Inhibitors Of Trypsin-like Enzymes Research Articles

A convenient synthesis of new α-aminoalkylphosphonates, aromatic analogues of arginine as inhibitors of trypsin-like enzymes

A simple and efficient protocol for the synthesis of new N-protected α-aminoalkylphosphonic diphenyl esters––aromatic analogues of arginine––is presented. The crucial, guanylating step was achieved using S-ethyl- N,N′-di(Boc)isothiourea in chloroform and in the presence of Et 3N and HgCl 2. Deprotection of the derivatives obtained was performed using trifluoroacetic acid in CH 2Cl 2 or hydrogenolysis over Pd/C. The products are potent inhibitors of trypsin.

Synthesis and kinetic studies of an amidine-containing phosphonofluoridate: a novel potent inhibitor of trypsin-like enzymes

The amidine-containing α-aminoalkylphosphonofluoridate 3 (Cbz-(4-AmPhGly)P(OPh)(F)) is a very potent inhibitor of trypsin-like enzymes. It was prepared by hydrolyzing the corresponding phosphonate diphenyl ester 4 followed by reaction of fluoride with the phosphonochloridate prepared from the intermediate phosphonic acid monoester 5. Compound 3 is the most potent amidine-containing organophosphorus inhibitor yet reported for trypsin-like enzymes. It inhibits trypsin and thrombin with second-order rate constants (kobs/[I]) of 2.6×105M−1s−1 and 1.0×105M−1s−1, respectively, showing a 130-fold and a 1250-fold rate enhancement over the corresponding diphenyl ester (4). It also inactivates trypsin 2 orders of magnitude more potently than simple phosphonofluoridates such as DFP,[1] Sarin and Soman. The phosphonofluoridate 3 does not inhibit other serine proteases such as porcine pancreatic elastase (PPE) and the esterase acetylcholinesterase (AChE). The phosphonofluoridate 3 is hydrolyzed rapidly in buffer solution and has a t1/2 of 4.5s at pH 7.5.

Fibrinogen-like substance and thrombin-like enzyme in seminal plasma: coagulation system of human semen.

This investigation was conducted to examine the presence of fibrinogen-like substance and thrombin-like enzyme in human semen (human seminal plasma) after liquefaction. The human seminal plasma contained small amounts of respective substances which are absorbed on anti-fibrinogen and anti-thrombin III affinity columns, respectively. The thrombin-like enzyme with Gly-Pro-Arg-pNA amidolytic activity was inhibited by human seminal plasma trypsin-like enzyme inhibitor (HSP-TI) and antithrombin III. The fibrinogen-like substance reacted with the thrombin-like enzyme, forming a fibrin-like substance. It would appear that certain aspects of the coagulation process in human semen constitute the same process as the final stage of the blood coagulation system.

5462964 Dipeptide boronic acid inhibitors of trypsin-like enzymes : Fevig John; Abelman Matthew M; Amparo Eugene; Cacciola Josep; Kettner Charles A; Pacofsky Gregory J; Wang Chia-Lin Lincoln University, PA, United States assigned to The Du Pont Merck Pharmaceutical Company

5462964 Dipeptide boronic acid inhibitors of trypsin-like enzymes : Fevig John; Abelman Matthew M; Amparo Eugene; Cacciola Josep; Kettner Charles A; Pacofsky Gregory J; Wang Chia-Lin Lincoln University, PA, United States assigned to The Du Pont Merck Pharmaceutical Company

Inhibition of a Tumour Protease with 3,4-Dichloroisocoumarin, Pentamidine-Isethionate and Guanidino Derivatives

Guanidinobenzoatase (GB) is a cell surface proteolytic enzyme capable of degrading fibronectin, and is associated with tumour cells and cells capable of migration. The location of active GB in sections has been demonstrated with 9-aminoacridine (9-AA), a competitive inhibitor of GB. 3,4-Dichloroisocoumarin (3,4-DCI) and pentamidine isethionate (PI) are inhibitors of trypsin-like enzymes. It has now been demonstrated that 3,4-DCI, PI, and guanidino derivative compounds are significant inhibitors of GB, on the surfaces of lung squamous cell carcinoma cells in frozen sections and free GB in solution. Dexamethasone acetate (DMA) and medroxy-progesterone (MP) did not show any significant inhibition of GB activity. These molecules lack a reactive chloride or guanidino groups and are thought to react at the nuclear level, rather than directly on this cell surface protease. Kinetic studies have shown that 3,4-DCI, PI and guanidino derivatives are reversible competitive inhibitors of GB, as determined in vitro on the purified enzyme. The inhibition resulting with 3,4-DCI was a time-dependent process. It is suggested that these inhibitors interact with GB by binding to its active site, resulting in the formation of enzyme-inhibiter complexes (GB-I). The GB-I complexes can be dissociated with SDS treatment, resulting in the regain of GB activity.

Trypsin inhibitors prevent the progesterone‐initiated increase in intracellular calcium required for the human sperm acrosome reaction

Inhibitors of trypsin-like enzymes, benzamidine hydrochloride and 4'-acetamidophenyl 4-guanidinobenzoate (also an inhibitor of other serine proteases), were tested for their effects on the acrosome reaction (AR) of human sperm initiated by progesterone or the calcium ionophore ionomycin. The AR was assayed by indirect immunofluorescence and transmission electron microscopy. The trypsin inhibitors, when added 10 min prior to stimulation by progesterone, significantly inhibited the AR in comparison with progesterone treatment alone. Transmission electron microscopic examination of the sperm after progesterone treatment indicated that the inhibitors blocked the membrane fusion events of the AR. By contrast, when ionomycin (at final concentrations of 3 microM) was added to sperm preincubated in inhibitors, sperm underwent morphologically normal AR, acrosomal matrix loss was not inhibited, and the percentage of acrosome-reacted sperm was the same as that obtained in the absence of inhibitors. Using the cell calcium indicator fura-2, we further demonstrated that both trypsin inhibitors prevented the progesterone-stimulated rise in intracellular Ca2+ ([Ca2+]int) required for the AR, but did not affect [Ca2+]int in unstimulated sperm. These results suggest that sperm trypsin-like activity may be directly or indirectly involved in increasing sperm [Ca2+]int during stimulation by progesterone.

Compound 48/80-induced secretion of histamine from rat peritoneal mast cells depends on a tryptase controlled step also leading to chymase activity

Compound 48/80 caused activation of rat mast cell tryptase and chymase along with the release of histamine. Following low speed centrifugation, tryptase remained essentially in the supernatant while chymase sedimented. Enzyme activities as well as histamine release were inhibited by phenylmethyl-sulfonyl fluoride and tosyl-lysine chloromethyl ketone, which are respectively unspecific and specific inhibitors of trypsin-like enzymes. Chymostatin inhibited chymase, but not the release of histamine by 48/80. Tryptase activity thus seems to be essentially involved in the mechanism of histamine release, while chymase activity may be merely one of its accompanying events.

Thioester Chromogenic Substrates for Human Factor VIIa: Substituted Isocoumarins Are Inhibitors of Factor VIIa and In Vitro Anticoagulants

Arginine thiobenzyl esters are convenient chromogenic substrates of factor VIIa (Z-Arg-SBzl, Kcat/KM = 1,600 M-1 s-1) and were used to study the kinetics of inhibition of factor VIIa by several mechanism-based isocoumarin inhibitors of trypsin-like enzymes. Isocoumarin derivatives substituted with a 7-guanidino or 3-isothiureidopropoxy group were good inhibitors of factor VIIa and acted as anticoagulants in human and rabbit plasma. With normal citrated human plasma, 4-chloro-3-ethoxy-7-guanidinoisocoumarin (3) and 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin (ACITIC, 6) prolonged the prothrombin time (PT) ca. two-fold and prolonged the activated partial thromboplastin time (APTT) more than 4.5-fold at 20-30 microM. Both compounds had smaller effects in rabbit plasma. The short half-life of ACITIC and related isocoumarins in plasma should make these compounds uniquely useful as anticoagulants in therapeutic situations where it is desirable to have anticoagulant effects for a short defined time period.

