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Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.

Tissue homogenates from mouse ear skin exposed to sulfur mustard (HD, which is a military designation and probably originated from a World War I slang term 'Hun Stuff') were assayed for serine and cysteine protease activities. Enzyme activity was measured using synthetic chromogenic thioester and fluorogenic 7-amino-4-methylcoumarin (AMC) substrates. The tissue samples were obtained from animals (n = 6) at 3, 6, 12 and 24 h post-exposure from the right ear (HD exposed), whereas control samples were obtained from the left ear (treated only with dichloromethane vehicle). The samples of naive control (left and right ear) were obtained from animals that received no HD treatment (n = 3). Elastase activity was assayed with t-butyloxycarbonyl-Ala-Ala-Ala-thiobenzylester, tryptase activity with benzyloxycarbonyl-Arg-AMC and benzyloxycarbonyl-Arg-thiobenzylester, chymase activity with succinylAla-Ala-Pro-Phe-thiobenzylester and succinyl-Ala-Ala-Pro-Phe-AMC, cathepsin B activity with benzyloxycarbonyl-Arg-Arg-AMC, cathepsin H activity with Arg-AMC and calpain activity with succinyl-Leu-Tyr-AMC. The HD-exposed skin homogenates obtained at 12 and 24 h post-exposure had higher elastase activity (670% and 1900% increase) than control samples. For tryptase and calpain activities, only HD-exposed skin homogenates at 24h post-exposure showed higher activities (220% and 170% increase) when compared to the control. No differences from control were observed for HD-exposed skin obtained at 3 and 6 h post-exposure for elastase, tryptase and calpain activities. Generally, both unexposed and HD-exposed skin had distinct cathepsin B and cathepsin H enzyme activities and small chymase activity. Enzymatic assays were also performed for other serine, cysteine and metalloproteases. These data document that proteases are involved in HD skin injury and continued assessment of proteolytic activity should be useful for identifying effective antiproteases with therapeutic use in reducing or eliminating tissue injury caused by HD cutaneous exposure.

Inflammatory reactions associated with sulfur mustard (bis(2-chloroethyl)sulfide, SM)-induced skin pathology, including the release of potent proteolytic enzymes capable of destroying cutaneous connective tissue proteins, are not well defined in vivo. In this study, protease activities were measured as potential indicators of SM-induced skin injury in a hairless mouse sulfur mustard vapor exposure model. We also investigated the effects of synthetic protease inhibitors on SM-induced proteolytic activity. Skin homogenates prepared from hairless mouse dorsal skin exposed to SM vapor (1.4 g/m3) were assayed for serine and cysteine protease activities using synthetic chromogenic and fluorogenic substrates. Skin samples were obtained 24 h after SM challenge from animals exposed for either 6 or 12 min, while controls were obtained from unexposed skin. With 6 min SM-exposed skin, significantly higher elastase, tryptase, cathepsin B, cathepsin L, and calpain II enzymatic activities were detected compared ...

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].

The role played by serine proteinases with trypsin-like specificity in chondrocyte-mediated cartilage proteoglycan breakdown was investigated by use of a selective proteinase inactivator, 7-amino-4-chloro-3-(-3-isothiureidopropoxy)isocoumarin, in explant culture systems. This compound was a rapid inactivator of urokinase-type plasminogen activator. It potently inhibited interleukin 1- and tumor necrosis factor-stimulated proteoglycan release from both nasal and articular cartilage. Its less potent inhibition of basal and retinoic acid-stimulated release appeared to be due to cytotoxic effects. The functional half-life of the inactivator in culture medium was 95 min, and its concentration in cartilage was 2.5-fold higher than in the surrounding medium. Following spontaneous hydrolysis the breakdown products of the inactivator were unable to inhibit proteoglycan release. Trypsin-like activity was demonstrated by enzyme histochemistry to be chondrocyte-associated and inhibited by the serine proteinase inactivator. Cell-associated and secreted plasminogen activator activity was detected by zymography. These results suggest the involvement of a serine proteinase(s) with trypsin-like specificity, possibly urokinase-type plasminogen activator, in chondrocyte-mediated cartilage proteoglycan breakdown occurring as a result of stimulation with proinflammatory cytokines. Basal proteoglycan breakdown may occur via a different pathway. Our findings point to a pathological role for serine proteinase(s) in the development of cartilage diseases such as arthritis, possibly in a cascade which results in the activation of the enzyme(s) directly responsible for proteoglycan breakdown. It remains to be shown whether the target serine proteinase is urokinase-type plasminogen activator.

Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37°C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.

Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (k cat /K m 45,000 M −1 s −1 ) comparable to purified human GranB. RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.

Skin homogenates of hairless guinea pigs exposed percutaneously to sulfur mustard (SM) were investigated by measuring four serine protease activities (elastase, tryptase, chymase, and Asp-ase) using sensitive chromogenic substrates. The homogenate samples from skin area directly exposed to S M showed enhanced elastase activities. The tryptase (trypsin-like enzyme) activity also increased slightly; however, the chymase (chymotrypsin-like enzyme) and Aspase (cleave after aspartic acid) activities did not show any increase. The enhanced elastase activities after SM exposure indicate that inflammation is present in the SM lesions. The inhibitory potency of MeO-Suc-Ala-Ala-Pro-Val-CH2Cl (an elastase inhibitor) and two amidine derivatives (inhibitors of trypsin-like enzyme) was tested against the activities present in samples from both exposed and control tissues. The elastase inhibitor decreased the hydrolysis of elastase substrate in the samples from SM-exposed skin to a greater extent than the samples from control tissues. The two trypsin inhibitors decreased the activity in samples from exposed and control tissues equally well. These substrate and inhibitor studies facilitate the characterization of various proteases affected by SM and may be useful for elucidating the mechanism of SM-induced vesication.

Exposure to [14C]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit. Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [14C]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits.

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable ofin vitroactivation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase, like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.

A series of 3-alkoxy-7-amino-4-chloroisocoumarins with various 3-alkoxy substituents have been prepared and evaluated as inhibitors of human leukocyte elastase (HLE). In addition, a new series of acyl, urea, and carbamate derivatives of 7-amino-4-chloro-3-methoxyisocoumarin (1), 7-amino-4-chloro-3-propoxyisocoumarin (3), and 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin (6) have been synthesized. Most of the synthesized compounds are very potent inhibitors of HLE with k obs /[I] values between 10 4 and 10 6 M -1 s -1 . Hydrophobic substituents on the 7-amino position of the isocoumarin ring afford the best selectivity and inhibitory potency for HLE. In the 2-bromoethoxy series, compound 24 with a PhNHCONH 7-substituent had a k obs /[I] value of 1.2 x 10 6 M -1 s -1 , was very selective for HLE, and was the most potent inhibitor of HLE tested. Of the extended chain L-phenylalanyl derivatives, the Bz-L-Phe compound 66 with a k obs /[I] value of 1.8 x 10 5 M -1 s -1 was the most potent inhibitor of HLE in the 3-methoxyisocoumarin series and was also very selective for HLE. Our results indicate that a high degree of selectivity, along with potency, can be introduced into mechanism-based isocoumarin inhibitors

Eight new biotinylated, mechanism-based isocoumarin serine protease inhibitors have been designed and synthesized to detect, localize, and isolate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy groups are inhibitors of various serine proteases including human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of spacers between the isocoumarin moiety and the biotin moiety enhanced enzyme inhibitory potency and may also promote binding of the enzyme-inhibitor complex to avidin. The 3-alkoxy groups conferred selectivity toward different serine proteases with chymotrypsin being inhibited effectively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE and moderate inhibitors of PPE. Full enzymatic activity was regained after the immediate addition of hydroxylamine to the inactivated chymotrypsin and PPE derivatives, which indicated that a simple acyl enzyme derivative is formed initially in the inhibition reaction. Egg avidin did not effect the rate of spontaneous enzyme reactivation rate while streptavidin accelerated the reactivation reaction. PPE inhibited by 7-[[6-(biotinylamino)caproyl]amino]-4-chloro-3- ethoxyisocoumarin (BIC 5) or 7-[[6-[[6-(biotinylamino)caproyl]amino] caproyl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobilized avidin columns. Most of inhibited PPE could be eluted from the monomeric or tetrameric avidin columns but only a portion (40-70%) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic measurements for the inhibition of porcine pancreatic elastase by 7-substituted 4-chloro-.3-ethox-yisocoumarins were performed. To obtain possible explanations for the kinetic results, structures resulting from energy minimizations of inhibitor-enzyme complex structures where each inhibitor was initially positioned in 64 locations within the active site were obtained. In keeping with solution NMR studies, a positive-charged His-57 was employed. The number of low energy complex structures with Ser-195 O γ -inhibitor benzoyl ester carbonyl carbon distances ≤ 2.9 A, Ser-195 Oγ-inhibitor benzoyl ester carbonyl carbon-inhibitor benzoyl ester carbonyl oxygen angles 7gt; 91°, and the inhibitor in the oxyanion hole exhibits a direct linear relationship to ln( K i / k 3 ). The proportion of those structures that show 7-substituent H-bonding between the inhibitor and porcine pancreatic elastase exhibits a direct relationship to k 3 . Assuming a direct linear relationship to ln( k 3 ) and k 3 , finer differences in k 3 than are experimentally observed are expected. The relationship with k 3 and that with K i / k 3 are shown to be useful tools for the design of more potent 7-substituted 4-chloro-3-ethoxyisocoumarins. A novel inhibitor of this class (4-chloro-3-ethoxy-7-[(2-methyl-2-butylcarbamoyl)amino]isocoumarin) expected to be more potent is synthesized and tested. Its potency within experimental error is as predicted. Although the relationship observed with K i / k 3 involves only a twofold increase in K i / k 3 (a statistically significant increase), results with the novel inhibitor show the relationship to be valid over a four- to fivefold increase.

Various amino acid and peptide thioesters were tested as substrates for human proteinase 3 and the best substrate is Boc-Ala-Ala-Nva-SBzl with a k cal / K m value of 1.0 × 10 6 nM it-1 s it-1 Boc-Ala-Ala-AA-SBzl (AA = Val, Ala, or Met) are also good substrates with k cal / K m values of (1-4) × 10 5 M −1 s −1 . Substituted isocoumarins are potent inhibitors of proteinase 3 and the best inhibitors are 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin and 3.4-dichloroisocoumarin (DCI) with k obs /[I] values of 4700 and 2600 M −1 s −1 , respectively. Substituted isocoumarins. peptide phosphonates and chloromethyl ketones inhibited proteinase 3 less potently than human neutrophil elastase (HNE) by 1-2 orders of magnitude.

We recently reported the purification of a lymphocyte granule protein called "fragmentin," which was identified as a serine protease with the ability to induce oligonucleosomal DNA fragmentation and apoptosis (Shi, L., R. P. Kraut, R. Aebersold, and A. H. Greenberg. 1992. J. Exp. Med. 175:553). We have now purified two additional proteases with fragmentin activity from lymphocyte granules. The three proteases are of two types; one has the unusual ability to cleave a tripeptide thiobenzyl ester substrate after aspartic acid, similar to murine cytotoxic cell protease I/granzyme B, while two are tryptase-like, preferentially hydrolyzing after arginine, and bear some homology to human T cell granule tryptases, granzyme 3, and Hanukah factor/granzyme A. Using tripeptide chloromethyl ketones, the pattern of inhibition of DNA fragmentation corresponded to the inhibition of peptide hydrolysis. The Asp-ase fragmentin was blocked by aspartic acid-containing tripeptide chloromethyl ketones, while the tryptase fragmentins were inhibited by arginine-containing chloromethyl ketones. The two tryptase fragmentins were slow acting and were partly suppressed by blocking proteins synthesis with cycloheximide in the YAC-1 target cell. In contrast, the Asp-ase fragmentin was fast acting and produced DNA damage in the absence of protein synthesis. Using a panel of unrelated target cells of lymphoma, thymoma, and melanoma origin, distinct patterns of sensitivity to the three fragmentins were observed. Thus, these three granule proteases make up a family of fragmentins that activate DNA fragmentation and apoptosis by acting on unique substrates in different target cells.

