The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Potential of pyrazolooxadiazinone derivatives as serine protease inhibitors.

As a part of an investigation on molecular hybrids as new serine protease inhibitors, the pyrazolo [4,3-c][1,2,5]oxadiazin-3(5H)-one ring system was selected as a model of potential mechanism-based inhibitors. Due to the inherent reactivity of this system an optimal balance between susceptibility to nucleophilic attack and stability in solvents was sought prior to development as therapeutic agents. Substitutions on N5 and C7 of the supporting pyrazole ring with either aliphatic or aromatic groups (compounds 2 a-m) and the replacement of the carbonyl oxygen on the reactive oxadiazinone ring with sulfur (compounds 3a,i) were explored. Two members (2i and 2k) of this class of inhibitors displayed time-dependent inhibition of HLE suggesting mechanism-based inhibition. The observation that HLE generated a product(s) from compound 2i which displayed an identical UV-Visible spectrum to that observed during non-enzymatic hydrolysis further supports this proposal. FlexX-based docking of these compounds into a model of the human leukocyte elastase (HLE) active site produced a molecular model of the inhibitor-enzyme interaction.

Structural determinants for potent, selective dual site inhibition of human pp60c-src by 4-anilinoquinazolines.

The kinetic mechanisms for the inhibition of pp60(c-src) tyrosine kinase (Src TK) by 4-anilinoquinazolines, an important class of chemicals as protein kinase inhibitors, were investigated. 4-Anilinoquinazolines with a bulky group at the 4'-position of the anilino group were shown to be competitive with both ATP and peptide, whereas molecules lacking such a bulky group only displayed an inhibition pattern typical of those competitive with ATP and noncompetitive with peptide. Modifications of the substituents on the carbocyclic ring did not perturb the inhibition pattern although the affinities of these modified inhibitors for Src TK were affected. Structural modeling of Src TK with inhibitor and peptide substrate bound indicated a direct atomic conflict between the bulky 4-position group and the hydroxy of the peptide tyrosyl to which the gamma-phosphate of ATP is transferred during the kinase reaction. This atomic conflict would likely prevent simultaneous binding of both inhibitor and peptide, consistent with the observed kinetic competitiveness of the inhibitor with peptide. The dual site inhibitors appeared to have both enhanced potency and selectivity for Src TK. One such inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline, had a 15 nM potency against Src TK and was selective over receptor tyrosine kinases VEGFR2 by 88-fold and C-fms by 190-fold.

The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo.

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.

SH2- and SH3-mediated interactions between focal adhesion kinase and Src.

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.

Planning and establishment of a high throughput screening site.

In 1996 and 1997, Glaxo Wellcome's US Research division planned and established their second generation research strategy. An important aspect of the strategy entailed development of two automated screening sites in Biochemistry in Research Triangle Park, NC. Development of the new operations required many decisions to be made very quickly, including automated process design, system selection and site preparation. Descriptions of the decision made in the development of one of the screening sites are presented in this paper.

Clinicopathologic findings in congenital aneurysms of the great vessels.

We describe the clinical, histopathologic, and angiographic findings in four children with congenital abnormalities of the great vessels of unknown cause, comprising either single or multiple arterial aneurysms, aortic/arterial dilatation, vessel tortuosity, or combinations of these abnormalities. Two children had early and severe respiratory distress due to aneurysmal compression of the trachea. All children had diffuse dilatation of several arteries, and two children also had tortuosity of multiple arteries. Progression of these abnormalities was clearly evident in one child, in whom diffuse vessel irregularity and tortuosity affected intra-abdominal, and intra and extra-cranial arteries. One child died at 5 years, while the other three have undergone successful surgical repair in the first 3 months of life and are now well, between age 2.5 and 7 years. The phenotype of each child appears unique but all have in common the rare finding of aneurysms of the aorta and main pulmonary artery. Congenital aortic aneurysms did not occur as an isolated finding in any of these children.

Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated.

Rapid digestion of pp60c-src tyrosine kinase (src TK) in combination with electrospray ionization mass spectrometry enabled the determination of the time course for autophosphorylation of three tyrosine sites (Y338, Y419, and Y530) and a correlation with src TK activity. A form of src TK was purified from baculovirus-infected cells which contains only Y338 partially phosphorylated. Incubation with MgATP increases the phosphorylation of all three sites. The autophosphorylation and dephosphorylation of Y419 are directly correlated with the level of src TK activity. The role of Y338 phosphorylation is unknown. Conditions resulting in complete autophosphorylation of Y530 were identified by electrospray ionization mass spectrometry. Surface plasmon resonance detection and size exclusion chromatography provide direct evidence for an intramolecular pY530-SH2 complex, supporting previous models [Matsuda, M., Mayer, B.J., Fukui, Y., & Hanafusa, H. (1990) Science 248, 1537-1539]. Contrary to these models, when the enzyme is fully phosphorylated on Y530, phosphorylated on Y419, and present only as the intramolecular pY530-SH2 complex, 20% of the kinase activity is retained. In addition, the k(m)'s for substrates are unaffected. Disruption of the pY530-SH2 interaction and activation of kinase activity by a high-affinity SH2 ligand yield a Kactivation which is 200-fold larger than the Kd for ligand binding to the uncomplexed src SH2 domain. These data suggest a Keq of 200 (unitless) for the intramolecular association of pY530 with the SH2 domain. We propose that the pY530-SH2 interaction modulates signal transduction by down-regulating src TK activity 5-fold, and perhaps more importantly by inhibiting protein-protein interactions with the SH2 domain. These results have significant implications relative to the development of SH2 ligands as therapeutics to control aberrant signal transduction. These ligands will be 200-fold less effective at inhibiting protein-protein interactions versus down-regulated src TK than versus activated src TK. This should minimize activation of src TK activity in normal cells and lead to an increased therapeutic index.

Proteoglycan-degrading activity of human stromelysin-1 and leukocyte elastase in rabbit joints. Quantitation of proteoglycan and a stromelysin-induced HABR fragment of aggrecan in synovial fluid and cartilage.

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.

Kinetic mechanisms of the forward and reverse pp60c-src tyrosine kinase reactions.

The kinetic mechanism of the pp60c-src tyrosine kinase (src TK) reaction was investigated in the forward and reverse directions. In the forward direction, initial velocities obtained by varying ATP and the peptide (FGE)3Y(GEF)2GD indicated a sequential addition of the two substrates. The peptide analog, (FGE)3F(GEF)2GD, was a competitive inhibitor versus the peptide substrate and a noncompetitive inhibitor versus MgATP. Interestingly, the tyrosine hydroxyl group imparts only a 6-fold increase in binding. AMP-PCP was a competitive inhibitor versus MgATP and a noncompetitive inhibitor versus the peptide substrate. These results prove that the addition of substrates is random. Furthermore, there appears to be little binding synergy as the KiMgATP approximately equal to 2.4KmMgATP. The phosphorylated peptide (FGE)3-pY-(GEF)2GD was a competitive inhibitor versus peptide and a noncompetitive inhibitor against MgATP, suggesting that a dead end complex can form between MgATP, the phosphorylated peptide product, and the enzyme. The reverse reaction was investigated by varying ADP and the phosphopeptide. (FGE)3-pY-(GEF)2GD. The initial velocity pattern was indicative of a sequential mechanism. There was even less binding synergy in the reverse direction as the KiMgADP approximately equal to 1.4KmMgADP. AMP-CP was a competitive inhibitor versus MgADP and a noncompetitive inhibitor versus the phosphopeptide. (FGE)3F(GEF)2GD was a competitive inhibitor versus the phosphopeptide and a noncompetitive inhibitor versus MgADP. These data prove that addition of the substrates in the reverse direction is random. (FGE)3Y(GEF)2GD was a competitive inhibitor against peptide substrate and a noncompetitive inhibitor against MgADP; therefore a dead end complex can form between MgADP, (FGE)3Y(GEF)2GD, and the enzyme. These results indicate that the src TK reaction follows a sequential bi-biequilibrium random mechanism in both directions, with dead end complexes forming when either MgATP and (FGE)3-pY-(GEF)2GD or MgADP and (FGE)3Y(GEF)2GD bind to the enzyme. The kinetic constants determined from the forward and reverse reactions were used in the Haldane equation to determine a K(eq) constant for the forward reaction of 10.1, corresponding to a delta G of -1.4 kcal/mol. This further confirms that the O-P bond of phosphotyrosine is similar in energy to that of the gamma-phosphoryl of MgATP.

Exploration of the sequence specificity of pp60c-src tyrosine kinase. Minimal peptide sequence required for maximal activity.

The minimum length required for phosphorylation of a peptide by pp60c-src tyrosine kinase (srcTK) was delineated in this work. Budde (M.D. Anderson University of Texas, personal communication) suggested that the peptide (FGE)3Y(GEF)2GD (peptide I) was a "good" srcTK substrate. Peptide I yielded a 251-fold higher kcat/Km than RRLIEDAEYAARRG, a peptide substrate based upon the autophosphorylation site of srcTK. This was due to a 38-fold lower Km and a 6.6-fold increase in kcat.N-terminal truncation of up to 8 residues in a series of peptides yielded only a 3-fold decrease in activity. Removal of the final N-terminal residue resulted in a 10-fold loss in substrate activity, primarily as a result of an increase in the Km. C-terminal truncations ending in the amide yielded no significant loss in activity until the Y + 3 residue was removed, which resulted in a 73-fold decrease in kcat/Km relative to peptide I. The latter was due primarily to an increase in Km. The results from peptides truncated on both termini suggest that subsite recognition N- and C-terminal relative to the site of phosphorylation can be examined independently. In addition, the observation that only 5 residues are required for significant substrate activity suggests that small molecule inhibitors based upon interactions with the phosphoacceptor site may be developed.

Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process.

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

Examination of the dephosphorylation reactions catalyzed by pp60c-src tyrosine kinase explores the roles of autophosphorylation and SH2 ligand binding.

pp60c-src tyrosine kinase (srcTK) catalyzes the dephosphorylation of phosphotyrosine-containing peptides, including phosphopeptides that bind with high affinity to the src SH2 domain. The mechanism for these dephosphorylation reactions was investigated. Dephosphorylation was inhibited by a competitive inhibitor for the ATP binding site. In the presence of ADP, dephosphorylation of phosphopeptide substrates is primarily due to the reversal of the kinase reaction. Autoactivated and unactivated srcTK both catalyzed the reverse of the kinase reaction; however, autoactivated srcTK displayed an increase in kcat of approximately 4-11-fold relative to unactivated srcTK, depending on the reaction conditions. Autoactivation of srcTK does not affect the Km's for MgADP or phosphopeptide (FGE)3-pY-(GEF)2GD. Unphosphorylated srcTK becomes phosphorylated during the reverse of the kinase reaction upon accumulation of free MgATP. In the presence of MgATP, srcTK also dephosphorylates peptide substrates, by first hydrolyzing MgATP to MgADP. Binding of phosphotyrosine peptide ligands to the src SH2 domain stimulated the rate of MgATP hydrolysis approximately 2-fold, but had not effect on the Km for MgATP. These data suggest that autophosphorylation of tyrosine 419 is not required for nucleotide or peptide binding, or catalysis involving small peptide substrates. In addition, these results suggest that both the forward and the reverse src tyrosine kinase reactions may be important in regulating the intracellular levels of protein tyrosine phosphorylation.

A cure for pulmonary arteriovenous fistulas?

Diffuse bilateral pulmonary arteriovenous fistulas developed in a cyanotic child with left atrial isomerism, consisting of an interrupted inferior vena cava with continuation of the hemiazygos vein to the isolated left superior vena cava, soon after she underwent a Kawashima operation that left the hepatic veins draining into the common atrium. The fistulas regressed after the hepatic veins were connected to the confluent pulmonary arteries with a left lateral atrial tunnel.

Determination of the kinetic parameters of Escherichia coli leader peptidase activity using a continuous assay: the pH dependence and time-dependent inhibition by beta-lactams are consistent with a novel serine protease mechanism.

Bacterial leader peptidase (LPase) is a potential target for the development of novel anti-infective agents, but to data only peptides based upon natural macromolecular substrates have been reported as inhibitors. In this work is described a continuous assay for Escherichia coli LPase activity, based upon Ac-WSASALAKI-AMC (I) as the substrate, that can be monitored either spectrophotometrically or spectrofluorometrically. The LPase reaction is coupled to the liberation of AMC (aminomethylcoumarin) via a nonspecific leucine aminopeptidase. LPase and a short form of the enzyme (LPase-sf) lacking the membrane spanning domains displayed saturable kinetics toward I. The second-order rate constants were approximately 2 x 10(5) M-1 h-1 at pH 7.5 and were comparable to those reported in the literature for peptide substrates based upon natural cleavage sites in preproteins. LPase was inhibited by beta-lactams. [S-(R*,S*)]-4-[(1-(((1-(5-toluoyl)butyl)amino)carbonyl)-3,3-dimethyl-4- oxo-2-azetidinyl)oxyl]benzoic acid (L-684,-248, 588 microM) inhibited the LPase-catalyzed hydrolysis of 50 microM I and 125 microM Ac-WLVP-Nleu-LSFAAEGDDPA-NH2 by 30% and 88% over 1 and 4 h, respectively. The inhibition of LPase by L-684,248 and its C-4 diasteromer was time dependent and yielded second-order rate constants (kinact/Ki) of 12 and 7.7 M-1 min-1, respectively. The process was structurally specific as the C-3 diethyl substituted beta-lactam (C-4 S-isomer) was inactive. The latter data correlate with the LPase preference for alanine at the P1 position of peptide substrates [Kuo et al. (1993) Arch. Biochem. Biophys. 303, 274-280].(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli leader peptidase: production of an active form lacking a requirement for detergent and development of peptide substrates.

The report by Zimmermann et al. (J. Biol. Chem. 257, 1982, 6529-6536) that the active site of Escherichia coli leader peptidase (LPase) is located in the periplasm led us to explore the possibility that soluble, active short forms of LPase could be produced. Detergent-free delta 2-75 mutant protein (LPase-sf) lacking the two N-terminal transmembrane spanning and the cytoplasmic domains was produced in high yield. The mass of the protein determined by electrospray ionization mass spectrometry was 27,952 amu. The increase of 42 amu over that predicted by the expected amino acid sequence indicates that the N-terminus of LPase-sf is acetylated. This is consistent with the inability to obtain an N-terminal sequence. LPase-sf lacks the site of autolysis present in LPase, thus circumventing problems associated with enzyme autocatalytic instability. LPase-sf and wild type LPase displayed comparable activity versus two peptide substrates. The peptides, based upon the cleavage sites of procoat and the precursor of maltose binding protein, were processed at the expected sites. In addition, the activity of both LPase's was not inhibited by classical inhibitors of the four classes of proteases. LPase-sf displayed similar activity in the presence and absence of detergent. Wild type LPase displayed specificity for alanine in the P1 subsite of the peptide WSASALX*KI and did not hydrolyze peptides with glycine, valine, or serine in that position. The availability of a detergent-free active form of LPase should facilitate mechanistic and structural studies.

Metastatic osteogenic sarcoma to the heart presenting as bacterial endocarditis.

A patient with metastatic osteogenic sarcoma involving the left atrium is described who presented with features of bacterial endocarditis. The source of infection was the adjacent esophagus into which the tumor had eroded. This case demonstrates that sarcomas metastasizing to the heart may result in a clinical condition indistinguishable from infective endocarditis. At post-mortem, careful dissection of cardiac metastases should be undertaken to check for possible esophageal involvement.

Orally active beta-lactam inhibitors of human leukocyte elastase. 2. Effect of C-4 substitution.

The effect of changing the C-4 substituent of 3,3-diethyl-1-[(benzylamino)carbonyl]-2-azetidinone on inhibition of HLE and in a model of HLE-induced lung damage in hamsters was explored. Substituents at this position do not appear to interact strongly with HLE with the most potent compounds having k(obs)/[I] = 6900 M-1 s-1. However, substituents at this position had a marked effect on in vivo activity. The greatest oral activity in the lung hemorrhage assay was achieved with C-4 aryl carboxylic acid ethers (60-85% inhibition at 30 mg/kg po). Based upon the established mechanism of inhibition by these compounds, the C-4 substituent would be released, and therefore, the pharmacological potential of these C-4 substituents was of considerable concern. Fortunately, compounds containing 4-hydroxybenzoic acid and 4-hydroxyphenylacetic acid ethers at C-4 were among the most active analogs. These phenolic acids are also found as urinary metabolites in healthy humans. Other heteroaryls at C-4 were also orally active in this model despite relatively modest enzyme activity.

Electrospray ionization mass spectrometry as a mechanistic tool: mass of human leucocyte elastase and a beta-lactam-derived E-I complex.

We have utilized liquid chromatography electrospray ionization mass spectrometry (ESI-MS) to probe the nature of the covalent E-I complex of human leucocyte elastase (HLE) and a beta-lactam. The mass spectrum of HLE isozyme 4 displayed one major and two minor components with masses of 25,202, 25,043, and 24,522 Da, respectively. Isozyme 3 displayed three components, with masses of 25,180, 24,030, and 24,523 Da. These data suggest that the isozymes differ in the type and not the content of carbohydrate. The minor components represent decreases in carbohydrate content. Inactivation of isozyme 4 with trans-4-(ethoxycarbonyl)-3-ethyl-1-[(4-nitrophenyl)sulfonyl]-azetidin -3-one increased the mass of the three components by that of the parent compound. Similar results were obtained with the mixture of HLE isozymes. These observations demonstrate that HLE does not catalyze the beta-elimination of p-nitrophenylsulfinate as Firestone et al. [(1990) Tetrahedron 46, 2255) suggested. In addition, it suggests that a "double hit" of both the active-site serine and histidine is not required to form a stable acyl-enzyme. Noncovalent complexes between HLE and either the tight-binding secretory leucoprotease inhibitor (SLPI) or a slow tight-binding peptide difluoroketone inhibitor were not observed by ESI-MS. SLPI displayed a mass of 11,710 Da in the absence and presence of HLE. These data demonstrate the utility of ESI-MS to probe the mechanism of inhibition of enzymes by mechanism-based inhibitors.

Comparison of the proteoglycanolytic activities of human leukocyte elastase and human cathepsin G in vitro and in vivo.

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.