hydrolysates showed that these enzymatic complexes completely digested the Kunitz trypsin inhibitor (60–70% of the total trypsin inhibitors) and had almost no effect on the Bowman-Birk trypsin and chymotrypsin inhibitor. All of the enzymatic complexes contain different sets of enzymes with different proteolytic specificity. This allow to make the conclusion that Bowman-Birk inhibitor is highly resistant to proteolysis and is not inactivated at enzymatic hydrolysis.

Nutritional quality of fresh and stored legumes sprouts – Effect of Lactobacillus plantarum 299v enrichment

Nutrient content and digestibility as well as factors with a potentially negative effect on these parameters were studied in legume sprouts enriched with L. plantarum 299v. The nutrient digestibility and contents were not strongly affected by the co-culture of the probiotic and sprouts. The highest digestibility of starch was observed for adzuki bean preparations (from 91.6% to 95.5%), while the lowest value was noted for soybean preparations (from 49.6% to 60.8%). A slight decrease in starch digestibility was observed in adzuki and soybean sprouts enriched with the probiotic (by about 5% and 7% respectively). An increase in starch digestibility was noted in lentil and mung bean sprouts. A key influence on protein digestibility was exerted by the activity of trypsin and chymotrypsin inhibitors. Generally, there was no negative effect of the studied factors on starch digestibility. Most importantly, the control and probiotic-rich sprouts retained high quality after cold storage.

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Influence of Different Cowpea (<i>Vigna unguiculata</i> (L.) Walp.) Genotypes from Burkina Faso on Proteases Inhibition

Cowpea is an important protein crop widespread in Africa. The purpose of this research was to determine the content of trypsin and chymotrypsin inhibitors in different genotypes of cowpea seeds. Trypsin percentage inhibition showed about 13.5-fold variation (5.12% ± 1.47 to 70.52% ± 6.58) between the cowpea genotypes respectively for KVx 421-2J and Kondesyoungo local. The chymotrypsin inhibitory activity varies among cowpea genotypes from 21.19 to 76.94%. The highest percent inhibitory activity was exhibited by KVx 396-4-5-2D on chymotrypsin. This study also showed significant correlations between type of cowpea genotype and trypsin inhibition potential and also between colour and chymotrypsin inhibition potential of cowpea seeds. Kondesyoungo local, a landraceae genotype exhibited the high potential to inhibit the trypsin enzyme. Coloured seeds of cowpea genotypes possess higher percentage inhibition of chymotrypsin than the colourless ones (p < 0.05). The calculated mean of trypsin and chymotrypsin inhibition activities showed that Labagela local genotype possess the highest inhibition activity of both protease enzymes. The PCA components analysis and the dendrogram performed basis on the protease inhibitory activities divided the thirty-one genotypes of cowpea used in this study into three classes. The results presented in this work can contribute greatly to the planning of a cowpea breeding program aimed at reducing the content of proteases inhibitors in order to improve the nutritional value of seeds or to increase PI content for tolerance to stored grain pests.

Molecular insight into chymotrypsin inhibitor 2 resisting proteolytic degradation

Chymotrypsin inhibitor 2 (CI2) is a special serine protease inhibitor which can resist hydrolysis for several days with a rapid equilibrium between the Michaelis complex and acyl-enzyme intermediate. The energies and conformational changes for subtilisin-catalyzed proteolysis of CI2 were examined in this paper for the first time by employing pseudo bond ab initio QM/MM MD simulations. In the acylation reaction, a low-barrier hydrogen bond between His64 and Asp32 in the transition state together with the lack of covalent backbone constraints makes the peptide bonds of CI2 break more easily than in other serine protease inhibitors. After acyl-enzyme formation, molecular dynamics simulations showed that the access of hydrolytic water to the active site requires partial dissociation of the leaving group. However, retention of the leaving group mainly by the intra- and inter-molecular H-bonding networks hinders the access of water and retards the deacylation reaction. Instead of the dissociation constant of inhibitors, we suggest employing the free energy at the acyl-enzyme state to predict the relative hydrolysis rates of CI2 mutants, which are testified by the experimental relative hydrolysis rates.