Diisopropyl Fluorophosphate Labeling of Sperm-Associated Proteinases1

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

Inhibition of Trypsin-Like Enzymes on Cells with Rhodamine-Aprotinin

Aprotinin, a polypeptide inhibitor of trypsin-like enzymes, has been labelled with rhodamine. Rhodamine-aprotinin inhibits trypsin in free solution in an identical manner to aprotinin. Rhodamine-aprotinin binds to trypsin-like enzymes on cells in formaldehyde fixed wax embedded sections. This technique has been used to locate cells possessing trypsin-like enzymes by means of fluorescent microscopy. In the present study we have used this technique to locate tumour cells.

The effect of serine esterase inhibitors on ionophore-induced histamine release from human pulmonary mast cells

The serine proteases tryptase and chymase are present in human pulmonary mast cells. About 10-100 times more tryptase than chymase is found in these cells. However, a clear physiological role for both enzymes remains to be elucidated; angiotensin processing has been proposed as one possible function of chymase. A dose-dependent inhibition of A23187-induced histamine release from dispersed human lung mast cells was observed after pretreatment with the serine protease inhibitor diisopropylfluorophosphate (DFP) or the chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) but not with the trypsin-like enzyme inhibitor N-tosyl-L-lysine chloromethylketone (TLCK). These results indicate that a chymase is probably an important factor in a late phase of human lung mast cell activation.

Cloning, Expression and Release of a Vibrio alginolyticus SDS-resistant Ca2+-dependent Exoprotease in Escherichia coli

SUMMARY: A Vibrio alginolyticus SDS-resistant, Ca2+-dependent serine exoprotease gene, cloned on a 7-1 kb DNA fragment in the recombinant plasmid pVP100, was expressed from its own promoter in Escherichia coli. Although active exoprotease was produced by late stationary phase E. coli(pVP100) cultures after 15 h incubation in proteinaceous medium containing Ca2+, transcription and translation of the exoprotease occurred before 6 h, during exponential growth. The cloned exoprotease was synthesized as a pool of inactive precursor molecules during exponential growth, and released as active exoprotease 8 h later by a process which did not require protein synthesis or involve cell lysis. Release of the exoprotease by E. coli(pVP100) was not inhibited by o-phenanthroline, quinacrine or cerulenin. Supernatant samples from E. coli(pVP100) cultures contained two SDS-resistant exoproteases with apparent M r values of approximately 54000 and 39000. The cloned exoprotease activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes.

Putative irreversible inhibitors of trypsin-like enzymes: analogues of basic amino acids bearing a carbodi-imide moiety

Putative irreversible inhibitors of trypsin-like enzymes: analogues of basic amino acids bearing a carbodi-imide moiety

Production and Activation of an SDS-resistant Alkaline Serine Exoprotease of Vibrio alginolyticus

Vibrio alginolyticus produced an extracellular SDS-resistant protease (protease A) with an apparent M r of approximately 54000 when cultured in complex, proteinaceous media. Ca2+ was required for the activation and stability of this protease. Its activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes. Optimum protease activity occurred under alkaline conditions. Two SDS-resistant exoproteases, B and C, with apparent M r values of approximately 41000 and 37000 respectively, were also produced in complex proteinaceous media. Dialysis of cell-free supernatant samples, which contained predominantly protease A, against distilled water, resulted in increased B and C activity. Production of protease A, B and C activities was inhibited by o-phenanthroline, quinacrine and lack of aeration.

Detection and characterization of extracellular proteases in Butyrivibrio fibrisolvens H17c

The detection and approximate molecular weights of extracellular serine protease isoenzymes produced by Butyrivibrio fibrisolvens H17c were determined by gelatin-PAGE. Nine bands of protease activity with apparent molecular weights of approximately 101000, 95000, 87000, 80000, 76000, 68000, 63000, 54000 and 42000 were detected after gelatin-PAGE of supernatants from exponential phase cultures. A tenth serine protease band with an apparent molecular weight of approximately 32000 was detected in stationary phase cells. The activities of all ten protease bands were inhibited by a serine protease inhibitor but their activities were not affected by inhibitors of trypsin-like enzymes or metallo-, sulphydryl-and carboxylproteases. The activity of all ten exoprotease bands was optimal between pH 6.0 and 7.5. The ten exoprotease bands were only detected in media containing trypticase or casamino acids as nitrogen sources. Production of the ten protease bands was not affected by the carbohydrate source.

