Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion.

The Bowman-Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman-Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P1 and P2' positions, as well as the C-terminal amidation on the structure-activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.

In vitro interaction of potential antiviral TMPRSS2 inhibitors with human serum albumin and cytochrome P 450 isoenzymes.

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.

Acacia seed proteins: Low or high quality? A comprehensive review.

The popularity of adding value to indigenous plant protein sources has increased due to the rise in the world population, high costs of animal protein as compared to plant proteins, and an increase in the consumer awareness of the nutritional and functional roles of dietary plant protein. Seeds of acacia plants (containing over 1,350 species) have considerable amount of protein (18.25% to 35.5%) and nutritionists have shown great interest in assessing the quality and functionality of proteins from these protein-rich plants. In this review, the overall nutritional and health-promoting properties of acacia seed (AS) species are introduced. Extraction, quality, and functional properties of proteins from different AS species are discussed. Furthermore, anti-nutritional components and protease inhibitors present in AS species and the effects of processing methods applied to lower the levels of anti-nutrients are also discussed. Previous applications of AS in food formulations are highlighted. This review aims to provide updated findings that have been reported on AS proteins and to highlight areas for further studies in order to increase the utilization potential of the seeds.

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Non­invasive proteome­wide quantification of skin barrier­related proteins using label­free LC­MS/MS analysis.

A number of epidermal proteins are closely related to skin barrier function, the abnormalities of which can lead to specific skin diseases. These proteins must be quantified to further investigate the changes in the skin barrier between healthy and disease states. However, the non­invasive and proteome­wide quantification of skin proteins without any labelling steps remains a challenge. In this study, 3M medical adhesive tapes were used to obtain skin samples from volunteers. Proteins were extracted from fresh skin samples and digested with trypsin. Each tryptic peptide was analysed in three replicates using liquid chromatography with tandem mass spectrometry analysis and label­free quantification. The data were searched against the Human Universal Protein Resource (UniProt) to match with known proteins. Using this method, 1,157 skin proteins recorded in the UniProt were quantified. A total of 50 identical proteins were identified in the three replicate analyses of all samples with no significant differences in abundance. The results provided an objective metric for further study of skin ageing and various skin diseases. Specifically, the non­invasive proteome­wide method used in this study can be applied to future studies of skin diseases related to barrier destruction by monitoring the changes in the levels of epidermal proteins.

Chemical composition, nutritional properties, and antioxidant activity of Licania tomentosa (Benth.) fruit.

Licania tomentosa is a Brazilian plant species that produces edible fruits, yet there is little information available concerning their nutritional and/or bioactive composition. This study aimed to evaluate the nutritional and polyphenol composition of L. tomentosa fruits (pulp and seeds) and measure antioxidant activity in ethanolic extracts.The pulp and seeds were excellent sources of fiber (25.62%-41.70%) as well as minerals and vitamins. L. tomentosa contained no lectins or protease inhibitors (chymotrysin and trypsin) and 12 polyphenol compounds were identified in the seed extracts with a predominance of flavonoids. The seeds also presented antioxidant activities using the DPPH (SC5010.30-15.87 µg/mL), TBARS (IC50 18.46-20.84 µg/mL), and FRAP (RC50 0.203-0.309 µg/mL) assays. Due to its nutrient and antioxidant content, L. tomentosa may be used for food applications.

[Determination of four protease inhibitors in chicken by ultra performance liquid chromatography-tandem mass spectrometry].

A method was developed for the determination of four protease inhibitors (saquinavir, ritonavir, nelfinavir and indinavir) in chicken using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted by shaking with 30% (v/v) acetonitrile aqueous solution (containing 1% (v/v) trichloroacetic acid), and purified by using mixed-mode cationic-exchanger (MCX) cartridges. The samples were separated on a Luna® C8 column (150 mm×2 mm, 3 µm) using 0.2% (v/v) formic acid aqueous solution (containing 5 mmol/L ammonium acetate) and acetonitrile as the mobile phases with gradient elution. The determination was carried out by using an electrospray ion source in the positive and multiple-reaction monitoring (MRM) modes. The calibration curves showed good linearities in the range of 0.1-20.0 µg/L, and the correlation coefficients (r2) were greater than 0.99. The limits of quantification (LOQs, S/N=10) of the four protease inhibitors varied from 0.20 µg/kg to 0.90 µg/kg. At the spiked levels of 1.0, 2.0, and 10.0 µg/kg, the average recoveries of the four protease inhibitors were ranging from 69.0% to 106.0%. The intra-day and inter-day relative standard deviations (RSDs) were 2.2%-13.8% (n=6) and 3.6%-14.6% (n=3), respectively. The method is simple, efficient, sensitive and accurate, and it can be used to detect residues of saquinavir, ritonavir, nelfinavir and indinavir in chicken.

[Diagnostic possibilities of modern biochemical study of sputum from patients with cystic fibrosis (literature review).]

The review presents the pathobiochemical and molecular mechanisms of sputum formation in patients with cystic fibrosis associated with the pathophysiological features of the disease. Statistical data on the prevalence of this pathology in the world and in the Russian Federation are presented. The mechanisms of sputum formation and disorders of the mucociliary apparatus, leading to the accumulation of viscous bronchopulmonary secret in cystic fibrosis, are considered. The principles of the relationship between the rheological properties of sputum and the formation of inflammation in the lungs with the addition of a concomitant specific microflora in the bronchopulmonary system in patients with cystic fibrosis are presented. Describes the opportunities for biochemical studies of sputum of patients with this pathology: determining the activity of enzymes (myeloperoxidase), the content of proteinase inhibitors (α2-macroglobulin and α1-antitrypsin) and proinflammatory cytokines (IL-8 and TNFa), concentrations of iron and ferriferous proteins (lactoferrin and ferritin), which makes biochemical studies of sputum available, non-invasive, quick and cost-effective method of diagnosis, which can be widely used as an auxiliary laboratory method and makes it possible to use these metabolites as diagnostic markers to assess the severity of inflammation and infection of the lower respiratory tract and predict the development of respiratory complications in patients with cystic fibrosis.

Transcriptome analysis of the spider Phoneutria pertyi venom glands reveals novel venom components for the genus Phoneutria.

Phoneutria nigriventer spider venom has been studied for more than 40 years and several components with pharmacological potential have been described in it. However, studies on venoms from other species of the Phoneutria genus are scarce. In this work, a conventional cDNA library from the species Phoneutria pertyi venom glands was constructed, aiming to identify novel putative cysteine-rich peptide toxins for the genus Phoneutria. 296 unique sequences were identified and 51 sequences corresponded to putative cysteine-rich peptide toxins. Besides cysteine-rich peptide toxins, other putative venom components such as protease inhibitors, defensins and serine proteinases were identified. Furthermore, by manual curation of the sequences with no match at UniProt, we were able to identify glycine-rich proteins (GRP), a class of venom component never described in Phoneutria genus. This work describes the first complete sequences of toxins from the venom of P. pertyi and reveals that, despite most of the retrieved toxins show a high identity to toxins identified in Phoneutria genus, novel putative toxins remains to be described.

Molecular Docking and 3D Qsar Studies of C000000956 as a Potent Inhibitor of Bace-1.

BACKGROUND: BACE-1 is an aspartate protease that is responsible for the proteolysis of amyloid precursor proteins (APP) into beta-amyloid (Aß), a neurotoxic peptide in patients with Alzheimer's disease (AD). As such, BACE-1 is a prime pharmacological target in the control of Aß in the brain and its inhibition will be a sound approach in AD therapy. METHODS: The computational pipeline which comprised molecular docking (MD), Quantitative Structure Activity Relationship (QSAR) modelling and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) studies enabled the prediction of molecular interaction and relative inhibitory potentials of the hit compound. RESULTS AND DISCUSSION: The current study reports a naturally sourced small molecule inhibitor of BACE1 (C000000956) which was obtained through a computational pipeline. Also, pharmacological constraints such as pH dependent activity of the enzyme and blood brain barrier permeation which have been associated with the efficacy of previous BACE-1 inhibitors were well catered for. Our results suggest that orally delivered C000000956 is a potential small molecule inhibitor of BACE-1 which may find usefulness in AD-therapy.

Ultrafast Single-Scan 2D†NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

Simultaneous quantification of simeprevir sodium: A hepatitis C protease inhibitor in binary and ternary mixtures with sofosbuvir and/or ledipasvir utilizing direct and H-point standard addition strategies.

Simeprevir sodium (SMV); a novel hepatitis C inhibitor, quells hepatitis C viral replication by binding to and repressing the protease, hepatitis C infection (HCV) NS3/4A. In this way, it is known as a prompt acting antiviral agent. Calibration curves of SMV were built in various solvents; ethanol, methanol, acetonitrile, chloroform and dichloromethane. It is obeyed up to 60.0 µg/mL; in all solvents at two maximum wavelengths (280 and 327 nm). Several investigations show that, SMV might be present in a mixture of Sofosbuvir (SOF) and/or Ledipasvir (LDP). So far as that is concerned, H-point standard addition strategy (HPSAS) is made to identify it in binary or ternary mixtures. Recovery studies are in the prevalent range (93.0-107.0%) with relative standard deviation <1.5%. A correlation between the developed techniques is carried out and it demonstrates that these strategies are effectively applied for the simultaneous analysis of SMV, SOF and LDP in several synthetic samples and pharmaceutics. Statistical treatment of the acquired data is carried out against a newly published HPLC technique using F- and t-treatments.

Fast and Cost-Effective Biochemical Spectrophotometric Analysis of Solution of Insect "Blood" and Body Surface Elution.

Using insect hemolymph ("blood") and insect body surface elutions, researchers can perform rapid and cheap biochemical analyses to determine the insect's immunology status. The authors of this publication describe a detailed methodology for a quick marking of the concentration of total proteins and evaluation of the proteolytic system activity (acid, neutral, and alkaline proteases and protease inhibitors), as well as a methodology for quick "liver" tests in insects: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea and glucose concentration analyses. The meaning and examples of an interpretation of the results of the presented methodology for biochemical parameter determination are described for the example of honey bees.

Transcriptomic Analysis of Pseudoscorpion Venom Reveals a Unique Cocktail Dominated by Enzymes and Protease Inhibitors.

Transcriptomic and genomic analyses have illuminated the diversity of venoms in three of the four venomous arachnid orders (scorpions, spiders, and ticks). To date, no venom gland transcriptome analysis has been available for pseudoscorpions, the fourth venomous arachnid lineage. To redress this gap, we sequenced an mRNA library generated from the venom glands of the species Synsphyronus apimelus (Garypidae). High-throughput sequencing by the Illumina protocol, followed by de novo assembly, resulted in a total of 238,331 transcripts. From those, we annotated 131 transcripts, which code for putative peptides/proteins with similar sequences to previously reported venom components available from different arachnid species in protein databases. Transcripts putatively coding for enzymes showed the richest diversity, followed by other venom components such as peptidase inhibitors, cysteine-rich peptides, and thyroglobulin 1-like peptides. Only 11 transcripts were found that code for putatively low molecular mass spider toxins. This study constitutes the first report of the diversity of components within pseudoscorpion venom.

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

Determination of polymeric impurities in asunaprevir drug substance and product using size exclusion effect of reversed-phase columns.

This paper describes the development of a simple reversed-phase HPLC method that can quantitate trace amounts of a polymeric degradants (BMT-041910) in asunaprevir drug substance and formulated drug product with quantitation limits of ∼0.05% w/w. The method has overcome several challenges of polymer quantitation such as band broadening, peak coeluting and low sensitivity. The hydrophobic function group (BOC) of BMT-041910 is removed to increase its aqueous solubility by a simple sample treatment procedure (des-BOC). The des-BOC polymer (BMT-052076) is excluded from stationary phase pores and eluted as a single peak before solvent front, and then its peak area response can be used to determine BMT-041910 amount. The HPLC conditions were optimized using a 250 × 4.6 mm Waters XSelect CSH column maintained at 30 °C with a mobile phase of water-acetonitrile-trifluoroacetic acid (20:80:0.1 v/v/v). The feasibility of other HPLC approaches including size exclusion chromatography and normal phase chromatography were also investigated and found to be less suitable for this particular application. Validation data for this method in terms of precision, linearity, accuracy and sensitivity are also presented.

Biochemical characterization of the YBPCI miniprotein, the first carboxypeptidase inhibitor isolated from Yellow Bell Pepper (Capsicum annuum L). A novel contribution to the knowledge of miniproteins stability.

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 µg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance.

Total proteolytic activity and concentration of alpha-1 antitrypsin in meconium for assessment of the protease/antiprotease balance.

BACKGROUND: During intrauterine life, various proteolytic enzymes and their main inhibitor, alpha-1 antitrypsin, accumulate naturally in meconium. A protease/antiprotease balance is required to maintain the biological stability of the environment in which the fetus develops. METHODS: The pool of active proteases was determined using the EnzChek Protease Assay Kit. The concentration of alpha-1 antitrypsin in meconium was measured by enzyme-linked immunosorbent assay. Serial portions of meconium (n=80) were collected from healthy full-term neonates (n=19). RESULTS: Mean concentrations of active proteases and alpha-1 antitrypsin were 1.55 [standard deviation (SD) 1.3]mgg-1 (range 0.15-6.17) and 3.72 (SD 1.78)mgg-1 (range 0.76-8.55), respectively, with significant correlation (Rs=0.32, p=0.004). A significant increase in the concentration of active proteases was found between the first and last meconium portions (p<0.05). The proteases in the last meconium portions had a higher reaction velocity and affinity for the substrate than the proteases in the first meconium portions. The active protease:alpha-1 antitrypsin ratio was <0.5 in all first meconium portions, but was higher in the last meconium portions. CONCLUSIONS: Strong correlation between the concentrations of active proteases and alpha-1 antitrypsin in meconium may indicate their mutual interaction in the intrauterine environment. Alpha-1 antitrypsin maintains the protease/antiprotease balance during fetal development.

Reverse Zymography: Overview and Pitfalls.

Reverse zymography is a technique by which protease inhibitor(s) in a sample could be electrophoretically separated in a substrate-impregnated acrylamide gel and their relative abundance could be semi-quantified. The gel after electrophoresis is incubated with a protease when the impregnated substrate and all other proteins of the sample are degraded into small peptides except the inhibitor(s) that show clear bands against a white background. Since reverse zymography cannot distinguish between a protease inhibitor and a protein that is resistant against proteolysis, the results should be confirmed from inhibition of protease activity by solution state assay.