Activating KRAS, NRAS, and BRAF Mutants Enhance Proteasome Capacity and Reduce Endoplasmic Reticulum Stress in Multiple Myeloma, Thereby Promoting Plasma Cell Survival and Proteasome Inhibitor Resistance

Introduction:Multiple myeloma, a malignant proliferation of differentiated plasma cells, is the second most commonly diagnosed hematologic malignancy, and the number of cases may grow by almost 60% between 2010 and 2030. Recent therapeutic advances, including the use of proteasome inhibitors (PIs), have contributed to a doubling of the median overall survival in myeloma patients. This has been paralleled by an increased understanding of the mutational spectrum in this disease, which was first noted almost three decades ago to harbor KRAS and NRAS mutations. KRAS, NRAS, and BRAF mutations which induce p44/42 Mitogen-activated protein kinase (MAPK) signaling are found in about half of myeloma patients, and seem to contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms still remains elusive.Methods:ANBL-6 and U266 human-derived myeloma cell lines have endogenous wild-type (WT) KRAS, NRAS, and BRAF, and were used in this study. All cell lines were validated through The MD Anderson Cancer Center Characterized Cell Line Core Facility. We established lines stably expressing WT, constitutively active (CA)(G12V/G13D/Q61H), or dominant negative (DN)(S17N) KRAS and NRAS mutants, or V600E or DN BRAF. Cell viability was evaluated using the WST-1 tetrazolium reagent, while the chymotrypsin-, trypsin- and caspase-like activities were determined using fluorogenic substrates.Results:CA KRAS, NRAS, and BRAF mutants reduced the sensitivity of ANBL-6 and U266 cells to bortezomib and carfilzomib, while their DN variants sensitized cells to both PIs. This was associated with an induction by these CA mutants of the proteasome chymotrypsin-, trypsin- and caspase-like activities, while the DN variants reduced proteasome activity. These activity changes occurred in parallel with increased expression at both the mRNA and protein levels of catalytically active Proteasome subunit beta (PSMB)-8, PSMB9, and PSMB10, and of the proteasome assembly chaperone Proteasome maturation protein (POMP). Mechanistic studies showed that MAPK induction by the CA mutants caused activation of the ETS transcription factor (ELK1), which was found to have consensus binding sites in the promoters of PSMB8, PSMB9, PSMB10, and POMP. Notably, ELK1 suppression reduced PSMB8, PSMB9, PSMB10, and POMP expression, directly linking RAS/RAF/MAPK signaling to proteasome biology, and this suppression enhanced PI sensitivity. Inhibition of MAPK signaling with either the MAPK kinase (MEK) inhibitor selumetinib or the pan-RAF inhibitor TAK-632 showed synergistic activity with either bortezomib or carfilzomib that was more consistent in cell lines harboring CA mutants as opposed to the DN or WT constructs. Combination regimens of selumetinib or TAK-632 with either bortezomib or carfilzomib induced greater inhibition of the proteasome chymotrypsin-, trypsin- and caspase-like activities than the PIs as single agents. Finally, CA KRAS, NRAS, and BRAF mutants reduced expression levels of genes and proteins involved in the unfolded protein response (UPR), including Activating transcription factor (ATF)-4, -5, and -6, as well as C/EBP homologous protein transcription factor (CHOP) and the spliced variant of X-box binding protein 1 (XBP1s). In contrast, their dominant negative counterparts enhanced expression of the UPR effectors, consistent with an increase in endoplasmic reticulum (ER) stress.Conclusion:Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue that these mutations promote plasma cell survival by reducing cellular stress, thereby distancing myeloma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma. DisclosuresLee:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dick:Takeda Oncology: Employment, Equity Ownership. Chattopadhyay:Takeda Oncology: Employment. Orlowski:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The impact of four processing methods on trypsin-, chymotrypsin- and alpha-amylase inhibitors present in underutilised legumes

The global trend in increasing plant-based protein diets due to health and ideological reasons, has created an increased demand for food legumes that exceeds current production. To meet this demand, it is timely to reduce relying solely on soybean, and explore the potential of the underutilised legumes that are cultivated regionally. Underutilised legumes are rich in protein, carbohydrates and other nutrients that are essential for consumer. However, relatively little is known about their anti-nutritional properties and processing methods. Anti-nutritional factors (ANFs) such as enzyme inhibitors are prevalent in legumes and may interfere with digestibility and nutrient absorption. Nevertheless, an optimised food processing method will overcome this challenge and warrant a safe inclusion of legume in plant-based protein diets. Hence current study aimed to optimise the food processing methods (soaking, wet heating, autoclaving and freezing) and evaluate their efficiency in eliminating the enzyme inhibitors [trypsin, chymotrypsin (CIA) and α-amylase (AIA) inhibitors] present in seven underutilised legumes. Current study showed that autoclaving at 121°C for 15min reduced the AIA in all underutilised legumes tested. The AIA and CIA of bambara groundnut were successfully inactivated by wet heating at 50°C for 60min, and by autoclaving at 121°C for 15min. While the CIA of chickpea was successfully inactivated by freezing at - 80°C for 24h.