The crystal structure of the acyl enzyme formed upon inhibition of porcine pancreatic elastase (PPE) by 4-chloro-3-ethoxy-7-guanidinoisocoumarin has been determined at a 1.85-A effective resolution. The chlorine atom is still present in this acyl enzyme, in contrast to the previously reported structure of the 7-amino-4-chloro-3-methoxyisocoumarin-PPE complex where the chlorine atom has been replaced by an acetoxy group. The guanidino group forms hydrogen bonds with the carbonyl group and side-chain hydroxyl group of Thr-41, and the acyl carbonyl group has been twisted out of the oxyanion hole. Molecular modeling indicates that the orientation of the initial Michaelis enzyme-inhibitor complex is quite different from that of the acyl enzyme since simple reconstruction of the isocoumarin ring would result in unfavorable interactions with Ser-195 and His-57. Molecular models were used to design a series of new 7-(alkylureido)- and 7-(alkylthioureido)-substituted derivatives of 3-alkoxy-7-amino-4-chloroisocoumarin as PPE inhibitors. All the 3-ethoxyisocoumarins were better inhibitors than those in the 3-methoxy series due to better interactions with the S1 pocket of PPE. The best ureido inhibitor also contained a tert-butylureido group at the 7-position of the isocoumarin. Due to a predicted interaction with a small hydrophobic pocket on the surface of PPE, this isocoumarin and a related phenylthioureido derivative are among the best irreversible inhibitors thus far reported for PPE (kobs/[I] = 8100 M-1 s-1 and 12,000 M-1 s-1). Kinetic studies of the stability of enzyme-inhibitor complexes suggest that many isocoumarins are alkylating the active site histidine at pH 7.5 via a quinone imine methide intermediate, while at pH 5.0, the predominant pathway appears to be simple formation of a stable acyl enzyme derivative.

SummaryArginine thiobenzyl esters are convenient chromogenic substrates of factor Vila (Z-Arg-SBzl, kcat/KM = 1,600 M−1 s−1) and were used to study the kinetics of inhibition of factor Vila 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 Vila 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-isothiurei-dopropoxy) 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 pM. 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.

In granules of hematopoetic cells, dipeptidyl peptidase I (DPPI) processes inactive proenzymes into active enzymes, e.g., lymphocyte progranzyme A. Our goal was to develop irreversible inhibitors of intracellular DPPI. First, we identified inhibitors with aqueous stability. Then we determined which inhibitors were nontoxic, could enter cells and inactivate intracellular DPPI. We screened nine dipeptide vinyl sulfone (VS) inhibitors (kobs/[I]72 M-1 s-1) and found six that were nontoxic. Four affected intracellular DPPI at25 microM. These compounds contained only uncharged amino acid residues; the two less reactive compounds contained charged Glu residues. The best one, Leu-Phe-VS-CH3, inactivated DPPI in cells with an ID50 of approximately 5 microM. This inhibitor was not the best inhibitor of purified DPPI. Longer aqueous stabilities were important predictors of cellular efficacy. Leu-Phe-VS-CH3 had a half life of 97 min at the pH of the extracellular medium (7.5) and 1302 min at pH 5.5 (the intracellular environment of DPPI). This VS had no direct effect on granzyme activities. In contrast, the diazomethyl ketone inhibitor Gly-Phe-CHN2 inhibited chymase activity. Several good intracellular DPPI VS inhibitors lacked reactivity with cathepsins B, H and L. In conclusion, we have identified DPPI inhibitors suitable for cellular applications.

Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed granzymes. Both perforin and granzymes are involved in the lytic activity. Although the biological functions of most granzymes remain to be resolved, granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other granzymes. The first granzyme crystal structure remains to be resolved, but in the interim, molecular models of granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of granzymes. The different granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.

Serine proteases (granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant granzyme H has chymotrypsin-like (chymase) activity, the first report of a human granzyme with this proteolytic specificity. Recombinant 32-kDa granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length granzyme H cDNA. The granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind granzyme B. This suggests a hitherto unsuspected intracellular function for granzyme H.

