Vol. 297, No. 1, August 15, pp. 144-146, 1992
Enantioselective Inhibition of Human Leukocyte Elastase William C. Groutas,’ Michael J. Brubaker, Radhika Venkataraman, and Michael A. Stanga Department
Wichita State University,
Wichita, Kansas 67208
Received March 10, 1992
(RS)-Diethyl-2-benzylsuccinate was resolved using achymotrypsin. The two enantiomers were then elaborated to yield (S)-(+) and (IQ-(-)-3-benzyl-N-[(methylsulfonyl)oxy]succinimide and the inhibitory activity of the two enantiomers toward human leukocyte elastase was subsequently determined. The k,/K, values for the R and S isomers were found to be 330 and 1500 M-’ s-l, 0 1992 Academic Press, Inc. respectively.
Human leukocyte elastase (HLE)’ has been implicated in the degradation of lung elastin (1) and basement membrane components (2). The unrestrained proteolytic action of HLE on lung elastin is believed to be the likely cause for the pathogenesis of pulmonary emphysema (3). Modulation of the activity of HLE through the use of inhibitors has been the focus of many recent investigations (4, 5). The inhibition of HLE and chymotrypsin (a-CT) by (RS)-3-benzyl-N-[(methylsulfonyl)oxy]succinimide and related compounds, as well as pertinent mechanistic and stability studies, has been reported by us recently (6-8). The typically enantioselective nature of enzyme-substrate and enzyme-inhibitor interactions has prompted us to investigate the inhibitory activity of the optically active isomers of 3-benzyl-IV-[(methylsulfonyl)oxy]succinimide toward HLE and a-CT, and the results of our studies are reported herein. EXPERIMENTAL
General. Human leukocyte elastase (EC 220.127.116.11) was purchased from Elastin Products Co., (Ownesville, MO). Chymotrypsin, methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide, and N-benzoyl-L-tyrosine ethyl ester were purchased from Sigma Chemical Co. (St. Louis, MO). The ir and NMR spectra were recorded on a Perkin-Elmer 1330 infrared
’ To whom correspondence should be addressed. Fax: (316)689-3431. ‘Abbreviations used: HLE, human leukocyte elastase; o-CT, chymotrypsin. 144
spectrophotometer and a Varian XL-300 nuclear magnetic resonance spectrometer, respectively. A Gilford uv/vis spectrophotometer was used in the enzyme assays and inhibition studies. A Jasco DIP-360 automatic digital polarimeter was used to measure the optical rotations. Chemical and biochemical studies. Racemic 2-benzyl diethyl succinate was resolved using a-chymotrypsin (9). The optically active monoester and diester were then hydrolyzed to yield the optically P-benzyl succinic acids. The R and S diacids were then transformed into the desired enantiomers (R)-(-)-1 and (S)-(+)-2 according to Scheme I and using synthetic methodology previously described by us (6,7). (R)-(-)-3-Benzyl-N-[(methylsulfonyl)oxy] succinimide 1 was obtained as a white solid, mp 73-74”C, [~]~o -52.7 (c 1.59, EtOAc). ‘H NMR (dimethyl sulfoxide4): 2.55 (dd, lH), 2.79 (dd, lH), 2.89 (dd, lH), 3.14 (dd, lH), 3.37 (m, lH), 3.55 (s, 3H), 7.3 (m, 5H). Infrared (Nujolk1725 cm-i (C=O). (S)(+)-3-Benzyl-N-[(methylsulfonyl)oxy] succinimide 2, mp 75-76”C, [011z50 +56.9 (c 1.5, EtOAc), has ir and NMR spectra identical to those of 1. The optical purity of compounds 1 and 2 was determined using a chiral shift reagent, as described in the literature (10, 11). The enzyme assays and inhibition studies were carried out as described in detail previously (6, 7). The kinetics data were analyzed according to Kitz and Wilson (12). The apparent pseudo-first-order inactivation rate constants were determined from the slopes of the semilogarithmic plots of enzymatic activity remaining vs time using In (E,/E,) = kob t, where E,/E, is the amount of active enzyme remaining after time t. The k,/K, values for the two enantiomers were computed by determining the keb values at various inhibitor concentrations and replotting the data according to (12)
where KI and kz are the dissociation constant for the enzyme-inhibitor complex and the limiting rate constant for the irreversible loss of enzymatic activity, respectively.
RESULTS AND DISCUSSION The observation that racemic 3-benzyl-N-[(methylsulfonyl)oxy] succinimide is an effective inhibitor of human leukocyte elastase and a-chymotrypsin (6), prompted us to probe the nature of the interaction of (S)-(+)- and (R)-(-)-3-benzyl-N-[(methanesulfonyl)oxy] succinimide with HLE. The present investigation aimed at determining the relative inhibitory activity of each enantiomer toward HLE, in order to gain some insight about the enantioselective inhibition of HLE by derivatives of 3alkyl-N-hydroxysuccinimide (6, 7). 0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
1. (a) 1 N NaOH,
Incubation of HLE with either the (S)-(+)- or the (R)(-)-isomer lead to time-dependent inactivation of the enzyme (Fig. 1). Analysis of the kinetics data according to Eq. (l), and as illustrated in Fig. 1, yielded the limiting rate constant (k2) for the loss of enzymatic activity and the dissociation constant of the enzyme-inhibitor complex (Ki) for each enantiomer. The k2 and K1 values, and the corresponding bimolecular rate constants ( k,/I&, M-l s-l), are summarized in Table I. It is evident from Table I that both enantiomers are effective inhibitors of HLE. (S)-(+)-2 inactivates the enzyme efficiently with a bimolecular rate constant that is about five times greater than that of the (R)-(-)-1 isomer. This difference is primarily due to the higher stability of the enzyme-inhibitor complex formed with (S)-(+)-2. The Kr of the (S)-isomer 2 is 22-fold lower than that of the (R)-isomer 1.
Inhibitory Activity of (S)-(+)- and (R)-(-)-3-Benzyl-N[(methylsulfonyl)oxy]succinimide toward Human Leukocyte Elastase Compound (W-)-l
k,/K, (M-l s-l)
0.15 mM 6.76 /.LM
(d) 10% Pd-C/H,,
2 (e) CH,SO,Cl/pyridine.
The inactivation of chymotrypsin by the (R)- and (S)isomers is much more efficient, and was too fast to measure by ordinary sampling methods. The higher efficiency of the two enantiomers toward chymotrypsin is not surprising and simply reflects the known preference of chymotrypsin for an aromatic amino acid as the Pi residue (13). The preferred Pi residue for HLE is valine; however, the enzyme exhibits broad substrate specificity and monomeric substrates or inhibitors having an aromatic ring can also be accommodated at the primary specificity site (S,) of the enzyme (14-16). The low enantioselectivity observed with compounds 1 and 2 can be rationalized in terms of Cohen’s active site model for a-CT (9, I’?), whereby both enantiomers can fit into the active site in ways that place the aromatic ring at the hydrophobic primary specificity site (ar-site) of the enzyme and the C-2 carbonyl group of each inhibitor at the hydrolytic n-site. This is followed by nucleophilic attack by the active site serine, leading to irreversible inactivation of the enzyme, via a cascade of steps previously shown by high-field NMR to include an enzymeinduced Lossen rearrangement (8). Theoretical calculations indicate that compounds 1 and 2 assume a minimum energy geometry that is complementary to the horseshoelike geometry of the active site cleft of HLE (18). In conclusion, this study has demonstrated that inhibitors derived from 3-alkyl-N-hydroxysuccinimide, such as, for example, 3-benzyl-N-[(methylsulfonyl)oxy]-
Time (min) FIG. 1. Kinetics of inactivation of leukocyte elastase by (R)-(-)-3-benzyl-N-[ (methylsulfonyl)oxy]succinimide, 1. Leukocyte elastase (288 nM) was incubated with compound 1 (2.88 PM); 4.32 MM; 5.76 pM; 7.2 pM) in 0.1 M Hepes buffer, pH 7.2, 25°C and 1% dimethyl sulfoxide. Aliquots were withdrawn periodically and assayed for enzymatic activity using methoxysuccinyl-Ala-Ala-Pro-Valp-nitroanilide (84.8 PM). Inset: Kitz and Wilson analysis of the kinetics data [see text and Ref. (12) for details].
succinimide, possess structural features that allow them to associate with HLE and a-CT in a way that leads to inhibition of the enzyme by both enantiomers.
8. Groutas, W. C., Stanga, M. A., and Brubaker, Chem. Sot. 111,1931-1932. 9. Cohen, S. G., and Milanovic,
M. J. (1989) J. Am.
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ACKNOWLEDGMENT The financial assistance of the National 38048) is gratefully acknowledged.
10. Whitesides, 6979-6980.
G. M., and Lewis, D. W. (1970) J. Am. Chem. Sot. 92,
11. Parker, D. (1991) Chem. Rec. 91, 1441-1457. 12. Kitz, R., and Wilson, I. B. (1962) J. Biol. Chem. 237, 3245-3249.
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