The molecular mechanism of protein denaturation in supercritical CO2: The role of exposed lysine residues is explored

Although there are many interests to use proteins in supercritical CO2, protein destabilization in this solvent is the major drawback. This is because of the non-natural CO2 environment which imposes essential instability on the protein structure. Due to the limitations of the experimental techniques to obtain in situ molecular levels information about protein microstructure at high pressures of the supercritical state, the molecular level reasons of the protein destabilization remains controversial and relatively unknown in supercritical CO2. While there is a consensus about the probable roles of surface lysine residues in destabilization of proteins in supercritical CO2, no clear mechanism is presented about the way that these amino acids impose instabilities on the protein conformation. Here, using molecular dynamics simulations, we extensively examined the structural behavior of the surface lysine residues of chymotrypsin inhibitor 2 as a model protein in supercritical CO2. In this way, a new molecular mechanism is proposed which states that the inappropriate interactions of polar side chain of lysine residues with the non-polar nature of supercritical CO2 enforce these exposed residues to return from the solvent bulk to the protein exterior and also to form new non-native hydrogen bonds with the acidic residues such as glutamic acid. This induces some surface regions to be very labile and dynamics. These regions are the starting sequence in the process of protein denaturation in supercritical CO2. Denaturation is extended by collapsing the structure due to the formation of multiple non-native interactions between different residues. To evaluate the proposed mechanism, surface exposed lysine residues were replaced to leucine amino acids with the hydrophobic side chains. Interestingly, the protein remains intact by this replacement even at 200 bar in supercritical CO2. However, replacing of lysine residues by negatively charged glutamic acid and also by glycine residues cannot prevent protein denaturation process. These molecular levels explorations not only address some unknown aspects of protein denaturation mechanism in supercritical CO2, but it also can essentially help to design new CO2-tolerant proteins which can be used in popular processes such as biocatalysis in supercritical CO2.

Inducible defense responses in Populus alba berolinensis to Pb stress

To better understand the effects of heavy metal stress on inducible defenses of woody plants, contents of nutritional substances and secondary metabolites, plus the activities of defense enzymes and protease inhibitors in the leaves of Populus alba berolinensis seedlings that were planted in either non-contaminated soil (control) or Pb-contaminated soil (at 300, 500 and 700mg/kg) were studied. Our results showed that protein and soluble sugar contents in leaves 30–50 days after the Pb stress were significantly lower than those in the control group in a dose-dependent manner, whereas the lignin and flavonoid contents were significantly higher. Polyphenol oxidase (PPO) and phenylalnine ammonialyase (PAL) activities in leaves were significantly enhanced by the low Pb concentration stress (300 mg/kg) for all three sampling dates (30, 40 and 50 days after the Pb treatments), while at the two higher Pb concentrations (500 and 700 mg/kg), PPO activities were always inhibited and PAL activities presented a tendency of “promoting first and then restraining” with the increase of exposure duration. The response patterns of trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities to Pb stress were similar to that of PPO, and presented a “low-promotion, high-inhibition” effect of Pb concentrations in leaves. These results suggest that the inducible defense responses in P. alba berolinensis leaves to Pb stress depend on both the Pb concentrations and the exposure duration, and the low Pb concentration at 300 mg/kg seemed to induce the strongest defense responses in the poplar seedlings, much stronger than those at the higher Pb concentrations.

Dinghupeptins A-D, Chymotrypsin Inhibitory Cyclodepsipeptides Produced by a Soil-Derived Streptomyces.

