Protease-inhibiting Properties Research Articles

Valorization of protein from by-product of purified rice starch industry: Protein hydrolysates exhibit a potential heat resistance and protease inhibition besides foam formation and stability properties

This study aimed to enhance the added value of protein from by-products of the purified starch industry (BPSI). Proteins from BPSI were extracted with 0–660 UI α-amylase before being hydrolyzed by neutrase from 0 to 175 UI for 0–150 min at 80 °C, pH 5.3. The results showed that with 264 UI of α-amylase treatment for 60 min, the extraction of proteins improved the content of protein recovery up to 87.1%. The kinetic parameters of neutrase were Vmax = 0.8982 μMol tyrosine/min and Km = 0.9103%. The most effective condition for neutrase hydrolysis was 150 UI, 120 min, and 20% protein concentration. The highest hydrolysis efficiency reached 16%. The recovered protein has few free amino acids compared with those in the hydrolysates. The protein hydrolysate (PH) showed high foam forming and stabilizing properties for 30 min compared with those of the natural protein recovered. Furthermore, the PH exhibited heat resistance and protease inhibition properties. Thus, the purity and natural state of PH extracted by α-amylase could be improved. Foam formation and stabilization together with heat resistance and proteinase K inhibition exhibited by the PH indicate the potential application of this in the rice beer industry.

Phytochemical screening, in-vitro antioxidant and anti protease activities of various solvent extracts of Muehlenbeckia platyclada root (F. Muell.) Meisn

The objective of this study was focussed on phytochemical screening, in vitro antioxidant and antiprotease activities of various solvent extracts of Muehlenbeckia platyclada root. The roots were washed thoroughly, shade dried and coarsely powdered. The powdered material of Muehlenbeckia platyclada was successively extracted with hexane, chloroform and methanol using soxhlet apparatus. Preliminary phytochemical screening for carbohydrates, proteins, alkaloids, phytosteroids, flavonoids, glycosides, polyphenolics, saponins, and tannins were done by following standard procedure. In vitro antioxidant activities of three solvent extracts were assessed using DPPH, ABTS and total antioxidant capacity and flavonoids were estimated using aluminum chloride colourimetric assay. In vitro anti-protease activity of the root was evaluated using trypsin as enzyme and BAEE (N-benzoyl-L-arginine ethyl ester) as a substrate. The results showed that phytochemicals such as carbohydrates, proteins, flavonoids and glycosides, which present in the methanolic extract, were absent in hexane and chloroform extract of the root. The in vitro antioxidant and antiprotease activities of the Muehlenbeckia platyclada root clearly showed that the plant root has prominent antioxidant and protease inhibiting properties. From this work, it can be concluded that Muehlenbeckia platyclada root has the potential to be a powerful antioxidant and protease inhibitor.

Hexamidine salts - applications in skin health and personal care products.

Hexamidine (HEX) has been used as a preservative in topical preparations since the 1950s. A number of studies also indicate that the molecule plays a beneficial role in skin homoeostasis. In this review, we describe the physicochemical properties of hexamidine diisethionate (HEX D) and the corresponding hydrochloride salt (HEX H). The biocidal and protease inhibition properties of HEX are outlined as well as the effects of HEX on lipid processing enzymes, corneocyte maturity, stratum corneum thickness and transepidermal water loss (TEWL). Skin permeation properties of HEX D and HEX H are summarized, and formulation approaches for effective dermal targeting of HEX are discussed.

Antidermatophytic and Protease-inhibiting Activities of Zerumbone: A Natural Sesquiterpene from the Rhizome of Zingiber zerumbet (L.) Roscoe ex J.E; Smith.

Context:Due to increase in the number of patients with impaired immunity, incidence of dermatophytoses has increased considerably. Antidermatophytic agents with anti-inflammatory and protease-inhibiting activities will help in restricting inflammatory response associated with dermatophytoses.Aims:The present study aims to evaluate antidermatophytic and protease-inhibiting activities of zerumbone. Cytotoxicity was tested using Chang liver cell line as a preliminary step in toxicity study.Methods and Materials:Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of zerumbone purified from the rhizome of Zingiber zerumbet were determined against Epidermophyton floccosum var. nigricans, Microsporum canis, Microsporum gypseum, and Trichophyton rubrum. MIC was determined according to Clinical and Laboratory Standards Institute (CLSI) method M38-A2. Protease-inhibiting property was tested using trypsin as the enzyme. In vitro cytotoxic effect was studied using the MTT assay.Results:MIC of zerumbone was 8 mg/L against E. floccosum and M. canis and 16 mg/L for M. gypseum and T. rubrum. MFC of zerumbone was 64 mg/L against E. floccosum and M. canis and 128 mg/L for M. gypseum and T. rubrum. Zerumbone exhibited remarkable protease-inhibiting activity. In the MTT assay, IC50 values were 150 and 0.31 µg, respectively, for zerumbone and reference drug.Statistical Analysis Used:For protease inhibition, assay and cytotoxicity assay control and tests were done in triplicate and the results are expressed as mean ± SD, where n = 3.Conclusions:Zerumbone is a novel candidate for use in dermatophytoses therapy because of the combined antifungal, anti-inflammatory (unpublished results), and protease-inhibiting properties. Cytotoxicity of zerumbone was found to be very low compared with the reference drug.KEY MESSAGES Zerumbone possesses antidermatophytic, anti-inflammatory, and protease-inhibiting activities. Hence, it is a novel candidate for the development of new antidermatophytic drug.Cytotoxicity of zerumbone against Chang liver cell line was found to be very low compared with the reference drug doxorubicin. SUMMARY Zerumbone isolated from the rhizome of Zingiber zerumbet exhibited antidermatophytic activity against E. floccosum and M. canis (MIC 8 mg/L) and M. gypseum and T. Rubrum (MIC 16 mg/L).Zerumbone exhibited remarkable protease-inhibiting activity.Zerumbone is a novel candidate for the development of new antidermatophytic drug.Cytotoxicity of zerumbone against Chang liver cell line was found to be very low compared with the reference drug doxorubicin. Abbreviations used: CFU: colony forming unit, CLSI: Clinical and Laboratory Standards Institute, COX: cyclooxygenase, DMSO: dimethyl sulphoxide, EDTA: ethylene diamine tetra acetic acid, FT-IR: Fourier transform–infra red spectroscopy, HPLC: high-performance liquid chromatography, LOX: lipoxygenase, IMTECH: Institute of Microbial Technology, LCMS: liquid chromatography mass spectrometry, MTT: 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTCC: microbial type culture collection, MFC: minimum fungicidal concentration, MIC: minimum inhibitory concentration, MPO: myeloperoxidase, NMR: nuclear magnetic resonance spectroscopy, PAR: proteinase-activated receptor, PBS: phosphate-buffered saline, TCA: trichloro acetic acid

APD3: the antimicrobial peptide database as a tool for research and education.

The antimicrobial peptide database (APD, http://aps.unmc.edu/AP/) is an original database initially online in 2003. The APD2 (2009 version) has been regularly updated and further expanded into the APD3. This database currently focuses on natural antimicrobial peptides (AMPs) with defined sequence and activity. It includes a total of 2619 AMPs with 261 bacteriocins from bacteria, 4 AMPs from archaea, 7 from protists, 13 from fungi, 321 from plants and 1972 animal host defense peptides. The APD3 contains 2169 antibacterial, 172 antiviral, 105 anti-HIV, 959 antifungal, 80 antiparasitic and 185 anticancer peptides. Newly annotated are AMPs with antibiofilm, antimalarial, anti-protist, insecticidal, spermicidal, chemotactic, wound healing, antioxidant and protease inhibiting properties. We also describe other searchable annotations, including target pathogens, molecule-binding partners, post-translational modifications and animal models. Amino acid profiles or signatures of natural AMPs are important for peptide classification, prediction and design. Finally, we summarize various database applications in research and education.

BF9, the First Functionally Characterized Snake Toxin Peptide with Kunitz-Type Protease and Potassium Channel Inhibiting Properties

Although numerous Kunitz-type toxins were isolated from snake venom, no bifunctional Kunitz-type snake toxins with protease and potassium channel inhibiting properties have been reported till now. With the help of bioinformatics analyses and biological experiments, we characterized Kunitz-type snake toxin BF9 as a bifunctional peptide. Enzyme and inhibitor reaction kinetics experiments showed that BF9 inhibited α-chymotrypsin with Ki value of 1.8 × 10⁻⁸ M. Electrophysiological experiments showed that BF9 inhibited the Kv1.3 potassium channel with an IC₅₀ of 120.0 nM, which demonstrated that serine protease inhibitor BF9 could also inhibit potassium channels. In addition, the key amino acids of BF9 responsible for the unique bifunctional mechanism are further investigated. To the best of our knowledge, BF9 is the first Kunitz-type snake peptide with the unique bifunctionality of potassium channel and serine protease inhibiting properties, providing novel insights into divergent evolution and functional applications of snake Kunitz-type peptides.

