Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases.
Eight new biotinylated, mechanism-based isocoumarin serine protease inhibitors have been designed and synthesized to detect, localize, and isolate serine proteases. Isocoumarins that contain a 4-chloro group, a biotinylated substituent at the 7-position, and different 3-alkoxy groups are inhibitors of various serine proteases including human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), trypsin, human recombinant granzyme A, chymotrypsin, and cathepsin G. Insertion of spacers between the isocoumarin moiety and the biotin moiety enhanced enzyme inhibitory potency and may also promote binding of the enzyme-inhibitor complex to avidin. The 3-alkoxy groups conferred selectivity toward different serine proteases with chymotrypsin being inhibited effectively by compounds with 3-phenylethoxy groups while derivatives with 3-methoxy, ethoxy, or propoxy groups were potent inhibitors of HLE and moderate inhibitors of PPE. Full enzymatic activity was regained after the immediate addition of hydroxylamine to the inactivated chymotrypsin and PPE derivatives, which indicated that a simple acyl enzyme derivative is formed initially in the inhibition reaction. Egg avidin did not effect the rate of spontaneous enzyme reactivation rate while streptavidin accelerated the reactivation reaction. PPE inhibited by 7-[[6-(biotinylamino)caproyl]amino]-4-chloro-3- ethoxyisocoumarin (BIC 5) or 7-[[6-[[6-(biotinylamino)caproyl]amino] caproyl]amino]-4-chloro-3-methoxyisocoumarin (BIC 7) was bound to immobilized avidin columns. Most of inhibited PPE could be eluted from the monomeric or tetrameric avidin columns but only a portion (40-70%) of the enzyme was active due to the partial formation of a stable alkylated enzyme derivative during the isolation process.(ABSTRACT TRUNCATED AT 250 WORDS)
Kam
C M
CM
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Abuelyaman
A S
AS
Li
Z
Z
Hudig
D
D
Powers
J C
JC
D000818:Animals; D001360:Avidin / Q000494:pharmacology; D001426:Bacterial Proteins / Q000494:pharmacology; D001710:Biotin / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D003374:Coumarins / Q000138:chemical synthesis* / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases / Q000032:analysis / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D019809:Streptavidin; D013329:Structure-Activity Relationship; D013552:Swine
D001426:Bacterial Proteins; D003374:Coumarins; D004793:Enzyme Reactivators; D006898:Hydroxylamines; D015842:Serine Proteinase Inhibitors; D001360:Avidin; D019811:Hydroxylamine; D001710:Biotin; D019809:Streptavidin; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase