Vol. 294, No. 1, April, pp. 144-146, 1992

Inhibitors of Human Neutrophil Cathepsin G: Structural and Biochemical Studies William C. Groutas,l Michael J. Brubaker, Rhadika Venkataraman, Michael A. Stanga, and Jerald J. McClenahan Department

of Chemistry,

Wichita State University,

Received October 16,1991, and in revised form November

Wichita, Kansas 67208


The interaction of a series of sulfonate and phosphate esters derived from N-hydroxysuccinimide with human leukocyte cathepsin G was investigated. The synthesized compounds were found to be time-dependent inhibitors of the enzyme. The composite interplay of steric and electronic effects leads to the formation of acyl enzymes of variable stability, ultimately resulting in partial or full recovery of enzymatic activity. Compounds acting via phosphorylation of the active site serine inactivated 0 1992 Academic the enzyme rapidly and irreversibly. Prees,


Polymorphonuclear leukocytes serve as an important host defense against bacterial and fungal infections through the intracellular release of an assortment of antibacterial proteins and proteolytic enzymes (l-3). The group of antibiotic proteins, collectively termed serprocidins (4, 5), includes the serine proteinases elastase, cathepsin G (cath G),’ proteinase 3, and azurocidin. These are stored in azurophil granules and are released extracellularly following neutrophil activation. Although azurocidin is closely related to elastase, cath G, and PR-3 in terms of sequence homology, basic pl, and spectrum of antibiotic activity, it lacks proteolytic activity. Several lines of evidence suggest that neutrophil-derived mediators play an important role in noninfectious inflammatory diseases (6). Tissue damage results from the unrestrained degradative action of proteolytic enzymes released extracellularly. Currently, there is an incomplete understanding of the fundamental biochemical mechanisms underlying the pathogenesis of these diseases. Furthermore, the relative importance and role of each mediator have not been fully ascertained. ’ To whom correspondence should be addressed. * Abbreviations used: cath G, cathepsin G; HLE, human leukocyte elastase. 144

Jeffrey B. Epp,

There is ample literature evidence in support of the hypothesis that cathepsin G may play a prominent role in inflammatory states. For example, cath G is known to activate matrix metalloproteinase 3 (stromelysin) (7), collagenase (8), and platelets (9). It also degrades basement laminin (lo), mediates glomerular injury in uiuo (ll), and stimulates airway gland serous cell secretion (12). Thus, specific inhibitors of cath G are of value as probes and as potential therapeutic agents. Our research endeavors have focused on the development of inhibitors of neutrophil-derived proteolytic enzymes based on the 3-alkyl-N-hydrosuccinimide system I (13-16). The Ri group in structure I (Fig, 1) serves as a recognition element that meets the specificity requirements of the target proteinase and is accommodated at the S1 subsite (16) of the enzyme. The main goal of the present study was the development of specific and potent inhibitors of cathepsin G through the structural manipulation of structures I and II. This work describes the inhibitory activity of a series of compounds toward cathepsin G. EXPERIMENTAL


General. Human neutrophil cathepsin G was obtained from Athens Research and Technology Co. Methoxysuccinyl-Ala-Ala-Pro-Phe-pnitroanilide was purchased from Sigma Chemical Co. (St. Louis, MO). A Gilford uv/vis spectrophotometer was used in the enzyme assays and inhibition studies. The synthesis and characterization of compounds 1, 2,4,6,9, 11, and 13-16, have been reported previously (13, 16). All new compounds, i.e., 3,6,7,8, and 10 were synthesixedusingprocedures similar to those described earlier (16). Enzyme assays and inhibition studies. Human cathepsin G was assayed by mixing 20 pl of a 63.5 pM enzyme solution (in 0.05 M sodium acetate buffer, pH 5.5), 10 nl dimethyl sulfoxide, and 970 ~1 Hepes buffer, pH 7.5, and placed in a thermostated test tube. A lOO-nl aliquot was transferred to a thermostated cuvette containing 660 nl Hepes buffer and 20 pl of a 46 mM solution of methoxysuccinyl-Ala-Ala-Pro-Phep-nitroanilide and the change in absorbance was monitored at 410 nm for 1 min. In a typical inhibition run, 10 ~1 of a 6.34 X lob4 M solution of the inhibitor in dimethyl sulfoxide was mixed with 20 ~1 63.5 pM enzyme 0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.










1 J-I


solution and 970 rlO.1 M Hepes buffer, pH 7.5, and placed in a constant temperature bath. One-hundred microliters was withdrawn at different time intervals and transferred to a cuvette containing 20 pl of a 46 mM substrate solution and 860 pl Hepes bufl’er. The absorbance was monitored at 410 nm for 1 min. The pseudo-first-order inactivation rate constants (&) were obtained from plots of ln(u&-,) vs t and expressed in terms of the apparent second-order inactivation rate constants, kJ [I] M-’ s-l (Table I). These are the average of duplicate determinations.




Cathepsin G (EC is a chymotrypsin-like serine proteinase possessing broad-spectrum antibacterial activity that is independent of its proteolytic activity (17). This 30-kDa glycoprotein has an extended binding site that has been mapped by Powers and co-workers using peptidyl p-nitroanilide substrates (18, 19). The enzyme shows a strong preference for hydrophobic substrates with a Phe residue at the S, subsite and Pro and Val or Thr at the SZ and S3 positions, respectively. Consequently, structures I and II were modified accordingly and the interaction of the resulting compounds with cathepsin G was then investigated. All the compounds listed in Table I were found to affect the activity of the enzyme in a time-dependent fashion. The pseudo-first-order rate constants were obtained by using the linear portion of plots of ln(uJu,) vs time and expressed in terms of the second-order inactivation rate constants k,b/[I] M-l s-l. The time course of the inhibition appeared to be greatly dependent on subtle structural variations in I and II. In the case of compounds l12, rapid acylation of the enzyme was followed by partial (compounds 11,12) or total (compounds l-10) recovery of enzymatic activity. Figure 2 depicts the time course of the inactivation of cathepsin G by compounds 2,12, and 13. The interaction of compounds l-10 with cath G was similar to that of 2, the half lives of reactivation with these compounds being in the neighborhood of 6-8 h. The results suggest that compounds l-10 form acyl enzymes of variable stability that deacylate slowly, and therefore appear to function as alternate substrate inhibitors (20-22).

G by Compounds

Compound 1 2 3

L = -0SOyR2

of Cathepsin


4 5 6 7 8 9 10 11 12

of Structure


Rz Methyl trans-styry1 m-Trifluoromethyl phenyl Methyl trawstyry1 Methyl tmwstyry1 Phenyl Methyl trarwstyry1 Methyl trwwstyry1

Benzyl Benzyl Benzyl o-Trifluoromethylbenzyl o-Trifluoromethylhenzyl m-Trifluoromethybenzyl m-Trifluoromethylbenzyl m-Trifluoromethylbenzyl m-Fluorobenzyl m-Fluorobenzyl p-Trifluoromethylhenzyyl p-Trifluommethylbenzyl

a Values in parentheses correspond to those obtained leukocyte elastase and are taken from Ref. (16).

2260 (1050)" 3780 (9430) 1200 (320) 4360 (190) 5470(1860) 2370(220) 2670 1110 2390 (370) 3120 265 (SO) 340

using human

Only a partial regain in enzymatic activity was observed with compounds 11 and 12 after 24 h (40 and 30%, respectively). The longer lasting inhibition of cathepsin G by compounds 11 and 12 suggests that the enzyme is inactivated by a mechanism similar to the one established for the inactivation of HLE by compound I, namely, acylation of the active site serine leads to the formation of a highly reactive isocyanate via an enzyme-induced Lossen rearrangement. Subsequent trapping of the electrophilic species by an active site nucleophile, such as His57, results in irreversible inactivation of the enzyme (15,








FIG. 2. Time dependence of enzymatic activity. Human leukocyte elastase (HLE) was incubated with each inhibitor under following conditions: (a) HLE, 1.27 pM, compound 2, 13.9 pM; (b) HLE, 1.26 jiM, compound 12,62.7 pM; (c) HLE, 1.26 pM, compound 13,13.0 pM; Hepes buffer, pH 7.2, 0.5 M NaCl, 1% DMSO.



TABLE II Inhibition

Compound 13 14 15 16

of Cathepain

G by Compounds



Hydrogen Isobutyl Hydrogen Isobutyl

Benzyl Benzyl Ethyl Ethyl


of Structure


hibitors of proteolytic enzymes, and that specificity for a target proteinase can be achieved through appropriate structural manipulations.


M-’ 8-l b b 10 30

(3170)” (6180) (970) (2740)

a Values in parentheses correspond to those obtained using human leukocyte elastase and are taken from Ref. (13). * Inactivation was too fast to measure by ordinary sampling techniques.

ACKNOWLEDGMENT The generous financial support of this work by the National of Health is gratefully acknowledged (HL 38048).


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Agents Actions 8,3-10.

16). It is evident from these observations that the stability of the acyl enzyme intermediate is greatly affected by small structural changes in each inhibitor. The -CF3 and -F groups increase the hydrophobic character of the compounds and at the same time influence the rate of deacylation of the acyl enzyme. This, in turn, affects the relative partitioning of the reactive species (20-22). It was observed earlier that the presence of a trurzsstyryl (Ph-CH=CH-, where Ph = phenyl) in an inhibitor increases its inhibitory activity toward leukocyte elastase (HLE) and proteinase 3 considerably (compare, for example, the ,&,,/[I] values obtained with HLE, and shown in parentheses in Table I, for compounds 1 and 2, and compounds 4 and 5). The interaction between the aromatic ring of the truns-styryl group, and an as yet unidentified aromatic residue located in the vicinity of the active site was proposed as a likely reason for this (13-16, 23). The modest beneficial effect of the trunsstyryl group observed with cath G suggests the absence of an effective aromatic-aromatic binding interaction, and is probably due to the greater hydrophobicity of the compounds. We have recently shown that phosphate esters of Nhydroxysuccinimide II inactivate HLE irreversibly by phosphorylating the active site serine, and that they do so by binding to the active site with the & group in the primary specifity pocket (SJ (13). As anticipated, the introduction of a benzyl group into structure II gave highly potent compounds (Table II). For example, the inactivation of cathepsin G by compounds 13 and 14 was rapid and irreversible (Fig. 2). Compounds with Rr = ethyl (15 and 16) were either inactive or marginally active, in accord with the known preference of the enzyme for an aromatic residue at its Sr subsite. In conclusion, the results of the present study indicate that structures I and II represent general classes of in-

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Inhibitors of human neutrophil cathepsin G: structural and biochemical studies.

The interaction of a series of sulfonate and phosphate esters derived from N-hydroxysuccinimide with human leukocyte cathepsin G was investigated. The...
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