SjAPI, the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

BackgroundSerine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.Principal FindingsHere, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues “AAV” and might be a useful template to produce new serine protease inhibitors.Conclusions/SignificanceTo our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

BackgroundWe have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function.ResultsWe identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas.ConclusionsThe similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions.

Pharmacologically active boranes

Abstract Novel methods are described for the preparation of alkyldimethylamine cyanoboranes and β-hydroxylalkyldimethylamine cyanoboranes by C-lithiation of trimethylamine cyanoboranes followed by reaction with alkyl halides, aldehydes, and ketones. Lithiation of the monobromo derivatives of amine cyanoboranes led to the synthesis of the first examples of diborane derivatives of amine cyanoboranes. Bromo derivatives of amine cyanoboranes and amine carboxyboranes have been synthesized by new simple and efficient methods. Amine fluorocyanoboranes and amine fluorocarboxyboranes, new classes of compounds, have been prepared from the bromo precursors by fluorine/bromine exchange using fluorinating reagents such as AgF and Et3N.3HF. Eight different derivatives of oxazaborolidines were synthesized and evaluated for their affect on Streptococcus mutans viability, adhesion, and biofilm formation using 3[H]-thymidine labeled bacteria, and fluorescent stained bacteria. This is the first reported antibacterial activity of this class of compounds. The minimal inhibitory concentration (MIC) values ranged from 0.26 to 10 mM. Structure-activity relationship was observed. The B-butyl moiety of the oxazaborolidines contributed an anti-adhesion effect for all derivatives, while its effect diminished when the boron atom was incorporated in a fused heterocyclic ring. The B-phenyl group induced bacterial adhesion in all tested compounds. In a separate study for boronated saccahrides and enzymatic inhibition, the complex formation between N-butylboronic acid and a series of monosaccharides was investigated by 1H, 13C, and 11B NMR spectroscopy and gas chromatography-mass spectrometry (GC-MS). Then, a series of boronic acid compounds with protease inhibition properties were prepared. The effect of added mono-, di-, and polysaccharides on the inhibitory activity of these compounds was studied. Potassium organotrifluoroborates were found to be reversible competitive inhibitors of α-chymotrypsin and trypsin. Based on 19F NMR, it was speculated that they inactivate the enzymes as a result of the formation of hydrogen bonds between fluorine atoms of the inhibitors and the serine protease.

A zinc metalloprotease inhibitor, Inh, from the insect pathogen Photorhabdus luminescens.

The entomopathogen Photorhabdus luminescens secretes many proteins during the late stages of insect larvae infection and during in vitro laboratory culture. The authors have previously characterized and purified a 55 kDa zinc metalloprotease, PrtA, from culture supernatants of P. luminescens. PrtA is secreted via a classical type I secretory pathway and is encoded within the operon prtA-inh-prtBCD. The 405 bp inh gene encodes a 14.8 kDa pre-protein that is translocated to the periplasm by the classical signal-peptide-dependent sec pathway, yielding the mature 11.9 kDa inhibitor Inh. Inh is a specific inhibitor of the protease PrtA. This study describes the purification of Inh and the initial characterization of its in vitro protease inhibition properties.

Potent HIV protease inhibitors containing a novel (2-phenylsulfanyl-1-hydroxyethyl)amide isostere

Based on the concept of bioisosterism, we report the design and synthesis of new protease inhibitors. These new compounds incorporate in their backbone the synthon 2-N-Acyl-2-Phenylsulfanyl-1-Hydroxyethyl (I) which confers on the resulting compounds both in vitro activity on MT 4 infected cells and HIV-1 protease inhibition properties.

Short and unexpectedly potent 3-pyrrolidinone type inhibitors of HIV-1 replication

Based on the specific PhePro proteolytic cleavage of the HIV protease, short pseudo-peptides incorporating a 3-pyrrolidinone ring have been synthesized. Their potencies to inhibit HIV-1 in MT 4 cell culture have been evaluated and compared to that of the bioisostere dipeptide BocPhePro. Analogues incorporating an aromatic residue have shown to inhibit HIV-1 infection in MT 4 human lymphoid cell with an IC 50 ranging from 1 to 10 μM. Further experiments are in progress to determine their HIV protease inhibition properties.

Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin.

Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.