Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases.
The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar K(i)s, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents.
Zani
Marie-Louise
ML
Inserm U618 Protéases et Vectorisation Pulmonaires, IFR 135 Imagerie Fonctionnelle, University of Tours, France.
Baranger
Kévin
K
Guyot
Nicolas
N
Dallet-Choisy
Sandrine
S
Moreau
Thierry
T
D000595:Amino Acid Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery; D053492:Elafin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008171:Lung Diseases; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053146:Myeloblastin / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism*; D055672:Static Electricity; D011503:Transglutaminases / Q000378:metabolism
D053492:Elafin; D008565:Membrane Proteins; C506806:PI3 protein, human; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D053481:Secretory Leukocyte Peptidase Inhibitor; C572915:trappin-2 A62L; D011503:Transglutaminases; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D053146:Myeloblastin