Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-Length NS3 (protease-helicase/NTPase): a comparative study of different C-terminals

Synthesis and inhibitory potencies of three types of protease inhibitors of the hepatitis C virus (HCV) full-length NS3 (protease-helicase/NTPase) are reported: (i) inhibitors comprising electrophilic serine traps (pentafluoroethyl ketones, alpha-keto acids, and alpha-ketotetrazoles), (ii) product-based inhibitors comprising a C-terminal carboxylate group, and (iii) previously unexplored inhibitors comprising C-terminal carboxylic acid bioisosteres (tetrazoles and acyl sulfonamides). Bioisosteric replacement with the tetrazole group provided inhibitors equally potent to the corresponding carboxylates, and substitution with the phenyl acyl sulfonamide group yielded more potent inhibitors. The hexapeptide inhibitors Suc-Asp-D-Glu-Leu-Ile-Cha-Nva-NHSO(2)Ph and Suc-Asp-D-Glu-Leu-Ile-Cha-ACPC-NHSO(2)Ph with K(i) values of 13.6 and 3.8 nM, respectively, were approximately 20 times more potent than the corresponding inhibitors with a C-terminal carboxylate and were comparable to the carboxylate-based inhibitor containing the native cysteine, Suc-Asp-D-Glu-Leu-Ile-Cha-Cys-OH (K(i)=28 nM). The acyl sulfonamide group constitutes a very promising C-terminal functionality that allows for prime site optimization.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Reversed hydroxamate-bearing thermolysin inhibitors mimic a high-energy intermediate along the enzyme-catalyzed proteolytic reaction pathway

A series of inhibitors that bear a reversed hydroxamate moiety have been evaluated as transition state analogue inhibitors for thermolysin. A linear correlation is observed between the K(i) values of these inhibitors and the kinetic parameters (K(M)/k(cat)) of the parallel series of related substrates, satisfying the criterion stipulated for transition state analogue inhibitors by Bartlett and Marlowe. Furthermore, examination of the binding mode of a related reversed hydroxamate bearing thermolysin inhibitor, in comparison with a transition state postulated for the enzyme-catalyzed proteolytic reaction revealed that the inhibitors under study mimic the electronic as well as the geometric characteristics of the transition state. On the basis of these results it may be concluded that the hydroxamate-bearing zinc protease inhibitors are a new type of transition state analogue inhibitors.     

Structure-activity relationship within the serine protease inhibitors of the pacifastin family

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display species selectivity fall in the same structural group.     

Synthesis and biological evaluation of hydroxamate-Based inhibitors of glutamate carboxypeptidase II

A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1' residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC(50) value of 220nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition.     

The squash family of serine proteinase inhibitors. Amino acid sequences and association equilibrium constants of inhibitors from squash, summer squash, zucchini, and cucumber seeds

Six amino acid sequences for trypsin inhibitors isolated from squash, summer squash, zucchini, and cucumber seeds were determined. All these inhibitors along with the two previously sequenced squash inhibitors (1) form the squash inhibitor family. The striking characteristic of the family is that its member inhibitors are very small (29-32 residues, 3 disulfide bridges). The association equilibrium constants with bovine beta trypsin for 6 squash family inhibitors were determined and range from 5.9 X 10(10) to 9.5 X 10(11) M-1.     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P2 position

Recently, we reported substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. These inhibitors showed potent inhibitory activities in enzymatic and cell assays. We also designed and synthesized non-peptidic and small-sized inhibitors possessing a heterocyclic scaffold at the P(2) position. By studying the structure-activity relationship of these inhibitors, we found that the σ-π interaction of an inhibitor with the BACE1-Arg235 side chain played a key role in the inhibition mechanism. Hence, we optimized the inhibitors with a focus on their P(2) regions. In this Letter, a series of novel BACE1 inhibitors possessing a 5-nitroisophthalic scaffold at the P(2) position are described along with the results of the related structure-activity relationship study. These small-sized inhibitors are expected improved membrane permeability and bioavailability.     

Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

Sodium transport inhibition by selective mitochondrial inhibitors in the urinary bladder of the toad

The effects of selective mitochondrial inhibitors on the short-circuit current and oxygen consumption displayed by the isolated urinary bladder of the toad was studied. Three types of compounds were used: (a) electron transfer inhibitors, Amytal, Cyanide and Antimycin A; (b) energy transfer inhibitors Guanidine, Oligomycin and Rutamycin; and (c) uncoupling agents, Carbonyl cyanide m-chlorophenylhydrazone and 2–4 dinitrophenol. The kinetics of inhibition of oxygen consumption indicated that the inhibitors tested were effectively reaching the mitochondria of the bladder cells. Different kinetics of inhibition of short-circuit current were obtained with the various inhibitors tested. Uncouplers and electron transfer inhibitors rapidly blocked the short-circuit current; energy transfer inhibitors only produced a slow and partial inhibition. A site of energy-coupling, tentatively identified with the intermediate formed in the energy transfer reactions closest to the electron transfer chain, is proposed.     

