Inhibition of serine proteases by peptidyl fluoromethyl ketones

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Abstract
We have synthesized peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases .alpha.-chymotrypsin and porcine pancreatic elastase. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the .gamma.-OH group of the active site serine to form a stable hemiacetal [Lowe G., and Nurse, D. (1977) J. Chem. Soc., Chem. Commun., 815; Chen, R., Gorestein, D. G., Kennedy, W. P., Lowe, G., Nurse, D., and Schultz, R. M. (1979) Biochemistry 18, 921; Shah, D. O., Lai, K., and Gorenstein, D. G. (1984) J. Am. Chem. Soc. 106, 4272]. 19F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF31 and Ac-ambo-Phe-CF3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrate or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of Ki concomitant with the change from a dipeptide analogue to a tetrapeptide analogue (Ac-Pro-ambo-Ala-CF3, Ki = 3 .times. 10-3 M; Ac-Ala-Ala-Pro-ambo-Ala-CF3, Ki = 0.34 .times. 10-6 M) correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues [Bartlett, P. A., and Marlowe, C. K. (1983) Biochemistry 22, 4618; Thompson, R. C. (1973) Biochemistry 12, 47]. In addition to chain length, the number of fluorine substituents also affects the Ki. In the case of chymotrypsin, the Ki for Ac-Leu-ambo-Phe-CF3 is 30-fold lower than that for Ac-Leu-ambo-Phe-CF2H (0.88 .times. 10-6 M vs. 25 .times. 10-6 M). With elastase this trend is not as profound. In all cases, however, the difluoro- and trifluoromethyl ketones are better inhibitors than the monofluoromethyl and nonfluorinated analogues. This improvement must be associated with both the degree of hydration of the fluoromethyl ketones and the significant effect that fluorine substitution has on lowering the first pKa of the hemiacetal hydroxyl. The latter change would cause the more fluorinated inhibitor to be able to interact better with the anionic hole near the active site. Fluorine substitution also lowers the koff values for the inhibitors. With elastase the trifluoromethyl ketone tetrapeptide has a koff of 1.25 .times. 10-4 s-1, while the corresponding difluoromethyl compound has a koff of 0.007 s-1. The monofluoromethyl ketone inhibitor of chymotrypsin, Ac-Leu-ambo-Phe-CFH2, is a weak competitive inhibitor (Ki = 200 .times. 10-6 M). It also demonstrates time-dependent irreversible inhibition with a second-order rate constant of 1.7 M-1 s-1. The irreversible inhibition is accompanied by covalent modification of a histidine residue and by fluoride ion release as detected by 19F NMR spectroscopy.