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PubMed1981

Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin.

Epitopes↗
PubMed2005

Characterization of the protease domain of Rice tungro bacilliform virus responsible for the processing of the capsid protein from the polyprotein.

BACKGROUND: Rice tungro bacilliform virus (RTBV) is a pararetrovirus, and a member of the family Caulimoviridae in the genus Badnavirus. RTBV has a long open reading frame that encodes a large polyprotein (P3). Pararetroviruses show similarities with retroviruses in molecular organization and replication. P3 contains a putative movement protein (MP), the capsid protein (CP), the aspartate protease (PR) and the reverse transcriptase (RT) with a ribonuclease H activity. PR is a member of the cluster of retroviral proteases and serves to proteolytically process P3. Previous work established the N- and C-terminal amino acid sequences of CP and RT, processing of RT by PR, and estimated the molecular mass of PR by western blot assays. RESULTS: A molecular mass of a protein that was associated with virions was determined by in-line HPLC electrospray ionization mass spectral analysis. Comparison with retroviral proteases amino acid sequences allowed the characterization of a putative protease domain in this protein. Structural modelling revealed strong resemblance with retroviral proteases, with overall folds surrounding the active site being well conserved. Expression in E. coli of putative domain was affected by the presence or absence of the active site in the construct. Analysis of processing of CP by PR, using pulse chase labelling experiments, demonstrated that the 37 kDa capsid protein was dependent on the presence of the protease in the constructs. CONCLUSION: The findings suggest the characterization of the RTBV protease domain. Sequence analysis, structural modelling, in vitro expression studies are evidence to consider the putative domain as being the protease domain. Analysis of expression of different peptides corresponding to various domains of P3 suggests a processing of CP by PR. This work clarifies the organization of the RTBV polyprotein, and its processing by the RTBV protease.

Amino Acid Sequence↗
PubMed1994

Human fibroblasts secrete a serine protease that cleaves insulin-like growth factor-binding protein-5.

We have previously reported the presence of proteolytic activity in conditioned medium from human fibroblast cultures that cleaves insulin-like growth factor-binding protein-5 (IGFBP-5) into non-IGF-I-binding fragments. Coincubation of IGF-I or IGF-II and IGFBP-5 with fibroblast cultures decreased proteolysis. The protease was purified by heparin-Sepharose affinity chromatography. The purified protease cleaved IGFBP-5 into 22-, 20-, and 17-kilodalton non-IGF-I-binding fragments. Protease inhibitor profiles obtained using partially purified enzyme showed that it was a calcium-dependent serine protease. After chelation with EDTA, the activity could only be partially restored with zinc, indicating that it was probably not a metalloprotease. The protease was specific for IGFBP-5 and did not cleave pure IGFBP-1, -2, -3, or -4. IGF-I and IGF-II caused minimal inhibition of proteolysis in vitro. This suggests that the IGF-I-induced increase in IGFBP-5 in fibroblast medium is only partially due to direct protease inhibition. Heparin, antithrombin-III (AT-III), and heparin cofactor-II had inhibitory activity, and heparin potentiated the activity of AT-III. Synthetic peptides, that contained the active sites of AT-III and alpha 1-antichymotrypsin, were also inhibitory. Peptides containing sequences found in two basic regions of IGFBP-5 were tested, and one had inhibitory activity. In summary, fibroblasts secrete a serine protease that cleaves IGFBP-5 and is specific for this form of IGFBP. The protease has properties that are similar to kallikreins, a family of serine proteases that is known to cleave epidermal and nerve growth factor-binding proteins.

Antithrombin III↗
PubMed1995

Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation.

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is uniquely regulated throughout MC3T3-E1 osteoblast differentiation: IGFBP-5 is first detectable in conditioned medium (CM) of replicating preosteoblasts (day 5); IGFBP-5 levels peak between culture days 8-12, then decline to almost undetectable levels in mature osteoblast cultures (> day 18) despite the persistence of IGFBP-5 messenger RNA. These observations suggest that IGFBP-5 concentrations may be regulated by posttranslational mechanisms. To determine whether proteolysis contributes to the disappearance of IGFBP-5 in CM of mature osteoblasts, serial samples of MC3T3-E1 cell CM obtained during a 30-day culture period were analyzed for IGFBP-5-degrading protease activity. Using [125I]recombinant human IGFBP-5 substrate zymography, we demonstrated that proteases with M(r) of 52-72 and 97 kilodaltons (kDa) were present in CM, and protease activity increased in concentration as cultures matured. The 52- to 72-kDa proteases were cation dependent and were inhibited by tissue inhibitor of metalloproteinase 1, a specific inhibitor of matrix metalloproteinases (MMPs), identifying them as MMPs. Furthermore, antisera to human MMP-1 and -2 immunoprecipitated IGFBP-5-degrading proteases with M(r) of 52 and 69/72 kDa, respectively, suggesting that homologous murine MMPs degrade IGFBP-5. Finally, MC3T3-E1 cell CM contained immunoreactive MMP-1 and -2, and MMP-2, in particular, increased significantly throughout differentiation. In contrast, the 97-kDa protease was neither inhibited by tissue inhibitor of metalloproteinase 1 nor immunoprecipitated by antisera to MMPs, suggesting that the 97-kDa protease is not a MMP. Together, these data suggest that MMPs along with an unidentified 97-kDa protease degrade IGFBP-5 in MC3T3-E1 cell cultures. Because truncated forms of IGFBP-5 have been shown to enhance the action of IGF in bone cells, IGFBP-5 proteases may be instrumental in IGF-mediated bone morphogenesis.

Animals↗
PubMed1998

Role of Nudel protease activation in triggering dorsoventral polarization of the Drosophila embryo.

The establishment of embryonic dorsoventral polarity in Drosophila depends on a signaling mechanism in which the signal for ventral development is locally produced. This mechanism requires the activity of the nudel gene in ovarian follicle cells, which provide dorsoventral positional information for the embryo. The nudel gene product, a large mosaic protein with a central serine protease domain, has been proposed to function in locally triggering a protease cascade that produces the ventral signal. Here we provide evidence that the serine protease activity of the Nudel protein is essential for embryonic dorsoventral polarity and that the active Nudel protease is generated by autoproteolytic cleavage of a zymogen form. Activation of the Nudel protease is independent of the other known proteases involved in dorsoventral polarity establishment and appears to occur symmetrically on the surface of the embryo. Our findings suggest that Nudel protease activation initiates the protease cascade that produces the ventral signal, but that spatial regulation occurring downstream of Nudel protease activation localizes the cascade to the ventral side of the embryo.

Animals↗
PubMed1998

Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi.

Cruzain, the major cysteine protease of the protozoan parasite Trypanosoma cruzi, is a target of rational drug design for chemotherapy of Chagas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cysteine protease inhibitors are still unclear. Here we report biological and ultrastructural alterations caused by cysteine protease inhibitors in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits the Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy utilizing either a biotinylated cysteine protease inhibitor to tag the active site, or a specific anti-cruzain antibody. Radiolabeled or biotinylated cysteine protease inhibitors bound exclusively to cruzain in intact epimastigotes confirming that cruzain is accessible to, and is targeted by the inhibitors. Treatment of T. cruzi epimastigotes with specific cysteine protease inhibitors arrested growth, altered the intracellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction in gold-labeled cruzain transported to lysosomes. Cisternae abnormalities in the Golgi compartment were followed by distention of ER and nuclear membranes. Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine protease inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alterations. Our results suggest that cysteine protease inhibitors prevent the normal autocatalytic processing and trafficking of cruzain within the Golgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These major alterations of the Golgi complex parallel the death of T. cruzi epimastigotes.

Animals↗
PubMed1985

Effect of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease from pronase on glucose metabolism in mice.