Inhibition of phorbol-ester-induced adhesion of differentiating human myeloid leukemic cells by pentamidine-isethionate

Human myeloid leukemia cells can be induced to differentiate into macrophage-like cells by various phorbol esters, particularly 12-O-tetradecanoyl-phorbol-14-acetate (TPA). In this study, the effect of several known protease inhibitors on TPA-induced differentiation of human acute promyelocytic leukemia cells (line HL-60) was tested. Among the tested compounds, only pentamidine-isethionate (PI), an inhibitor of trypsin-like enzymes, prevented one early marker of differentiation, e.g. cell adherence to plastic and glass surfaces. However, PI failed to affect other markers of differentiation and did not inhibit readherence of scraped and resuspended TPA-treated cells. Exposure to TPA resulted in a decrease in the cellular alkaline proteolytic activity and an increase in the acid proteolytic activity. PI further inhibited the residual activity of the alkaline protease in the 36,000 g pellet fraction of the TPA-treated cells, but did not reduce this activity in control cells. The present results indicate, on the basis of the differential effects of PI, that the emergence of differentiation markers in HL-60 cells following exposure to TPA is independent of the induction of adherence.

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus.

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

Phorbol ester-induced adhesion of murine erythroleukemia cells: possible involvement of cellular proteases.

Phorbol ester tumor promoters produce a rapid increase in adhesiveness of murine erythroleukemia (MEL) cells. Following treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and other tumor promoters, these cells adhere to the surface of the culture dish or become agglutinated to each other. Structurally related compounds which are devoid of tumor promoting activity failed to induce agglutination of MEL cells. Pentamidine isethionate (PI) and tosylamide-phenylethyl-chloromethyl ketone, two known inhibitors of trypsin-like enzymes, prevent the phorbol esters-induced adherence and agglutination. A short exposure to TPA results in an increase in protease activity at the alkaline pH range. This TPA-induced proteolytic activity is inhibited by PI. Induction of erythroid differentiation by hexamethylene-bisacetamide is associated with a decrease in TPA-induced cell adhesion and TPA-induced proteolytic activity. Taken together, these results suggest the participation of an alkaline proteolytic activity in the membranal changes evoked by phorbol esters.

CHANGES OF THE OPIOID PEPTIDE CONTENT BY NOXIOUS STIMULI IN RAT INCISOR PULP

It has been reported that many cells with granules containing met-enkepha-lin(ME)-like peptides were observed in dog canine pulp (Kudo et al.,1981). Since incisor pulp of the rat also contained ME-like peptides, it was examined whether noxious stimuli caused an increase of the ME-like peptide content in the pulp as suggested in the dog canine pulp. Although electric stimulation(ES) to the pulp markedly increased the peptide content in pulp, only cavity preparation (C.P.) without ES also clearly increased the peptide content. The increased content of peptides by CP. was not influenced by cycloheximide, but decreased by FOY-305, a trypsin-like enzyme inhibitor, and enhanced by Captopril and decreased by infusion of saline into the pulp cavity. The peptide content of crude extract from intact pulp markedly increased with trypsin but not with carboxypeptidase B digestion, suggesting that processing of ME-like peptides in pulp is different from that of opioid peptides in adrenal medulla. On the other hand, all fractions(fr.) of crude extract from intact pulp by HPLC were digested with both enzymes. While HPLC profile of the non-digested fr. showed three peaks of ME-immuno-reactivity(ME-IR), ME, ME-Arg-Phe and unidentified one, that of the digested fr. did not only show a new peak of ME-IR but also an increase of ME-IR at ME-Arg-Phe peak. Furthermore, by comparison of HPLC profiles of crude extract from CP. pulp with that from intact pulp, it was shown that the contents of ME, ME-Arg-Phe and leu-enkephalin in CP. pulp increased much more that those contents in intact pulp. From these results, it was suggested that noxious stimuli led to activation of trypsin-like enzyme(s) followed by processing from precursor protein to opioid peptides, which were degraded by angiotensin converting enzyme, in pulp of the rat.