Crystal structure of mung bean inhibitor lysine active fragment complex with bovine beta-trypsin at 1.8A resolution.

The crystal structure of the complex of mung bean inhibitor lysine active fragment with bovine beta-trypsin has been determined by X-ray crystallographic analysis at a resolution of 1.8 A. Refinement of the model of the complex converged at a final R value of 0.16. From the resulting electron density map, about one-third of the residues of the inhibitor were identified and two residues, at position P4 and P2' respectively, were found to be inconsistent with the sequence reported previously. The peptide chain of the inhibitor at the trypsin active site turns back sharply at Pro23I and forms a 9-residue reactive loop, which interacts with trypsin in a similar manner to the other families of inhibitors, suggesting an important and common role of these regions in exhibiting inhibitory activity.     

Design of peptide inhibitors for furin based on the C-terminal fragment of histone H1.2

The mammalian proprotein convertase furin has been found to play an important role in diverse physiological and pathological events, such as the activation of viral glycoproteins and bacterial exotoxins. Small, non-toxic and highly active, furin inhibitors are considered to be attractive drug candidates for diseases caused by virus and bacteria. In this study, a series of peptide inhibitors were designed and synthesized based on the C-terminal fragment of histone H1.2, which has an inhibitory effect on furin. Replacing the reactive site of inhibitors with the consensus substrate recognition sequence of furin has been found to increase inhibitory activity greatly. The most potent inhibitor, I4, with 14 amino acid residues has a Ki value of 17 nM for furin. Although most of the synthesized peptides were temporary inhibitors, the inhibitor I5, with nine amino acids, retained its full potency, even after a 3 h incubation period with furin at 37 degrees C. These inhibitors may potentially lead to the development of anti-viral and anti-bacterial drug compounds.     

Prostaglandin inhibitors and the development of mung bean seedlings

The effect of cortisol and prostaglandin inhibitors on the growth and development of germinating mung bean, Vigna radiata L. Wilzek, cv. Jumbo was investigated. Cortisol, indomethacin, and a mixture of cortisol with aspirin, or benoxaprofen significantly increased radicle length and the number of lateral roots as compared with non-treated controls. A mixture of cortisol and indomethacin significantly increased growth of hypocotyls.     

Effects of guanidine inhibitors on mung bean mitochondria

The effects of phenylethylbiguanidide, decamethylenediguanidide, and octylguanidine have been studied with mung bean hypocotyl mitochondria (Phaseolus aureus var. Jumbo) supplied with malate, reduced nicotinamide adenine dinucleotide, succinate, or ascorbate-tetramethyl-p-phenylenediamine as substrates. The guanidines act as energy transfer inhibitors, all three inhibiting all three phosphorylation sites. Phenylethylbiguanidide causes only partial inhibition even at relatively high concentrations. Decamethylenediguanidide inhibits about 70% of the malate respiration, 55% of the succinate respiration, and 35% of the ascorbate-tetramethyl-p-phenylenediamine respiration.     

Association properties of trypsin inhibitors from lima bean

The molecular-weight properties of three purified proteinase inhibitors from lima bean were studied by using high speed sedimentation equilibrium. Two isoinhibitors [fraction I and II, nomenclature from Jones et al., (18)]do not self-associate at moderate pH and concentration (<4 g/liter). Fraction IV exists as a monomer at pH 2.0 and polymerizes at higher pH values. The molecular-weight data fit a monomer ? dimer equilibrium at pH 7.0, and a monomer ? dimer ? trimer equilibrium at pH 4.65.     

Reconstructed mung bean trypsin inhibitor targeting cell surface GRP78 induces apoptosis and inhibits tumor growth in colorectal cancer

Glucose regulated protein 78 (GRP78) has been reported to be present on cell membranes of cancer cells but not the normal cells, serving as a potential anti-cancer target. In the present study, a fusion protein containing the GRP78 binding peptide WIFPWIQL and the active fragment of mung bean trypsin inhibitor was constructed, and its targeted anti-tumor effects were investigated both in vitro and in vivo. The results showed that the fusion protein specifically inhibited growth and induced apoptosis in colorectal cancer cells but not in the normal cells. Mechanistically, these anti-tumor effects were attributed to induction of G1 phase arrest and activation of multiple apoptotic pathways. Importantly, the fusion protein could also suppress the growth of xenografted human colorectal carcinoma in vivo. Our study reveals that this fusion protein may be developed as a therapeutic agent for treatment of colon cancer, and holds important implications for developing other anti-cancer peptide drugs.     

Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans

The mung bean (Vigna radiata (L.) Wilczek) trypsin inhibitor (MBTI) is rapidly modified by limited proteolysis during the early stages of seedling growth. Using an electrophoretic assay that separates the unmodified inhibitor (MBTI-F) and the first two modified species (MBTI-E and -C), a pH optimum of approximately 4 was found for the modification reaction. The inhibitor modifying activity is initially low in ungerminated seeds, with the reaction F leads to E being the primary reaction catalyzed. Activity catalyzing the production of MBTI-C appears on the first day of germination. This activity (F leads to E leads to C) increases up to 6 days after inhibition, at which time the cotyledons begin to abscise. The activity converting MBTI-F and -E to MBTI-C was strongly inhibited by phenylmethylsulfonyl fluoride (3.3 mM) but only weakly by iodoacetate (9 mM) and not at all by pepstatin A (9 microM), leupeptin (18 microM), or EDTA (5 mM). These results suggest the involvement of proteinases other than the major endopeptidase of the germinating seed, vicilin peptidohydrolase. This conclusion is further supported by gel filtration of the extracts of cotyledons on Sephacryl S-200. At least three proteinases are present in germinated cotyledons capable of modifying MBTI-F to MBTI-C and/or -E. All are distinguishable from vicilin peptidohydrolase on the basis of their molecular weight and inhibition by low molecular weight organic reagents.     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Crystallization and preliminary studies of lima bean trypsin inhibitor

Crystals of lima bean trypsin inhibitor (LBTI) were obtained by using the vapor phase equilibration technique with sodium/potassium tartrate as the precipitating agent. The space group was determined to be cubic, I2₁3 with a= 110.2 Å. These crystals diffract to about 1.9 Å resolution. Preliminary analysis of self-rotation maps (calculated from native x-ray intensity data) suggests the presence of two monomers in the asymmetric unit. LBTI is very thermostable and retains activity even after boiling for 10 minutes. This property is exploited as part of its purification procedure. © 1997 Wiley-Liss, Inc.     

Natural plant enzyme inhibitors: isolation and properties of a trypsin inhibitor from jack bean (Canavalia ensiformis)

A protease inhibitor which is equally active on bovine and porcine trypsins was isolated in a homogenous form from jack bean (Canavalia ensiformis). The preparation with a molecular weight of 18 kDa was found to be a glycoprotein with a high half cysteine content. Isoleucine and tyrosine were found to be absent. The inhibitor was heat-stable and stable at pH 2.0 and 11.0. It was ten times less active on bovine alpha-chymotrypsin and pronase than on trypsin. It displayed weak action on subtilisin BPN, porcine elastase and pepsin. The inhibitor was most effective in blocking the total proteolytic, tryptic and chymotryptic activities of rabbit pancreatic preparation. The relative ratios of inhibitions of the three activities on rabbit, bovine and human systems were respectively 1250:100:1, 600:100:1 and 46:18:1. While different substrates (except denatured serum albumin) did not significantly alter the magnitude of inhibition of bovine trypsin, the extent of inhibition of bovine alpha-chymotrypsin by the jack bean inhibitor was highly dependent on the substrate used in the assay.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsin inhibitor (Schistocerca gregaria chymotrypsin inhibitor) were designed and synthesized by convergent peptide synthesis. For each model peptide, the inhibitory constant (Ki) on chymotrypsin and the solution structure were determined. In addition, molecular dynamics calculations were performed for all of them. Two models containing approximately half of the parent inhibitor (17 of 35 residues) were designed and subsequently found to have no substantial inhibitory activity (Ki values in the mM range). The third model composed of 24 amino acid residues proved to be an effective (Ki approximately 10(-7)) inhibitor of bovine chymotrypsin. Both the solution structure properties determined by NMR spectroscopy and the dynamic behaviour of the latter model system are comparable to the native inhibitor. In contrast, the structure and dynamics of the first two related model peptides show characteristic differences. We suggest that the conformation and flexibility of the modelled protease inhibitor are crucial for its biological efficiency. Moreover, the structural and dynamic features of the binding loop (28-33) and those of the rest of the molecule appear to be interdependent. Most importantly, these structural characteristics can be rationally modified, at least partially, by peptide design.     