Effect of an N-succinyl-L-trialanine p-nitroanilide-hydrolyzing protease (STA-protease) purified from Pronase on glucose metabolism was investigated by an intravenous injection into fasted mice. The maximum decrease in blood sugar level by STA-protease was observed 2 h after the injection of a dose of 5.0 mg/kg. The hypoglycemic activity was observed with other microbial proteases, such as Pronase E and subtilisin BPN', whereas no activity was found with the modified enzymes which almost wholly lost their proteolytic activities by the treatment with diisopropyl fluorophosphate or guanidine HCl. The increase in blood sugar level by epinephrine and the conversion of [1-14C] pyruvate into blood glucose were distinctly suppressed with STA-protease as well as 5-methoxyindole-2-carboxylic acid (MICA), an inhibitor of gluconeogenesis. A slight increase in glycogen content in diaphragm of mice was observed during 2-4 h after the injection of STA-protease, though it was markedly increased by insulin. In test of glucose tolerance, the increase in blood sugar was distinctly suppressed by insulin but not by STA-protease. Blood lactate level was not subjected to change by STA-protease, in Therefore, STA-protease may affect the in vivo metabolism of glucose in a different way from the in vitro action which has been reported to react with cell surface and consequently to mimic the actions of insulin.

Aluminum Silicates↗
PubMed2003

Participation of caspase-3-like protease in necrotic cell death of myocardium during ischemia-reperfusion injury in rat hearts.

This experimental study was designed to determine if caspase-3-like protease is activated during a short period of ischemia - reperfusion (I-R) that did not induce apoptosis, and whether protease-3-protease inhibitor could prevent myocardial I-R injury, especially necrotic cell death. The subjects were 20 isolated rat hearts; 10 were pretreated for 20 min with 100 micromol/L of the protease-3-protease inhibitor, peptide antagonist Asp-Glu-Val-Asp-CHO (DEVD) (Group D), and compared with the 10 no-pretreated hearts (Group C). The hearts were then subjected to 20, 30, 45, and 60 min of normothermic global ischemia followed by 30 min of reperfusion. Caspase-3-like protease was significantly elevated after 45 min and 60 min in ischemic hearts. Group D had reduced levels of caspase-3-like protease activity after 45 min and 60 min (302+/-58%, 378+/-69% of pre-ischemic control, respectively), as compared with Group C (542+/-74%, 689+/-85%, respectively) (p<0.05, p<0.05, respectively). Histological analysis also demonstrated a decrease in cellular damage in Group D, as the count ratio of necrotic cells with total cardiomyocytes was 38%, as compared with 78% in the control group (p<0.05). Caspase-3-like protease participated in I-R injury in rat hearts and inhibition of this protease resulted in a reduction of necrotic cell death.

Animals↗
PubMed1996

Bacillus stearothermophilus cell shape determinant gene, mreC and mreD, and their stimulation of protease production in Bacillus subtilis.

Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19. The cloned fragment stimulated production of a 37-kDa protease in B. subtilis. The nucleotide sequence of the genes and their flanking regions were identical to the B. subtilis cell shape determinant genes mreC and mreD [J. Bacteriol., 176, 6729-6742 (1992); J. Bacteriol., 176, 6717-6728 (1992)]. The mreC and mreD genes in B. subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24 h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli. The protease productions in B. subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes. The extracellular protease was purified, and the molecular mass of the enzyme was 37,000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.0 and 50 degrees C, respectively, and the enzyme was stable at pH 7-10. The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate.

Amino Acid Sequence↗
PubMed1997

Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.

Amino Acid Sequence↗
PubMed2006

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion.

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.

Amino Acid Sequence↗
PubMedDate not supplied

Extracellular proteases of Staphylococcus spp.

Bacterial proteases secreted into an infected host may exhibit a wide range of pathogenic potentials. Staphylococci, in particular Staphylococcus aureus, are known to produce several extracellular proteases, including serine-, cysteine- and metalloenzymes. Their insensitivity to most human plasma protease inhibitors and, even more, the ability to inactivate some of these make the proteases potentially harmful. Indeed, several recent studies have shown that staphylococcal proteases are able to interact with the host defense mechanisms and tissue components as well as to modify other pathogen-derived virulence factors. A tight, cell density-dependent control of proteolytic activity expression, similar to that of the well-defined virulence determinants, further suggests the role of staphylococcal proteases in the infection process. Consistently, alterations in coordinated expression of extracellular proteins markedly diminished the virulence. However, despite these data and the fact that a strain deficient in sspABC operon coding for serine (sspA) and cysteine (sspB) proteases was highly attenuated in virulence in the animal infection model, it was impossible to unambiguously demonstrate the importance of any particular protease as a virulence factor. Therefore, it can be assumed that the orchestrated expression and interaction of a variety of extracellular and cell surface proteins rather than any particular one is responsible for the staphylococcal pathogenicity and that the proteases apparently play an important role in this complex process. Such redundant mechanism is very well suited for promoting the survival of staphylococci under diverse environmental conditions encountered in the infected host.

Animals↗
PubMedDate not supplied

Protease and virulence of the extracellular products produced by Vibrio carchariae after growth on various media.

Protease and virulence of the extracellular products (ECP) of Vibrio carchariae strain EmI82KL, a causative agent of gastroenteritis in Epinephelus coioides, cultured on different media were studied. The bacteria grown on peptone agar, nutrient agar or brain heart infusion agar produced higher protease activities than that grown on tryptic soy agar (TSA) in terms of protein content. The addition of ethylenediamine di(o-hydroxyphenylacetic acid) or horse serum in TSA enhanced the ECP protease production while the addition of grouper serum apparently reduced the enzyme activity indicating the presence of protease inhibitor(s) in the fish serum. Furthermore, the use of grouper meat or peptone as a single nutrient source remarkably enhanced the production of ECP protease. Adding proteinaceous materials from animal sources (horse serum, grouper meat or peptone) on agar manifested higher ECP protease activity than that from plant source (TSA), indicating the intestine of carnivorous groupers might favour the existence, survival or infection of the bacterium. The protease was a metal-chelator-sensitive serine protease since it was inhibited by 3,4-dichloroisocoumarin and phenylmethanesulfonyl fluoride while about 80% of its activity inhibited by chelating agents (ethylene-diaminetetraacetic acid and ethylene glycol-bis(beta-amino-ethylether) N,N,N',N'-tetraacetic acid). The ECP obtained from each medium was not lethal to the groupers suggesting that the bacterium is low virulent. As grouper is carnivorous, therefore, the role of the protease played in the fish intestine probably is competing for nutrients and/or associated with the cause of edema leading to gastroenteritis.

Animals↗
PubMedDate not supplied

A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis.

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.

Animals↗
PubMed2002

The determination and use of optimized protease substrates in drug discovery and development.

There is an increasing need to rapidly determine the specificity of proteases that potentially play a role in human and animal disease. Substrates for novel proteases can be discovered by testing standard protease substrates such as oxidized insulin B-chain, by screening commercially available substrates for other proteases, or by preparing derivatives of known biological targets. The relative importance of each substrate residue can be determined through alanine-scanning, or by preparing incremental changes at one or more positions within the known substrate. More efficient methods such as coupled liquid chromatography - mass spectrometry (LC-MS) or C-terminal/N-terminal sequencing of reaction products allow the selection of improved substrates from mixtures of peptides. In other cases mixtures of substrates can be spatially segregated prior to protease treatment during chemical synthesis on beads or membranes. Positional scanning libraries can be used to find substrates for proteases with interdependent subsites, while minimizing required synthetic and screening effort. As proteases catalyze both hydrolysis and amide bond formation, acyl transfer from protease-substrate intermediates to mixtures of peptide nucleophiles provide substrate sequence information. Genetic methods including substrate phage display, retroviral display, bacterial display, and yeast alpha-halo assays combine selection with the ability to propagate selected sequences and directly deconvolute the cleaved peptide via sequencing of substrate-coding DNA. This review describes various methods for optimizing protease substrates for biological activity and the use of optimized substrates in pharmaceutical discovery.

Animals↗
PubMed2002

Cysteine proteases of malaria parasites: targets for chemotherapy.

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.

Animals↗
PubMed2004

Coumarin and isocoumarin as serine protease inhibitors.

Serine proteases are attractive targets for the design of enzyme inhibitors since they are involved in the etiology of several diseases. Within the class of serine proteases, HLE is one of the most destructive enzymes in the body. It is implicated in the promotion or exacerbation of a number of diseases including pancreatitis, acute respiratory syndrome, rheumatoid arthritis, atherosclerosis, pulmonary emphysema, and cystic fibrosis. Thrombin, a trypsin-like serine protease, plays a dual role in thrombogenesis, including fibrin formation and platelet activation. As a result, thrombin constitutes one of the most widely studied targets for antithrombotic strategy. Numerous inhibitors of serine proteases have been reported during the past three decades. Among them, coumarin-type molecules displayed a high inhibitory potency towards various serine proteases. At that time, halomethyl dihydrocoumarins have been shown to behave as the first general suicide inhibitors of serine protease. These molecules inhibit several proteases such as human leucocyte elastase, porcine pancreatic elastase, thrombin, urokinase and human plasmin. Isocoumarins are very effective as mechanism-based inhibitors of serine proteases. Pharmacomodulation on the 3-alkoxy-4-chloroisocoumarins and the 3-alkoxy-7-amino-4-chloroisocoumarins led to strong inhibitors of numerous serine proteases such as HLE, human factor XIa and XIIa, thrombin, urokinase and kallikrein. Recently, a series of coumarins characterised by an alkyl, aryl ester, amide, thioester or ketone in the position 3 and an electrophilic chloromethyl moiety in the position 6 have been developed. These compounds were found to be high inhibitors of alpha-chymotrypin, HLE and human thrombin.

Binding Sites↗
PubMed2005

Screening for protease substrate by polyvalent phage display.

Proteases are key regulators of many physiological and pathological processes [1,2], and are recognized as important and tractable drug candidates. Consequently, knowledge of protease substrate recognition and specificity promotes identification of biologically relevant substrates, helps elucidating a protease's biological function, and the design of specific inhibitors. Traditional methods for establishing substrate recognition profiles involve the identification of the scissile bond within a given protein substrate by proteomic methods such as Edman degradation. Then, synthetic peptide variants of this sequence can be screened in an iterative fashion to arrive at more optimized substrates. Even though it can be fruitful, this iterative strategy is biased toward the original substrate sequence and it is also tremendously cumbersome. Furthermore, it is not amenable to high throughput analysis. In 1993, Matthew & Wells presented a method for the use of monovalent "substrate phage" libraries for discovering peptide substrates for proteases, in which more than 10(7) potential substrates can be tested concurrently [3]. A library of fusion proteins was constructed containing randomized substrate sequences placed between a binding domain and the gene III coat protein of the filamentous phage, M13, which displays the fusion protein and packages the gene coding for it inside. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). This method allows one to rapidly survey the substrate recognition and specificity of individual or closely related members of proteases. Over the past decade, substrate phage screening has shown terrific utility in rapidly determining protease specificity and characterization of substrate recognition profile of proteases. In some cases, the structural insights of the catalytic domain were obtained from comparison of substrate specificity among closely related family of proteases [4-6]. The number of proteases (from various classes) characterized by this approach testifies to its power. Since the initial development of substrate phage library, different versions of the substrate phage cloning vectors have been constructed to further improve the utility of substrate phage display. This review will provide an overview of the construction of substrate phage display libraries, screening of substrate phage libraries, examples of application, summary and future directions.

Antibody Affinity↗