Amino acid and peptide thioesters which contained Arg or Lys in the P 1 position were tested as substrates for rat skin tryptase, and the kinetic constants k cat / K M for the better substrates such as Z-Aba-Arg-SBzl, and Z-Gly-Arg-SBzl were over 5,000,000 M −1 s −1 . The inhibitory potency of arginine fluoroalkyl ketones, benzamidine derivatives, and substituted isocoumarins containing basic functional groups was studied with rat skin tryptase, human lung tryptase, human skin tryptase, and bovine trypsin. 1-Naphthoyl-Arg-CF 3 was the best arginine fluoroalkyl ketone reversible inhibitor for rat skin tryptase with a K I of 0.9 μM. 1-(4-Amidinophenyl)-3-(4-phenoxyphenyl) urea showed competitive inhibition against bovine trypsin and rat skin tryptase with K I values of 2 and 4 μM, respectively. Isocoumarin derivatives with isothioureidoalkoxy substituents at the 3 position were potent irreversible inhibitors of these three tryptases with k obs /[ I ] values of 10 4 -10 5 M −1 s −1 . 4-Chloro-3-(2-isothioureido)ethoxy-7-phenylcarbamoylaminoisocoumarin and 7-benzylcarbamoylamino-4-chloro-3-(3-isothioureido)propoxyisocoumarin inactivated trypsin and formed stable trypsin-inhibitor complexes which regained less than 8% of activity upon standing in the pH 7.5 buffer and regained 30-75% of activity in the presence of 0.3 M NH 2 OH after 1 day, In contrast, the complexes with rat skin tryptase regained activity rapidly, indicating differences in the inhibition mechanism and active site structures of these related enzymes.

Peptide thioesters are sensitive substrates of various serine peptidases and metalloendopeptidases. Thioester substrates generally have high enzymatic hydrolysis rates and low background hydrolysis rates, and the hydrolysis rates can be easily monitored in the presence of thiol reagents such as 4,4′-dithiodipyridine or 5,5′-dithiobis (2-nitrobenzoic acid). Peptide thioester substrates have been invaluable for the study of enzyme specificity and enzyme inhibitors, especially in cases where no other practical synthetic substrates are available. Tripeptide substrates of the type Boc-Ala-Ala-AA-SBzl, where AA is nearly all of the 20 common amino acids, have now been synthesized and should be useful for the subsite mapping of new serine peptidases and the study of crude cell preparations containing serine peptidases.

Arg-containing peptide chloromethyl ketones including D-Phe-Pro-Arg-CH2Cl derivatives have been synthesized and tested as inhibitors for thrombin and several blood coagulation enzymes. The parent compound, D-Phe-Pro-Arg-CH2Cl is still the best thrombin inhibitor in the series with kobs/[I] value of 10(7) M-1s-1. Extension by one amino acid (Phe or Gly), or a peptide moiety (ClCH2-Arg-Pro-D-Phe-CO-CO-, ClCH2-Arg-Pro-D-Phe-CO-(CH2)3-CO-, where-indicates a reversed amino acid residue, -CO-CHR-NH-) on the N-terminus of D-Phe-Pro-Arg-CH2Cl reduces the inhibition constant by 1-2 orders of magnitude, which indicates the importance of a free amino group at the N-terminus. The tripeptide D-Phe-Pro-Arg-CH2Cl and related tetrapeptide inhibitors inhibit thrombin more potently than factor IXa and plasma kallikrein by 2-5 orders of magnitude. Z-Arg-CH2Cl and Phe-Phe-Arg-CH2Cl which contain a large hydrophobic group at the P2 site inhibit thrombin poorly. All the peptide chloromethyl ketones inhibit plasma kallikrein moderately with kobs/[I] values of 10(2)-10(3) M-1s-1 but inhibit factor IXa poorly (kobs/[I]20 M-1s-1). Conjugates of albumin with the bis chloromethyl ketones [(CO-D-Phe-Pro-Arg-CH2Cl)2, (CH2)3-(CO-D-Phe-Pro-Arg-CH2Cl)2] were prepared and are potent thrombin inhibitors. These conjugates are model compounds for developing specific thrombus-bound thrombin inhibitors which may have therapeutic application in the treatment of coagulation disorders.

A series of dipeptides which contained phosphonate analogs of proline and piperidine-2-carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite specific. The best inhibitor in the series is Ala-PipP(OPh-4-Cl)2 (13), which has a k(inact) of 0.353 s-1 and KI of 236 microM. The DPP-IV inhibitors Ala-ProP(OPh)2 (6), Ala-ProP(OPh-4-Cl)2 (12), and Ala-PipP(OPh-4-Cl)2 (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds 12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline phosphonate residue (PipP) or a Pro phosphonate residue (ProP) at the P1 site are stable in a pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala-PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of excess inhibitor and incubation in buffer for 1 day. Since the phosphonate inhibitors are specific toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing the biological functions of DPP-IV and may be useful therapeutically in the prevention of the rejection of transplanted tissue.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

Reactivity of proteinase 3 (PR3) was tested against various amino acid and thioester substrates. The best substrate is Boc-Ala-Ala-Nva-SBzl with a kcat/Km value of 1.0 x 10(6) M-1.s-1. We also studied the effect of C-ANCA on PR3 proteolytic activity towards elastin and inactivation by alpha 1-antitrypsin (alpha 1AT). C-ANCA IgG from 8 patients with active Wegener's granulomatosis were tested and found to inhibit elastin degradation by PR3 and to prevent the inactivation of PR3 by alpha 1AT.

