TY - JOUR
T1 - Inhibition of human neutrophil elastase by erythromycin and flurythromycin, two macrolide antibiotics
AU - Gorrini, M.
AU - Lupi, A.
AU - Viglio, S.
AU - Pamparana, F.
AU - Cetta, G.
AU - Iadarola, P.
AU - Powers, J. C.
AU - Luisetti, M.
PY - 2001
Y1 - 2001
N2 - Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (kobs) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (Ka). A proflavine displacement assay was used to determine the deacylation rate constant (kd). Both Er and FE are good HNE inhibitors, showing a high ka and a low kd. Because the number of turnovers per inactivation of Er was ≅ 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a kd only, whereas for FE, in addition to the kd, an alkylation constant (K2) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.
AB - Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (kobs) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (Ka). A proflavine displacement assay was used to determine the deacylation rate constant (kd). Both Er and FE are good HNE inhibitors, showing a high ka and a low kd. Because the number of turnovers per inactivation of Er was ≅ 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a kd only, whereas for FE, in addition to the kd, an alkylation constant (K2) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.
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M3 - Article
C2 - 11694455
AN - SCOPUS:0034760267
VL - 25
SP - 492
EP - 499
JO - American Journal of Respiratory Cell and Molecular Biology
JF - American Journal of Respiratory Cell and Molecular Biology
SN - 1044-1549
IS - 4
ER -