Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

Recent advances in the synthesis,design and selection of cysteine protease inhibitors

Inhibition of cysteine proteases is emerging as an important strategy for the treatment of a variety of human diseases. Intense efforts involving structure-based inhibitor design have been directed toward several cysteine proteases, including cathepsin K, calpain, human rhinovirus 3C protease and several parasitic cysteine protease targets. Other successful recent efforts have involved combinatorial synthesis and screening for identification of new inhibitor templates.     

Dissecting and designing inhibitor selectivity determinants at the S1 site using an artificial Ala190 protease (Ala190 uPA)

A site-directed mutant of the serine protease urokinase-type plasminogen activator (uPA), was produced to assess the contribution of the Ser190 side-chain to the affinity and selectivity of lead uPA inhibitors in the absence of other differences present in comparisons of natural proteases. Crystallography and enzymology involving WT and Ala190 uPA were used to calculate free energy binding contributions of hydrogen bonds involving the Ser190 hydroxyl group (O(gamma)(Ser190)) responsible for the remarkable selectivity of 6-halo-5-amidinoindole and 6-halo-5-amidinobenzimidazole inhibitors toward uPA and against natural Ala190 protease anti-targets. Crystal structures of uPA complexes of novel, active site-directed arylguanidine and 2-aminobenzimidazole inhibitors of WT uPA, together with associated K(i) values for WT and Ala190 uPA, also indicate a significant role of Ser190 in the binding of these classes of uPA inhibitors. Structures and associated K(i) values for a lead inhibitor (CA-11) bound to uPA and to five other proteases, as well as for other leads bound to multiple proteases, help reveal the features responsible for the potency (K(i)=11nM) and selectivity of the remarkably small inhibitor, CA-11. The 6-fluoro-5-amidinobenzimidzole, CA-11, is more than 1000-fold selective against natural Ala190 protease anti-targets, and more than 100-fold selective against other Ser190 anti-targets.     

Partial purification and characterization of a cysteine protease inhibitor from the plerocercoid of Spirometra erinacei

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.     

Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: protease complex receptors increase during in vitro myogenesis

In the course of studies on the regulation of plasminogen activator-mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myoblast medium was compared to that of human PNI purified from human fibroblasts. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of a) increased PNI proteolytic inhibitory activity expression and b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle surface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.     

Investigating the role of histidine 157 in the catalytic activity of human cytomegalovirus protease

Herpesvirus proteases belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad, while classical serine proteases have an acidic residue as the third member. To gain a better understanding of the molecular basis for the functional role of the third-member His residue, we have carried out structural and biochemical investigations of human cytomegalovirus (HCMV) protease that bears mutations of the His157 third member. Kinetic studies showed that all the mutants have reduced catalytic activity. Structural studies revealed that a solvent molecule is hydrogen-bonded to the His63 second member and Ser134 in the H157A mutant, partly rescuing the activity of this mutant. This is confirmed by our kinetic and structural observations on the S134A/H157A double mutant, which showed further reductions in the catalytic activity. The structure of the H157A mutant is also in complex with the PMSF inhibitor. The H157E mutant has the best catalytic activity among the mutants; its structure, however, showed conformational readjustments of the His63 and Ser132 residues. The Ser132-His63 diad of HCMV protease has similar activity as the diads in classical serine proteases, whereas the contribution of the His157 third member to the catalysis is much smaller. Finally, structural comparisons revealed the presence of two conserved structural water molecules at the bottom of the S(1) pocket, suggesting a possible new direction for the design of HCMV protease inhibitors.     

Degranulation of human eosinophils induced by Paragonimus westermani-secreted protease

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.     

A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast

Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1‐ and S1‐type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ～45% and improving the accumulation of a murine diagnostic antibody, C5‐1, co‐secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5‐1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

Potent and selective inhibition of membrane-type serine protease 1 by human single-chain antibodies

Specific human antibodies targeting proteases expressed on cancer cells can be valuable reagents for diagnosis, prognosis, and therapy of cancer. To this end, a phage-displayed antibody library was screened against a cancer-associated serine protease, MT-SP1. A protein inhibitor of serine proteases that binds to a defined surface of MT-SP1 was used in an affinity-based washing procedure. Six antibodies were selected on the basis of their ELISA profiles and ability to serve as useful immunological reagents. The apparent K(i), indicative of the potency of the antibodies at inhibiting human MT-SP1 activity, ranged from 50 pM to 129 nM. Two of the antibodies had approximately 800-fold and 1500-fold selectivity when tested against the most homologous serine protease family member, mouse MT-SP1, that exhibits 86.6% sequence identity. Surface plasmon resonance was used as an independent means of determining the binding constants of the six antibodies. Association rates were as high as 1.15 x 10(7) s(-)(1) M(-)(1), and dissociation rates were as low as 3.8 x 10(-)(4) s(-)(1). One antibody was shown to detect denatured MT-SP1 with no cross reactivity to other family members in HeLa or PC3 cells. Another antibody recognized the enzyme in human prostate tissue samples for immunohistochemistry analysis. The mode of binding among the six antibodies and the protease was analyzed by competition ELISA using three distinctly different inhibitors that mapped the enzyme surface. These antibodies constitute a new class of highly selective protease inhibitors that can be used to dissect the biological roles of proteolytic enzymes as well as to develop diagnostic and therapeutic reagents.     