Serine proteases initiate several complex biochemical responses to sudden, life-threatening situations. The immediate and localized activation of coagulant serine proteases prevents mammals from bleeding to death. The immediate activation of complement serine proteases kills common bacteria and prevents bacterial sepsis. The granules of cytotoxic lymphocytes (CTL) are packages which contain a unique subfamily of serine proteases and the pore-forming protein, perforin. These sequestered serine proteases are induced during the differentiation of lymphocytes to become competent killer cells (Lobe et al., 1986; Gershenfeld and Weisman, 1986). In vivo, lymphocytes laden with toxic granules accumulate at sites of graft rejection (Mueller et al., 1988). Exocytosis exposes the granule contents to high extracellular concentrations of calcium, the cation required for cell lysis. In vitro, freshly isolated natural killer (NK) lymphocytes and lymphokine-activated T lymphocytes release these protease-packed granules during killing. Proteases alone do not mediate lysis. Perform is necessary for cell lysis. The proteases undoubtably perform significant functions immediately associated with cytolysis.

Analogs of Ep-475 (2a), designed to explore the role played by the carboxylate in epoxysuccinate thiol protease inhibitors, have been synthesized and tested as inhibitors of papain and cathepsin B. Papain and cathepsin B are rapidly inactivated by carboxylates 2a and 6a, but are inactivated much more slowly by 2b-2f, 6c, and 6f, in which the carboxylate is absent or replaced by an amide, ester, or ketone. This order of reactivity contrasts with the inherent reactivity of substituted epoxides toward a non-enzymatic thiolate, previously shown to decrease in the order: COCH3CO2CH3CONH2HCO2H. The results suggest that electrostatic attraction between the carboxylate of the inhibitor and protonated His159 of papain facilitates docking of the inhibitor in the active site of the enzyme, a conclusion reached previously from X-ray crystallographic structures of epoxysuccinates bound to papain. The most reactive isoleucine analog, 6a, was significantly less reactive than leucine-containing Ep-475 (2a), while the less reactive isoleucine derivatives, 6c and 6f, were similar in reactivity to the corresponding leucine derivatives, 2c and 2f, respectively.

A step leading to the formation of the covalent complexes between porcine pancreatic elastase (PPE) and 7-[(alkylcarbamoyl)amino]-4-chloro-3-ethoxyisocoumarins (alkylHNCO-EICs) is the formation of the noncovalent Michaelis complex. No average structures are available for the Michaelis complexes of PPE with alkylHNCO-EICs. We present the results of an initial step in obtaining these structures and have determined kinetic constants as well. The kinetic results indicate that formation of the Michaelis complex is what differentiates the effectiveness of these inhibitors in inactivating PPE. The structural and kinetic results together suggest that the structure of the Michaelis complex is necessary for the design of potent alkylHNCO-EIC inhibitors of PPE. Two novel alkylHNCO-EICs are predicted to be the best inhibitors of this series. An alternate mechanism for serine protease inhibition is also proposed. Evidence for, and studies that may add support to, the hypothesized mechanism are discussed.

Chronic Obstructive Pulmonary Disease (COPD), comprised of chronic bronchitis and pulmonary emphysema, is a serious health problem in the world. In the USA, COPD caused more than 70,000 deaths in 1986 and more than 10,000,000 Americans suffered from the disease.1 The association between severe α1protease inhibitor (α1PI) deficiency and emphysema has led to the hypothesis that an elastase-antielastase imbalance causes emphysema.2,3 Human neutrophil elastase (HNE) is most likely the cause of emphysema in both smokers and non-smokers, although the evidence supporting the elastase-antielastase hypothesis is largely indirect in smokers with normal protective levels of α1PI. Homogenates of leucocytes as well as highly purified preparations of HNE produce emphysema in experimental animals and only elastolytic enzymes will induce experimental emphysema. In addition, neutrophils are increased 4–5 fold in the lungs of smokers and it is postulated that α1PI is inactivated by powerful oxidizing agents in the cigarette smoke and PMNs contributing to the elastase-antielastase imbalance. Other proteases including human proteinase 3 and macrophage elastase may also play a role in lung destruction in emphysema. Nevertheless, the majority of investigators in the field believe that a human neutrophil elastase-antielastase imbalance plays the major role in the pathogenesis of emphysema.