A slow fluctuation in the molecular conformation of a soya bean trypsin inhibitor

The kinetics of the hydrogen-deuterium exchange reaction in a trypsin inhibitor (Kunitz) from soya bean have been followed by infrared absorption measurements in aqueous solutions at various temperatures and pH values. It was found that, in every case, 49% of the total peptide hydrogen atoms exchange relatively slowly. This amount corresponds to 83 peptide groups per molecule, and this is considered to be equal to the number of peptide NH groups involved in hydrogen bonds with the carbonyls of other peptide groups in the protein molecule in its native form. Each rate constant (k) determined at pH 2.75 for this category of the NH groups is in good agreement with the value expected from an idea that the breaking of the peptide-peptide hydrogen bonds takes place very slowly, and that this is the rate-determining process in the hydrogen-deuterium exchange reaction. Thus, by ultraviolet absorption measurements at 297 nm, the equilibrium constant of the native and denatured forms has been determined in the temperature range from 42 to 53.5 °C, as well as the reaction rate of reaching equilibrium from an off-equilibrium state. From these data the rate constant (k₁) of the denaturation reaction is determined, and the k₁ value is found to be practically equal to the hydrogen exchange rate constant (k). The Arrhenius plot of this rate constant (k) gives a straight line in the 25 to 55 °C region, and this gives a value of 48.6 kcal/mol for the activation energy of the denaturation reaction. The rate of this reaction is found to be very low at 25 °C; its half-life is about eleven days. Infrared absorption spectra observed in the amide I region suggest that the very slow denaturation of this protein is accompanied by a conformation change from an α-helix to a β-form. The number of the peptide groups involved in this α → β change is estimated to be 9 ± 3.     

Structure-based in-silico rational design of a selective peptide inhibitor for thymidine monophosphate kinase of mycobacterium tuberculosis

Tuberculosis still remains one of the most deadly infectious diseases. The emergence of drug resistant strains has fuelled the quest for novel drugs and drug targets for its successful treatment. Thymidine monophosphate kinase (TMPK) lies at the point where the salvage and de novo synthetic pathways meet in nucleotide synthesis. TMPK in M.tb has emerged as an attractive drug target since blocking it will affect both the pathways involved in the thymidine triphosphate synthesis. Moreover, the unique differences at the active site of TMPK enzyme in M.tb and humans can be exploited for the development of ideal drug candidates. Based on a detailed evaluation of known inhibitors and available three-dimensional structures of TMPK, several peptidic inhibitors were designed. In silico docking and selectivity analysis of these inhibitors with TMPK from M.tb and human was carried out to examine their differential binding at the active site. The designed tripeptide, Trp-Pro-Asp, was found to be most selective for M.tb. The ADMET analysis of this peptide indicated that it is likely to be a drug candidate. The tripeptide so designed is a suitable lead molecule for the development of novel TMPK inhibitors as anti-tubercular drugs.