The effect of various monoamine oxidase (MAO) inhibitors on the response of blood pressure of rats and cats to tyramine

The "cheese effect" is the clinically most important side effect of structurally different MAO inhibitors. It occurs mainly as a result of the interaction of MAO inhibitor with tyramine in foodstuffs. Anaesthetised rats and cats were used in order to investigate and compare the influence of the effect of tyramine by selective MAO type-B inhibitors with that produced by non-selective and A-selective MAO inhibitors on the one hand, and on the other hand, different MAO-B inhibitors with (-)deprenyl. (-)Deprenyl was the only one which inhibited the effect of tyramine in the experimental animals used, while other MAO inhibitors potentiated the tyramine effect. Therefore this study indicates that not only non-selective and A-selective inhibitors potentiate the effect of tyramine but selective inhibitors of B-type MAO as well. The inhibition of tyramine uptake by (-)deprenyl is a remarkable exception from the rule.     

Formation of Bowman-Birk inhibitors during the germination of horsegram (Dolichos biflorus)

Three Bowman-Birk type inhibitors (HGGI-I, II and III), which appear in the cotyledons of 120 h germinated horsegram (Dolichos biflorus) seeds have been purified to homogeneity by size-exclusion chromatography and ion-exchange chromatography. HGGI-I, HGGI-II and HGGI-III differ from each other and from the dormant seed inhibitors in amino acid composition, molecular size and charge. The amino-terminal sequence analyses indicate that these inhibitors are derived from the isoinhibitors of the dormant seed by a limited proteolysis and not by de novo synthesis. These inhibitors differ from each other at their amino-terminus. HGGI-II identical to HGGI-I except for the loss of a single amino-terminal aspartyl residue, where as HGGI-III shows the loss of a pentapeptide. All the three inhibitors are potent competitive inhibitors of trypsin and chymotrypsin. The dissociation constants (K(i)s) for trypsin inhibition indicate that amino-terminal tail of the inhibitors play a role in trypsin binding probably through electrostatic interaction.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.     

PARP inhibitors in ovarian cancer: Sensitivity prediction and resistance mechanisms

Poly (ADP‐ribose) polymerase (PARP) inhibitors have provided great clinical benefits to ovarian cancer patients. To date, three PARP inhibitors, namely, olaparib, rucaparib and niraparib have been approved for the treatment of ovarian cancer in the United States. Homologous recombination deficiency (HRD) and platinum sensitivity are prospective biomarkers for predicting the response to PARP inhibitors in ovarian cancers. Preclinical data have focused on identifying the gene aberrations that might generate HRD and induce sensitivity to PARP inhibitors in vitro in cancer cell lines or in vivo in patient‐derived xenografts. Clinical trials have focused on genomic scar analysis to identify biomarkers for predicting the response to PARP inhibitors. Additionally, researchers have aimed to investigate mechanisms of resistance to PARP inhibitors and strategies to overcome this resistance. Combining PARP inhibitors with HR pathway inhibitors to extend the utility of PARP inhibitors to BRCA‐proficient tumours is increasingly foreseeable. Identifying the population of patients with the greatest potential benefit from PARP inhibitor therapy and the circumstances under which patients are no longer suited for PARP inhibitor therapy are important. Further studies are required in order to propose better strategies for overcoming resistance to PARP inhibitor therapy in ovarian cancers.     

Optimization of a series of potent and selective ketone histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in the clinic. Herein we describe the optimization of a series of ketone small molecule HDAC inhibitors leading to potent and selective class I HDAC inhibitors with good dog PK.     

Evaluating scoring functions for docking and designing beta-secretase inhibitors

Several simple scoring methods were examined for 2 series of beta-secretase (BACE-1) inhibitors to identify a docking/scoring protocol which could be used to design BACE-1 inhibitors in a drug discovery program. Both the PLP1 score and MMFFs interaction energy (E(inter)) performed as well or better than more computationally intensive methods for a set of substrate-based inhibitors, while the latter performed well for both sets of inhibitors.     

Amino acid sequences of trypsin inhibitors from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds.

Three inhibitors (CMCTI-I, II, and III) were isolated from oriental pickling melon (Cucumis melo L. var. Conomon Makino) seeds by acetone precipitation, gel filtration, and reversed phase chromatography. The amino acid sequences of these inhibitors were: [table; see text] The reactive sites (P1 and P1' sites) of these inhibitors are presumed to be the Lys-Ile indicated by an arrow, comparing them with other squash family inhibitors. All three inhibitors can inhibit lysyl endopeptidase and trypsin at the enzyme-inhibitor ratio of 1:1.