Inhibition of the serine proteases leukocyte elastase, pancreatic elastase, cathepsin G, and chymotrypsin by peptide boronic acids

Three alpha-aminoboronic acid-containing analogs of good peptide substrates for serine proteases were synthesized, MeO-Suc-Ala-Ala-Pro-boro-Phe-OH, MeO-Suc-Ala-Ala-Pro-boro-Ala-OH, and MeO-Suc-Ala-Ala-Pro-boro-Val-OH. They were effective inhibitors of chymotrypsin, cathepsin G, and both leukocyte and pancreatic elastase at nanomolar concentrations (0.10-20 nM). Except for cathepsin G, inhibition was not simply competitive, but showed kinetic properties corresponding to the mechanism for slow-binding inhibition, i.e. E + I in equilibrium EI in equilibrium EI*, where EI and EI* are enzyme-inhibitor complexes and EI* is more stable than EI. This type of inhibition has not been observed previously for synthetic inhibitors or serine proteases and in this study it was observed only for peptide boronic acids which satisfy the primary specificity requirements of the protease.     

Inhibition of human leukocyte elastase, cathepsin G, chymotrypsin A alpha, and porcine pancreatic elastase with substituted isobenzofuranones and benzopyrandiones

Several 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones, 3-(1-haloalkylidene)-1(3H)-isobenzofuranones, and 3-bromomethyl-1H-2-benzopyran-1-ones containing masked halo ketone functional groups were synthesized and tested as inhibitors of several serine proteases including human leukocyte (HL) elastase and cathepsin G. While many of the 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones were quite potent inhibitors of the enzymes tested, the alkylideneisobenzofuranones and benzopyran-1-ones inhibited poorly or not at all. The 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones decomposed rapidly upon addition to buffer to give the corresponding 3-alkyl-1H-2-benzopyran-1,4(3H)-diones. The pure benzopyran-1,4-diones were extremely potent inhibitors of HL elastase and chymotrypsin A alpha but did not inactivate porcine pancreatic elastase or cathepsin G. Enzymes inhibited by the isobenzofuranones and benzopyran-1,4-diones regained activity slowly upon standing or after dialysis (t1/2 = 5-16 h) and more rapidly in the presence of 0.5 M hydroxylamine, which indicated the presence of labile acyl moieties in the inhibited enzyme. These results are consistent with a scheme in which the active site serine of the protease reacts with the lactone carbonyl of these inhibitors to give a stable acyl enzyme and alkylation of another active site residue by the unmasked halo ketone functional group does not occur.     

Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.

Five sensitive substrates of human leukocyte and porcine pancreatic elastase having the sequence MeO-Suc-Ala-Ala-Pro-Val-X where -X is -NA (4-nitroanilide), -SBzl (thiobenzyl ester), -OEt, -AMC (4-methyl-7-coumarylamide), or -NNapOMe (1-methoxy-3-naphthylamide), were synthesized. The kinetic constants for the enzymatic hydrolysis as well as the sensitivity of each substrate are reported. Hydrolysis of the peptide -AMC and -NNapOMe derivatives were followed by monitoring spectrofluorometrically the release of H-AMC and H-NNapOMe, respectively. Cleavage of the thiobenzyl ester yields benzyl mercaptan as the hydrolysis product. Its release was monitored at 412 nm by reaction with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the assay mixture to produce the 3-carboxy-4-nitrothiophenoxide anion or at 324 nm by reaction with 4,4′-dithiodipyridine to produce 4-thiopyridone. Hydrolysis of the ethyl ester was measured using a coupled assay with NAD⁺ and liver alcohol dehydrogenase. The NADH⁺ formed upon oxidation of the ethanol released from cleavage of the ester was followed at 340 nm. MeO-Suc-Ala-Ala-Pro-Val-SBzl, the best substrate of the series, was capable of detecting as little as 2.4 pm (0.072 ng/ml) of active-site titrated human leukocyte elastase and 5.8 pm (0.15 ng/ml) of active-site titrated porcine pancreatic elastase using 4,4′-dithiodipyridine. The corresponding values with Ellman's reagent were 5.0 pm (0.15 ng/ml) and 7.4 pm (0.19 ng/ml), respectively. Advantages of this substrate are its high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and ease of synthesis. One disadvantage is the interference of high concentration of thiols with the assay. The peptidyl-AMC is almost as sensitive as the thiobenzyl ester and can detect 11 pm of HL elastase and 18 pm of PP elastase. An advantage of this substrate is the fact that cleavage involves a peptide bond. Disadvantages are relatively low $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and greater difficulty in synthesis. The peptidyl-NNapOMe has possible utility in histochemical studies due to the low intrinsic fluorescence of the substrate relative to the peptidyl-AMC. For a rate assay it has a lower sensitivity than either the thiobenzyl ester or peptidyl-AMC. The coupled liver alcohol dehydrogenase-ethyl ester assay offers no advantages except in cases where the ester substrate is commercially available. The 4-nitroanilide assay enjoys moderate sensitivity and is extremely convenient for routine use. Except for the peptidyl ethyl ester assay, all of the human leukocyte elastase assays reported in this paper are vastly more sensitive than any other existing assay for this protease.