Four new cyclodepsipeptides, dinghupeptins A-D (1-4), possessing a rare N5-(2-hydroxylethyl)glutamine moiety, were isolated from cultures of the soil-derived Streptomyces sp. SC0581. Their structures were elucidated by spectroscopic and advanced Marfey's amino acid analysis, and their 3D structures were established by theoretical conformational analysis. Compounds 1 and 2, containing a 3-amino-6-hydroxypiperidone unit, displayed selective inhibition of chymotrypsin with IC50 values of 2.1 and 1.1 μM, respectively. Enzyme kinetic analysis and molecular docking experiments revealed they are competitive inhibitors binding to the active site of chymotrypsin.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

Loci and candidate genes controlling root traits in wheat seedlings-a wheat root GWAS.

Two hundred one hexaploid wheat accessions, representing 200years of selection and breeding history, were sampled from the National Small Grains Collection in Aberdeen, ID, and evaluated for five root traits at the seedling stage. A paper roll-supported hydroponic system was used for seedling growth. Replicated roots samples were analyzed by WinRHIZO. We observed accessions with nearly no branching and accessions with up to 132cm of branching. Total seminal root length ranged from 70 to 248cm, a 3.5-fold difference. Next-generation sequencing was used to produce single-nucleotide polymorphism (SNP) markers and genomic libraries that were aligned to the wheat reference genome IWGSCv1 and were called single-nucleotide polymorphism (SNP) markers. After filtering and imputation, a total of 20,881 polymorphic sites were used to perform association mapping in TASSEL. Gene annotations were conducted for identified marker-trait associations (MTAs) with - log10P > 3.5 (p value < 0.003). In total, we identified 63 MTAs with seven for seminal axis root length (SAR), 24 for branching (BR), four for total seminal root length (TSR), eight for root dry matter (RDM), and 20 for root diameter (RD). Putative proteins of interest that we identified include chalcone synthase, aquaporin, and chymotrypsin inhibitor for SAR, MYB transcription factor and peroxidase for BR, zinc fingers and amino acid transporters for RDM, and cinnamoyl-CoA reductase for RD. We evaluated the effects of height-reducing Rht alleles and the 1B/1R translocation event on root traits and found presence of the Rht-B1b allele decreased RDM, while presence of the Rht-D1b allele increased TSR and decreased RD.

Interface Interactions of the Bowman–Birk Inhibitor BTCI in a Ternary Complex with Trypsin and Chymotrypsin Evaluated by Semiempirical Quantum Mechanical Calculations

Black‐eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a small protein from Bowman–Birk protease inhibitor family, which simultaneously inhibits trypsin and chymotrypsin. Through the inhibition of trypsin‐ and chymotrypsin‐like sites on the 20S subunit of human proteasome, BTCI acts as a potent anticarcinogenic agent inducing apoptosis in breast cancer cells. Because of the lack of crystallographic information of the BTCI‐proteasome complex, we analyze here the BTCI‐chymotrypsin and BTCI‐trypsin interfaces using computations. We adopt the corrected semiempirical quantum‐mechanical methods in combination with implicit description of the water environment. Firstly, we carefully check the representativeness of smaller systems by fragmentation and analyzing the convergence of the overall interaction energies. Then, we use the “virtual glycine scan” technique to understand the binary complex formation, identifying the most contributing amino acid side chains in the interfaces. Besides detailed quantification of all important residue contributions, the importance of Lys26 and Phe53 in the BTCI and Asp186 (trypsin) and Ser195 (chymotrypsin) residues is confirmed. In summary, we have worked out an accurate and efficient in silico protocol for protein–protein interfaces, which can be later used for studying the inhibition of 20S proteasome.

Effects of ethanol on the secondary structure specific hydration properties of Chymotrypsin Inhibitor 2 in its folded and unfolded forms

ABSTRACTMolecular dynamics simulations were performed for the protein Chymotrypsin Inhibitor 2 in water–ethanol binary mixtures at several ethanol concentrations at two different temperatures to explore the differential role of ethanol on the hydration properties of the secondary structural segments of the protein. We find that, compared to the water structure around the secondary structure segments of the unfolded protein in pure water, water molecules preferred to be structured more in presence of ethanol. The structuration of ethanol is noticed to be higher around helix and sheet, as compared to that around the loop. The differential slow structural relaxations of the hydrogen bonds involving the secondary structural segments of protein in water–ethanol mixed solutions, as compared to that in pure water infer that ethanol alters the water properties in a remarkable manner. The slow relaxations of the hydrogen bonds formed between ethanol and the secondary structural segments of the protein, in general, suggests that ethanol interacts directly with the protein. However, the relatively faster relaxation of the ethanol-segment hydrogen bonds involving unfolded protein as compared to that involving the folded protein suggests that the unfolded protein surface can remain dynamically free to interact with water more.

Discovery of Specific Nonpeptide Probe for Chymotrypsin via Molecular Docking-Based Virtual Screening and the Application.

Chymotrypsin is a proteolytic enzyme associated with numerous biological processes. Moreover, it has been reported to be significantly involved in pancreatic diseases. Thus, its rapid and sensitive detection is of great significance for early diagnosis and related drug discovery. Herein, a new strategy of molecular docking-based virtual screening (MDVS) was developed for the identification of a new nonpeptide-based biosensor targeting chymotrypsin. The newly discovered probe, numbered probe 20, exhibits about 45-fold specificity toward chymotrypsin over the similar enzyme trypsin and produces about 250-fold higher increase of fluorescence intensity under the catalysis of chymotrypsin. Furthermore, the probe successfully allowed the characterization of the kinetics of chymotrypsin inhibitors. More importantly, the endogenous chymotrypsin in zebrafish was visualized by nonpeptide probe for the first time, demonstrating the potential of the probe 20 for future application in the mechanistic study or clinic diagnosis for pancreatic diseases. Accordingly, the MDVS strategy could be generally applied in the identification of specific fluorescent probe for a particular enzyme.

Cyanopeptolins with Trypsin and Chymotrypsin Inhibitory Activity from the Cyanobacterium Nostoc edaphicum CCNP1411.

Cyanopeptolins (CPs) are one of the most frequently occurring cyanobacterial peptides, many of which are inhibitors of serine proteases. Some CP variants are also acutely toxic to aquatic organisms, especially small crustaceans. In this study, thirteen CPs, including twelve new variants, were detected in the cyanobacterium Nostoc edaphicum CCNP1411 isolated from the Gulf of Gdańsk (southern Baltic Sea). Structural elucidation was performed by tandem mass spectrometry with verification by NMR for CP962 and CP985. Trypsin and chymotrypsin inhibition assays confirmed the significance of the residue adjacent to 3-amino-6-hydroxy-2-piperidone (Ahp) for the activity of the peptides. Arginine-containing CPs (CPs-Arg2) inhibited trypsin at low IC50 values (0.24–0.26 µM) and showed mild activity against chymotrypsin (IC50 3.1–3.8 µM), while tyrosine-containing CPs (CPs-Tyr2) were selectively and potently active against chymotrypsin (IC50 0.26 µM). No degradation of the peptides was observed during the enzyme assays. Neither of the CPs were active against thrombin, elastase or protein phosphatase 1. Two CPs (CP962 and CP985) had no cytotoxic effects on MCF-7 breast cancer cells. Strong and selective activity of the new cyanopeptolin variants makes them potential candidates for the development of drugs against metabolic disorders and other diseases.

A sensitive ratiometric fluorescence probe for chymotrypsin activity and inhibitor screening

Chymotrypsin (CHT) is involved in a series of physiological processes and disorders and it is known as a biomarker of pancreatic function. Thus, a sensitive and selective method for evaluating the relative CHT levels in different live cells is urgently needed to better understand the function of CHT. Herein, a ratiometric fluorescence probe (NI) with strong intramolecular charge transfer (ICT) has been rationally designed. The probe is constructed by incorporating a recognition group of 4-bromobutyryl into the fluorescent skeleton of 1,8-naphthalimide. The presence of CHT cleaves the 4-bromobutyryl group from naphthalimide and remarkably alters the probe's photophysical properties with a large Stokes shift, thus achieving ratiometric detection of CHT. With a detection limit of 8.4 ng/mL, NI shows high binding affinity and comparable catalytical efficiency against CHT and was successfully applied for the inhibitor characterization. The results also demonstrate that the designing strategy in the text provides a more promising application prospect for screening others CHT inhibitors.

Optimizing liposomes for delivery of Bowman-Birk protease inhibitors - Platforms for multiple biomedical applications.

