Potent inactivator of alpha-chymotrypsin: 2,2-dimethyl-3-(N-4-cyanobenzoyl)amino-5-phenyl pentanoic anhydride

We synthesized a novel potent alpha-chymotrypsin inactivator, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, which fulfilled the criteria of a mechanism-based inactivator: first-order kinetics, irreversibility, saturation kinetics and substrate protection. The inactivation rate constant (k(inact)) and the enzyme-inhibitor dissociation constant (K-I) were calculated to be 0.017 s(-1) and 0.071 mu M, respectively (k(inact)/K-I = 242000 M-1 s(-1)). These kinetic parameters indicate that this compound is one of the most powerful alpha-chymotrypsin inactivators ever reported. The average number of alpha-chymotrypsin turnovers per inactivation (partition ratio) was calculated to be 1, which indicates that it is a stoichiometrically ideal inactivator of alpha-chymotrypsin. We compared the IC50 values of this compound with those of several chymotrypsin-like serine proteinases (bovine alpha-chymotrypsin, recombinant human chymase and human neutrophil cathepsin G) and a metallo proteinase, rabbit angiotensin converting enzyme (ACE), Our compound, 2,2-dimethyl-3-(N-4-cyanobenzoyl) amino-5-phenyl pentanoic anhydride, inhibited bovine alpha-chymotrypsin potently (IC50 = 1.0 (+/- 0.2) x 10(-9) M) as well as other chymotrypsin-like serine proteinase; recombinant human chymase (IC50 = 7.0 (+/- 1.0) x 10(-8) M) and human neutrophil cathepsin G (IC50 = 1.8 (+/- 0.2) x 10(-7) M), However, rabbit ACE was not inhibited by this compound (IC50 > 1 X 10(-4) M). (C) 1997 Academic Press.