Biotinylated isocoumarins, new inhibitors and reagents for detection, localization, and isolation of serine proteases

Abstract

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)

Cathepsin A Is the Major Hydrolase Catalyzing the Intracellular Hydrolysis of the Antiretroviral Nucleotide Phosphonoamidate Prodrugs GS-7340 and GS-9131

“…In addition, the specific activities of the purified enzyme and the recombinant CatA towards GS-7340 were virtually identical (35 nmol ⅐ min Ϫ1 ⅐ g). DFP and 3,4-DCI are effective inhibitors of serine hydrolases, including CatA (6,11,17,22,26,32). Purified prodrug hydrolase and the recombinant CatA were both inhibited by 3,4-DCI and DFP with IC 50 s of ϳ0.5 M and 5 to 10 M, respectively (Table 2).…”

Activity-based protein profiling: The serine hydrolases

“…However, this inhibitor displayed remarkable specificity for NTE in these experiments and thus appeared too selective to be useful as a general probe for serine hydrolases. Powers and colleagues had generated isocoumarin inhibitors coupled to biotin as serine hydrolase inhibitors (19,20). Although these isocoumarins reacted with a greater range of serine hydrolases than the aforementioned saligenin phosphoramidate, the requirement that these compounds alkylate a second functional group in the enzyme active site to achieve stable irreversible inhibition suggested that a significant number of serine hydrolases might show poor sensitivity to such reagents.…”

Utilization of biotinylated diphenyl phosphonates for disclosure of serine proteases

“…a-Aminoalkyl diphenyl phosphonate esters have been shown to be superb inactivators of serine proteases with no activity against cysteine proteases, are nontoxic in vivo, and are stable under physiological conditions [1,2]. Biotinylated aminoacyl chloromethanes and isocoumarins have found use as active-site-directed affinity labels for the disclosure of serine proteases from a variety of biological milieu and using techniques ranging from SDS-PAGE and Western blotting to immunocytochemistry and affinity purification [3][4][5][6]. We have previously reported on the synthesis of N-protected diphenyl phosphonate analogues of ornithine and lysine [7] and in this paper we detail the synthesis of the biotinylated analogues and report on their kinetic characterization and utilization for the detection of trypsin-like serine proteases using SDS-PAGE and Western blotting.…”