title: Structure-activity studies of fluoroketone inhibitors of alpha-lytic protease and human leukocyte elastase.
abstract: We have synthesized a series of peptidyl fluoroketones that reversibly inhibit the serine proteases human leukocyte elastase (HLE) and alpha-lytic protease (alpha-LP). Ac-ambo-AlaCF3 (1) inhibits HLE and alpha-LP with Ki's of 2.4 and 15 mM, respectively. The effects of structural variations on this parent compound on Ki and the kinetics of inhibition were studied. The acetyl group was replaced by the tripeptide Z-L-Ala-L-Ala-L-Pro to yield the tetrapeptide trifluoroketone (TFK) Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF3 (2). This extension reduced Ki 3500-fold for HLE and 3000-fold for alpha-LP. Removal of a fluorine atom from a TFK decreases Ki about 15- to 30-fold with both enzymes. Replacement of one fluorine atom of 2 by a residue (-CH2-CH2-COLeuOMe) (6) which can interact with the S'1 and S'2 subsites decreased Ki 30-fold for HLE and 150-fold for alpha-LP compared to Z-L-Ala-L-Ala-L-Pro-ambo-AlaCF2H (3). The Ki of 6 for HLE is approximately equal to that of trifluoroketone 2. For alpha-LP Ki of 6 is 10-fold lower than that for the trifluoroketone 2. Inhibitors with Ki values less than 10(-7) M exhibit slow binding kinetics. By analogy to cholinesterases and chymotrypsin, it is likely that these enzymes combine with the keto form of the inhibitor to form the enzyme-inhibitor complex. Therefore, kon and Ki were corrected for the ketone concentration. The corrected kon values for the slow binding inhibitors are in most cases less than diffusion controlled, ranging between 8.2 X 10(4) and 4.68 X 10(6) M-1 s-1. An exception is Z-L-Ala-L-Ala-L-Pro-ambo-ValCF3 (8) where kon = 9 X 10(7) M-1 s-1, which is nearly diffusion controlled.
authors.affiliation: Brandeis University, Graduate Department of Biochemistry, Waltham, Massachusetts 02254.
authors.forename: C P
authors.initials: CP
authors.lastname: Govardhan
authors.forename: R H
authors.initials: RH
authors.lastname: Abeles
mesh_terms: D000595:Amino Acid Sequence; D005461:Fluorine; D006801:Humans; D007659:Ketones / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D019272:Leukocyte Elastase; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology*; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007659:Ketones; D009842:Oligopeptides; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D005461:Fluorine; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase