Chelator-induced iron excretion in iron-overloaded marmosets.

In order to test new orally active iron chelators in a predictive way, a primate model has been developed. This model makes use of the marmoset monkey (Callithrix jacchus) and its overall design is similar to a previously reported monkey model. However, this new model enables a higher compound throughput and requires lower amounts of test compound because the animals are much easier to handle and have much lower body weights. The marmosets were iron-overloaded by three intraperitoneal injections of iron (III) hydroxide polyisomaltose. For the iron-balance studies, the animals were kept in metabolic cages and were maintained on a low-iron diet in order to reduce faecal background. After compound administration, the excretion of iron in urine and faeces was followed for 2 d. A series of well-known chelators was tested for validation of the model. In particular, comparison of the iron-clearing properties of DFO, L1, CP94 and HBED in marmosets and humans demonstrated the predictive value of the model and justify our expectation that if iron chelators such as CGP65015, ICL670A and CGP75254A are active in marmosets, they will be active in humans as well.

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties.

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine alpha-chymotrypsin (alpha-chymotrypsin) [native and with the Met192 residue converted to the sulfoxide derivative (Met(O) alpha-chymotrypsin)] as well as to native bovine beta-trypsin (beta-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0 degrees C and 30.0 degrees C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methionyl residues present at the proteinase:inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation of methionyl residues of SLPI and alpha-chymotrypsin, by reaction with chloramine T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion of native SLPI and native alpha-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 A resolution.

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.

Selective oxidation of Met-192 in bovine alpha-chymotrypsin. Effect on catalytic and inhibitor binding properties.

Catalytic and inhibitor binding properties of bovine alpha-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 alpha-chymotrypsin), have been examined relative to the native enzyme (alpha-chymotrypsin), between pH 4.5 and 8.0 (mu = 0.1), and/or 5.0 degrees C and 40.0 degrees C. Values of kcat, k+2 and/or k+3 for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 alpha-chymotrypsin are similar, as well as values of Km for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, Ks and Km values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 alpha-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to serine (pro)enzymes: a comparative thermodynamic study.

The binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10 degrees C to 40 degrees C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu-proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] greater than human leucocyte elastase congruent to human cathepsin G congruent to subtilisin Carlsberg congruent to bovine alpha-chymotrypsin greater than bovine alpha-chymotrypsinogen A congruent to porcine pancreatic elastase congruent to bovine beta-trypsin. The serine (pro)enzyme-inhibitor complex formation is an entropy-driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift of the invariant hystidyl catalytic residue from approximately to 6.9 in the free serine proteinases and bovine alpha-chymotrypsinogen A to congruent to 5.1 in the serine (pro)enzyme-inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine (pro)enzyme-inhibitor contact regions.

Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor.

Secretory leukoprotease inhibitor (SLPI), a 12-kD nonglycosylated serine antiprotease with a high capacity for inhibiting neutrophil elastase (NE), is produced by cells of mucosal surfaces including the human lung. The molar concentrations of SLPI in total respiratory tract epithelial lining fluid (ELF) were 56 +/- 10% that of alpha 1-antitrypsin, suggesting SLPI may be more important for the anti-NE protection of the pulmonary epithelial surface than previously thought. However, evaluation demonstrated that SLPI in respiratory ELF was only one-third functional. Studies aerosolizing recombinant SLPI (rSLPI) to sheep demonstrated that in the short term, neither aerosolization and alveolar deposition nor the lavage procedure inactivated the SLPI molecule. In vitro studies with rSLPI demonstrated that exposure to oxidants did not modify the form of the molecule, while exposure to oxidants and NE caused the molecule to be cleaved from 12 to 8 kD. Consistent with this, evaluation of SLPI in lavage fluid of individuals with cystic fibrosis (a condition with oxidants and NE on the respiratory epithelium) showed that the SLPI was degraded. However, evaluation of SLPI in normal ELF by molecular sieve analysis and Western analysis demonstrated an intact 12-kD molecule, suggesting that the partial inactivation of SLPI in normals in vivo is not because it is complexed to NE or exposed to oxidants + NE. Together, these observations demonstrate that SLPI is present in large amounts in respiratory ELF, but since the majority of the SLPI is inactive, it likely does not play a significant role in protecting the normal respiratory epithelium, except perhaps in the upper airways where the levels of SLPI are the highest.

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium.

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution.

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.

Evaluation of the rapid plasma elimination of recombinant alpha 1-proteinase inhibitor: synthesis of polyethylene glycol conjugates with improved therapeutic potential.

Plasma-derived alpha 1-proteinase inhibitor (alpha 1PlI) is used for replacement therapy in patients with emphysema who have a deficiency of the protein. Future therapy with alpha 1PI with reactive site mutants of this inhibitor will probably require the use of recombinant-derived alpha 1PI (r alpha 1PI). However, the pharmacologic efficacy of r alpha 1PI in humans has been hindered, since r alpha 1PI is rapidly cleared from the circulation of experimental animals. Our studies of the metabolism of r alpha 1PI in mice demonstrate that the rapid clearance of r alpha 1PI is due to renal filtration. However, after preventing renal filtration by ligation of the renal arteries, we find that r alpha 1PI is metabolized in a manner similar to alpha 1PI. Conjugation of r alpha 1PI to polyethylene glycol of Mr approximately 4000 (PEG-4) greatly slows the clearance of r alpha 1PI. The PEG-4 r alpha 1PI conjugate is metabolized in a manner similar to alpha 1PI and has similar kinetic properties before and after oxidation with N-chlorosuccinimide. Moreover, proteinase complexes of PEG-4-r alpha 1PI are catabolized by the same hepatocyte receptor that binds alpha 1PI-proteinase complexes. The results of these studies lay the groundwork for the synthesis of pharmacologically effective r alpha 1PI derivatives with acceptable plasma retention times.

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis.

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5. The crystals belong to the monoclinic space group P2(1), with unit cell constant: a = 55.3 A, b = 59.4 A, c = 42.5 A, beta = 99.0 degrees; one complex moiety is present per asymmetric unit. The crystals diffract to 2.0 A resolution and are suitable for detailed X-ray crystallographic investigations.

Control of exogenous proteinases and their inhibitors at the macrophage cell surface.

The actions and availability of human neutrophil elastase and its protein inhibitor, Eglin, when co-incubated with macrophages were investigated. Eglin did not induce radical production by mouse peritoneal macrophages; nor were specific binding sites for Eglin detected on these cells. Mouse peritoneal macrophages could inactivate both elastase and Eglin extensively, when these targets were used at concentrations appropriate to the extravascular fluids. Two methods were used for assessing such inactivation: one, as in previous literature, only took account of molecules remaining in the supernatant after interaction with the cells; the other (lacking from most previous studies) took into account all target molecules, including those associated with the cells. From an analysis of both types of experiment, it was shown that the cell-derived inactivators were stable products, whose quantity was not significantly influenced by the induction of a macrophage oxidative burst and its associated free radicals. They were probably mainly proteinases and proteinase inhibitors. Thus, mouse peritoneal macrophages restrict the activity of proteinases and inhibitors by means of stable molecules, such as proteins. Other mononuclear phagocytes may use free radicals and oxidants more extensively in this respect.

Free radicals inactivate human neutrophil elastase and its inhibitors with comparable efficiency.

Free radicals produced in a Fenton reaction (H202/Cu), modelling some xenobiotic and cell-mediated inflammatory affronts, efficiently inactivated the elastase-inhibitor eglin, but equally, human neutrophil elastase itself. Elastase activity was not regenerated from proteinase/inhibitor complexes during radical attack. Three different elastase inhibitors, eglin, secretory leukocyte proteinase inhibitor and alpha-1-proteinase inhibitor were all similarly sensitive to inactivation. Unlike certain oxidants which can selectively inactivate alpha-1-proteinase inhibitor, free radicals may influence comparably the availability of both proteinase inhibitors and their targets.

Fibronectin-cleaving activity in bronchial secretions of patients with cystic fibrosis.

In cystic fibrosis, colonization of the airways with Pseudomonas aeruginosa follows colonization with Staphylococcus aureus and is related to accelerated deterioration of pulmonary function. Because P. aeruginosa adheres better to cell surfaces devoid of fibronectin, we searched for fibronectin-cleaving activity in bronchial secretions and saliva from 24 patients with cystic fibrosis who were followed up for 4.5 y and from two control groups. Proteolytic activity against 125I-labeled fibronectin was continuously present in cystic fibrosis bronchial secretions; significantly higher fibronectin-cleaving activity was found in older vs. younger patients, in patients with advanced disease stages determined by a five-stage scoring system, and in those colonized with P. aeruginosa. The fibronectin-cleaving activity was due to neutrophil elastase and cathepsin G. Cystic fibrosis bronchial secretions had proteolytic activity against surface fibronectin of airway mucosal cells. Thus fibronectin-cleaving activity of bronchial secretions rather than of saliva may favor P. aeruginosa colonization of the upper respiratory tract in individuals with cystic fibrosis.