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Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.

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A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7(N-Cbz-glycylglycylargininamido)4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 12SI-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese The enhanced production of plasminogen activator (PA) activity has been shown to be a characteristic of many different cell types. The intracellular and extracellular levels of PA have been demonstrated to be substantially elevated in malignant cells in culture (1-6), cells treated with a tumor promoter (7), activated macrophages (8, 9), established cell lines (10, 11), granulosa cells during ovulation (12), embryonic cells during differentiation (13,14), and hormone-treated uteri (15). The standard system used for measuring PA in these cells is an indirect, two-step assay in which plasminogen is incubated with a source of PA and the plasmin activity generated is quantitated by using fibrin, casein, or protamine as substrates (16)(17)(18)(19). There is a need, however, for a simple, sensitive, direct assay that allows both rapid measurement and kinetic analysis of PA, independent of plasmin generation. In addition, the presence of two proteases of similar specificities in the two-step assay precludes the screening of potential PA inhibitors.A series of synthetic fluorogenic substrates, specific for a number of serine proteases, utilizing the leaving group 7-amino-4-methylcoumarin (AMC) has been described (20,21). In a continuation of this approach, we have now prepared a synthetic peptide specific for the cleavage site of PA, incorporating the same leaving group. This compound is Cbz-GlyGly-Arg-AMC. We report here the use of this substrate in the direct fluorescent assay of PA from normal and malignant cells, some kinetic parameters of these enzymes, and the effect of various low and high molecular weight protease inhibitors on the various enzymes. The various PAs were analyzed by the direct fluorescent technique in parallel with the indirect standard 25I-labeled fibrin plate assay using purified plasminogens from both canine and bovine sources. (24). When the cultures had attained high cell density (1 X 107 cells per 100-mm culture dish), the plates were washed three times with minimal medium and further incubated in serum-free minimal medium. The medium was removed from the cultures every 12 hr and was a source of extracellular PA; it is referred to as harvest fluid (HF). After it was harvested, the HF was immediately centrifuged to remove cells and cellular debris and acidified to pH 3.5 by the addition of 1 M HCl. Ammonium sulfate was added to 70% saturation, and the resulting precipitate was recovered by centrifugation and resuspended in 1/100 the volume of the original HF in 0.05 M glycine.HCI buffer, pH 3.0. MATERIALS AND METHODSWI-38, HeLa, and Chinese hamster ovary cells were grown in serum-containing medium, washed, and incubated in serum-free Higuchi medium (25). This me...