Thrombin has a central regulatory role in hemostasis and is formed by both the intrinsic and extrinsic pathways of blood coagulation (Fenton, 1986, 1981). The major function of this serine protease is the cleavage of fibrinogen to form fibrin clots, but thrombin also activates factors V, VIII, XIII and protein C which are important in the control of hemostasis and thrombosis. In addition, thrombin can stimulate platelet secretion and aggregation in blood, and mediate other nonhemostatic cellular events. Our understanding of the various biological roles of thrombin has been facilitated by the development of convenient chromogenic and fluorogenic assays for the measurement of thrombin activity; and synthetic, low-molecular-weight inhibitors which are important probes for the study of thrombin’s mechanism, specificity, and function in the coagulation system.

We show that chymotrypsin-like, as well as trypsin-like, proteases are in granules isolated from cytolytic lymphocytes by the capacity of the granules to hydrolyze the peptide substrates Z-Phe-Leu-Phe-SBzl and Z-Ala-Gly-Arg-SBzl, respectively. We report protease inhibitors that can abrogate or delay granule-mediated cytolysis. Two mechanism-based isocoumarin serine protease inhibitors and Z-Gly-Leu-Phe-CH2Cl completely abrogated granule cytolysis. Lima bean and soybean trypsin inhibitors and chymostatin delayed but did not prevent this cytolysis. These data represent the first use of the powerful isocoumarin inhibitors as biological probes and indicate that lymphocyte serine proteases participate in the granule cytolytic process.

A series of new azapeptide p-nitrophenyl esters containing a variety of P1 aza-amino acid residues have been synthesized, and the reaction of these azapeptides with chymotrypsin A alpha, subtilisin BPN', subtilisin Carlsberg, and human leukocyte cathepsin G at pH 4-7 has been studied. These azapeptides were found to be very useful as active site titrants and inhibitors of serine proteases with chymotrypsin-like specificity. Stable acyl derivatives of serine proteases are formed in the reaction with azapeptides and can be used for future crystallographic investigations. The effects of changing the nature of the P1' leaving group (-ONp, -OPh, -OCH2CF3, -OEt) for these azapeptides was also investigated. N-Acetyl-L-alanyl-L-alanyl-alpha-azanorleucine p-nitrophenyl ester can be used as an active site titrant for human leukocyte cathepsin G and N-acetyl-L-alanyl-alpha-azaphenylalanine p-nitrophenyl ester is a suitable titrant for chymotrypsin A alpha, subtilisins, or cathepsin G.

Granules that are potently cytolytic in vitro can be obtained from cytotoxic lymphocytes that kill virally infected cells and tumor cells. These granules contain pore-forming proteins and several serine proteases. Here we indicate that at least two different proteases participate in the lysis mediated by granule proteins from RNK-16 rat leukemia cells. We report twelve different mechanism-based or “suicide” isocoumarin serine protease inhibitors which have different 3- and 7-substituents that confer selectivity and reactivity towards either the chymotrypsin- (“chymase”) or trypsin-like (“tryptase”) protease activities of RNK-16 cells. Second order inhibition rates of inactivation (k obsd /[I]) for the RNK-16 granule proteases ranged between 164 and 22,640 M −1 s −1 . These new, specific and highly reactive isocoumarin serine protease inhibitors also abrogated the cytolysis mediated by lymphocyte granule proteins. The eight inhibitors with large hydrophobic or basic substituents that conferred chymase or tryptase specificities were more effective at inactivating lytic function than the four elastase-directed inhibitors with smaller substituents. All twelve new isocoumarin inhibitors blocked cytolysis at lower concentrations than 3,4-dichloroisocoumarin, a potent general mechanism-based serine protease inhibitor that also blocks RNK-16 granule protease activities and lysis.