One of the major challenges in the administration of therapeutic proteins involves delivery limitations. Liposomes are well-known drug delivery systems (DDS) that have been used to overcome this drawback; nevertheless, low protein entrapment efficiency (EE) still limits their wide biomedical application on a commercial scale. In the present work, different methods for protein entrapment into liposomes were tested in order to obtain tailored DDS platforms for multiple biomedical applications. The protein used as model was the Black-eyed pea Trypsin and Chymotrypsin Inhibitor (BTCI), a member of the Bowman-Birk protease inhibitor family (BBIs), which has been largely explored for its potential application in many biomedical therapies. We optimized reverse-phase evaporation (REV) and freeze/thaw (F/T) entrapment methods, using a cationic lipid matrix to entrap expressive amounts of BTCI (∼100 μM) in stable liposomes without affecting its protease inhibition activity. The influence of various parameters (e.g. entrapment method, liposome composition, buffer type) on particle size, charge, polydispersity, and EE of liposomes was investigated to provide an insight on how to control such parameters in view of obtaining a high entrapment yield. In addition, BTCI liposome platforms obtained herein showed to be versatile vesicles, allowing surface modification with moieties/polymers of interest (e.g. PEG, transferrin). The aforementioned results are relevant to focusing on the entrapment of other promising BBIs or protein agents sharing similar structural features. These findings encourage future studies to investigate the advantages of using the liposome platforms presented herein to broaden the use of this type of DDS for BBI biomedical applications.

Biochemical properties of a bacterially-expressed Bowman-Birk inhibitor from Rhynchosia sublobata (Schumach.) Meikle seeds and its activity against gut proteases of Achaea janata

Crude proteinase inhibitors (CPIs) extracted from the seeds of Rhynchosia sublobata, a wild relative of pigeon pea showed pronounced inhibitory activity on the larval gut trypsin-like proteases of lepidopteran insect pest – Achaea janata. Consequently, a full-length cDNA of Bowman-Birk inhibitor gene (RsBBI1) was cloned from the immature seeds of R. sublobata. It contained an ORF of 360 bp encoding a 119-amino acid polypeptide (13.3 kDa) chain with an N-terminus signal sequence comprising of 22 amino acids. The amino acid sequence and phylogenetic analysis together revealed that RsBBI1 exhibited a close relation with BBIs from soybean and Phaseolus spp. A cDNA sequence corresponding to RsBBI1 mature protein (89 amino acid stretch) was expressed in E. coli. The recombinant rRsBBI1 protein with a molecular mass of 9.97 kDa was purified using trypsin affinity chromatography. The purified rRsBBI1 exhibited non-competitive mode of inhibition of both bovine trypsin (Ki of 358 ± 11 nM) and chymotrypsin (Ki of 446 ± 9 nM). Its inhibitory activity against these proteases was stable at high temperatures (>95 °C) and a wide pH range but sensitive to reduction with dithiothreitol (DTT), indicating the importance of disulphide bridges in exhibiting its activity. Also, rRsBBI1 showed significant inhibitory activity (IC50 = 70 ng) on A. janata larval gut trypsin-like proteases (AjGPs). Conversely, it showed <1% inhibitory activity (IC50 = 8 μg) on H. armigera larval gut trypsin-like proteases (HaGPs) than it has against AjGPs. Besides, in vivo feeding experiments clearly indicated the deleterious effects of rRsBBI1 on larval growth and development in A. janata which suggests it can be further exploited for such properties.

A Bowman-Birk type chymotrypsin inhibitor peptide from the amphibian, Hylarana erythraea

The first amphibian skin secretion-derived Bowman-Birk type chymotrypsin inhibitor is described here from the Asian green frog, Hylarana erythraea, and was identified by use of molecular cloning and tandem mass spectrometric amino acid sequencing. It was named Hylarana erythraea chymotrypsin inhibitor (HECI) and in addition to inhibition of chymotrypsin (Ki = 3.92 ± 0.35 μM), the peptide also inhibited the 20 S proteasome (Ki = 8.55 ± 1.84 μM). Additionally, an analogue of HECI, named K9-HECI, in which Phe9 was substituted by Lys9 at the P1 position, was functional as a trypsin inhibitor. Both peptides exhibited anti-proliferation activity against the human cancer cell lines, H157, PC-3 and MCF-7, up to a concentration of 1 mM and possessed a low degree of cytotoxicity on normal cells, HMEC-1. However, HECI exhibited higher anti-proliferative potency against H157. The results indicate that HECI, inhibiting chymotryptic-like activity of proteasome, could provide new insights in treatment of lung cancer.