Kinetic studies on the inhibitions of mast cell chymase by natural serine protease inhibitors: indications for potential biological functions of these inhibitors

We have found that degranulation from mast cells is specifically inhibited by the inhibitors of chymase (10). Among the natural serine protease inhibitors tested, Bowman-Birk soybean protease inhibitor, Eglin C, and human alpha 1-antichymotrypsin inhibited chymase more strongly than did chymostatin, Kunitz soybean protease inhibitor, and phosphatidylserine. Of the inhibitors tested, Bowman-Birk soybean protease inhibitor was the strongest inhibitor of chymase, its Ki value being 13.2 X 10(-9) M. Kinetic studies showed that these inhibitors were all noncompetitive inhibitors of chymase. Bowman-Birk and Kunitz soybean protease inhibitors inhibited both chymotrypsin-type and trypsin-type serine proteases but Eglin C specifically inhibited chymotrypsin-type proteases.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Human proteoglycan testican-1 inhibits the lysosomal cysteine protease cathepsin L.

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a Ki of 0.7 nM, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu.     

Activity in monomers of human cytomegalovirus protease.

Herpesvirus proteases require dimerization for activity, although crystallographic data indicate that each monomeric subunit possesses a well-separated and complete active site. This suggests that dimerization stabilizes the monomeric protease subunits in an active conformation. Chemical cross-linking with disuccinimidyl glutarate was used to capture human cytomegalovirus protease in its various conformations. The cross-linked protease retained activity under mildly chaotropic conditions (0.25 to 0.75 M urea) in contrast to non-cross-linked protease which lost activity. Identification of active protease species by incorporation of radioactive diisopropylfluorophosphate showed that in addition to cross-linked dimers, cross-linked protease monomers were responsible for a significant fraction of the total protease activity. These results are consistent with the hypothesis that herpesvirus protease activation occurs by stabilization of an active conformer in the dimer.     

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the K(m). Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.     

Predicting functional residues of the Solanum lycopersicum aspartic protease inhibitor (SLAPI) by combining sequence and structural analysis with molecular docking

The Solanum lycopersicum aspartic protease inhibitor (SLAPI), which belongs to the STI-Kunitz family, is an effective inhibitor of the aspartic proteases human cathepsin D and Saccharomyces proteinase A. However, in contrast with the large number of studies on the inhibition mechanism of the serine proteases by the STI-Kunitz inhibitors, the structural aspects of the inhibition mechanism of aspartic proteases from this family of inhibitors are poorly understood. In the present study, we have combined sequence and structural analysis methods with protein-protein docking to gain a better understanding of the SLAPI inhibition mechanism of the proteinase A. The results suggest that: i) SLAPI loop L9 may be involved in the inhibitor interaction with the proteinase A′s active site, and ii) the residues I144, V148, L149, P151, F152 and R154 are implicated in the difference in the potency shown previously by SLAPI and another STI-Kunitz inhibitor isolated from Solanum tuberosum to inhibit proteinase A. These results will be useful in the design of site directed mutagenesis experiments to understand more thoroughly the aspartic protease inhibition mechanism of SLAPI and other related STI-Kunitz inhibitors.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

Evidence that serpin architecture intrinsically supports papain-like cysteine protease inhibition: engineering alpha(1)-antitrypsin to inhibit cathepsin proteases

The closely related serpins squamous cell carcinoma antigen-1 and -2 (SCCA-1 and -2, respectively) are capable of inhibiting cysteine proteases of the papain superfamily. To ascertain whether the ability to inhibit cysteine proteases is an intrinsic property of serpins in general, the reactive center loop (RCL) of the archetypal serine protease inhibitor alpha(1)-antitrypsin was replaced with that of SCCA-1. It was found that this simple substitution could convert alpha(1)-antitrypsin into a cysteine protease inhibitor, albeit an inefficient one. The RCL of SCCA-1 is three residues longer than that of alpha(1)-antitrypsin, and therefore, the effect of loop length on the cysteine protease inhibitory activity was investigated. Mutants in which the RCL was shortened by one, two, or three residues were effective inhibitors with second-order rate constants of 10(5)-10(7) M(-)(1) s(-)(1). In addition to loop length, the identity of the cysteine protease was of considerable importance, since the chimeric molecules inhibited cathepsins L, V, and K efficiently, but not papain or cathepsin B. By testing complexes between an RCL-mimicking peptide and the mutants, it was found that the formation of a stable serpin-cysteine protease complex and the inhibition of a cysteine protease were both critically dependent on RCL insertion. The results strongly indicate that the serpin body is intrinsically capable of supporting cysteine protease inhibition, and that the complex with a papain-like cysteine protease would be expected to be analogous to that seen with serine proteases.