The reaction of a series of azapeptides with porcine pancreatic (PP) elastase and human leukocyte (HL) elastase has been studied and a series of new inhibitors and active site titrants were found for both PP elastase and HL elastase. Azapeptide p-nitrophenyl esters acylate both HL and PP elastase to form stable acylenzymes, which can be used for crystallographic studies. We have investigated the effect of a P1, P2, or P3 aza-amino acid residue on the reactivity of azapeptides with elastase. We have also studied the effect of changing the nature of the P1' leaving group and other portions of azapeptide structure. N-Acetyl-L-alanyl-L-alanyl-alpha-azaalanine p-nitrophenyl ester, N-acetyl-L-alanyl-L-alanyl-alpha-azanorleucine p-nitrophenyl ester, and N-acetyl-L-alanyl-L-alanyl-alpha-azanorvaline p-nitrophenyl ester are suitable titrants for either PP or HL elastase.

A new apparatus was constructed to tritiate biological molecules using mass analysed tritium ion beams. The magnetic sector of this device was constructed of high field Sm-Co permanent magnets. Mass analyzed ions of desired kinetic energy were impacted on an involatile solid and 3H exchanged for H. The 3H2+ ions were found to be more reactive than 3H2+ ions. The sensitive peptide aldehyde leupeptin was utilized to test the efficacy of mass analyzed ion beam tritiation and leupeptin was not decomposed by this process.

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.

Rat RNK-16 leukemia cells kill YAC-1, which are the cells lysed by rodent natural killer lymphocytes. We found chymotrypsin-like proteinase (‘chymase’) activity in the RNK-16 dense granules that also contain cytolytic activity. The chymase activity hydrolyzed the thiobenzyl peptide substrate Suc-Phe-Leu-Ohe-SBzl and, in comparison to RNK-16 tryptase activity, was selectively inhibited by three different types of serine proteinase inhibitors. The selective inhibitors were the fungal aldehyde chymostatin, the chloromethylketone Z-Gly-Leu-Phe-CH 2 Cl, and the mechanism-based or ‘suicide’ inhibitor 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin. These proteinase inhibitors also blocked RNK-16 granule-mediated cytolysis. Chymostatin, a reversible inhibitor, delayed granule-mediated cytolysis, whereas the irreversible chloromethylketone and isocoumarin proteinase inhibitors completely abrogated granule-mediated cytolysis. The two irreversible inhibitors displayed biphasic inhibition of the chymase activity, indicating that at least two chymases are present in the granules. By Northern blot analysis, we found that RNK-16 mRNA hybridized strongly with a cDNA probe of CCPI, a mouse cytotoxic T lymphocyte serine proteinase gene. These data imply that chymase activity in the cytotoxic granules is important for cytolytic function and is likely to belong to a new subfamily of serine proteinases.

A serine protease (Mr 70,000 to 75,000) appearing in sheep lung lymph after capillary damage induced by Escherichia coli endotoxin, oleic acid, or air emboli, was studied for its specificity toward a series of synthetic peptide and thioester substrates containing an Arg residue in the P1 position. High specificity constants (kcat/Km) were generally obtained with substrates having two or more basic amino acid residues, and with those having a Gln residues in the P2 position. Secondary enzyme-substrate interactions at sites more removed from the scissile bond are of importance, since a few peptides with two basic residues were hydrolyzed slowly, and the site of cleavage of natural peptides was influenced by the amino acid sequence beyond the immediate vicinity of the hydrolyzed bond. The properties of the enzyme and its pattern of specificity distinguish it from enzymes of the clotting cascade, from components of the complement system, and from lung and skin tryptase. The enzyme was inactivated by p-amidinophenylmethanesulfonyl fluoride and by a series of mechanism-based isocoumarin derivatives, the most potent inhibitor being 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Enzyme solutions inactivated by reaction with isocoumarin inhibitors could be completely reactivated after 30 h by treatment with hydroxylamine at neutral pH. Formation of a stable sheep lymph acyl enzyme--in contrast to thrombin and other trypsin-like enzymes--is not followed by alkylation of an active site nucleophile that leads to irreversible enzyme inactivation. The high activity toward substrates with two basic residues suggests that the enzyme may potentially function in processing of precursors of bioactive peptides.

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.