Oxidation resistant muteins of antileukoproteinase as potential therapeutic agents.
Native antileukoproteinase (ALP) and two oxidant resistant mutants ALP 242 and ALP 231 were synthesized by means of recombinant DNA technology. In the ALP 242 molecule the methionine residue located in the reactive centre of the binding loop is replaced by a leucine residue. In ALP 231 all four methionine residues of the second domain were substituted by leucine residues. The native inhibitor and the two oxidant resistant molecules show comparable inhibitory capacities towards human neutrophil elastase (HLE) and cathepsin G. All three inhibitors were treated with different reactive oxygen species. After incubation with chloramine T or supernatants of activated polymorphonuclear leukocytes (PMN's) a drastic drop of inhibitory capacity of the native molecule was observed. Compared to the native form of ALP the mutant ALP 242 was less inactivated, whereas ALP 231 was nearly totally resistant towards all reactive oxygen. (Heinzel-Wieland R. et al., Biomed Biochim Acta 50: 677-681 (1991)) The intratracheal administration of HLE into the lung of Syrian Hamsters induced mild to moderate emphysematous lesions. The inhibitory potencies of native ALP and the ALP mutants were determined in this animal model by means of intratracheal instillation of the different molecules one hour prior to the administration of HLE. The inhibitory effects of ALP 242 and ALP 231 towards HLE-induced emphysema were significantly better than that of the native molecule. Surprisingly no significant differences between the two mutants were observed. (Rudolphus A. et al., Clin Sci 81: 777-784 (1991)) In a second animal model the emphysema was induced by repeated intratracheal administration of lipopolysaccharides (LPS) into the hamster lungs. This model is characterized by a chronic process of inflammation probably caused by a continuous release of endogenous elastase from infiltrating PMN's. Repeated applications of 1 mg of ALP 242 reduced the LPS-induced emphysema by 70 to 80%. In contrast, equal amounts of the native molecule resulted in significantly lower inhibition of the LPS-induced emphysema, only 23-30% reduction was observed. Repeated applications of 1 mg of ALP 231 reduced the LPS-induced emphysema only about 50%. So far it is not yet clear, why the totally oxidant resistant ALP 231 was less effective than the ALP 242 molecule. (Stolk J. et al., Pulmonary Pharmacology in press (1992))
Steffens
G J
GJ
Department of Molecular Biology, Grunenthal GmbH, Aachen, Germany.
Heinzel-Wieland
R
R
Saunders
D
D
Wolf
B
B
Rudolphus
A
A
Stolk
J
J
Krarnps
J A
JA
Dijkman
J A
JA
D000818:Animals; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D008070:Lipopolysaccharides; D008647:Mesocricetus; D009154:Mutation; D010084:Oxidation-Reduction; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011656:Pulmonary Emphysema / Q000139:chemically induced / Q000188:drug therapy; D017382:Reactive Oxygen Species / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*
D008070:Lipopolysaccharides; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase