In vivo inhibition of cathepsin B by peptidyl (acyloxy)methyl ketones.

Peptidyl (acyloxy)methyl ketones, previously established as potent irreversible inhibitors of the cysteine proteinase cathepsin B in vitro, were investigated and optimized for their inhibitory activity in vivo. Incorporation of polar or charged functional groups in the inhibitor structure afforded effective cathepsin B inhibition, following dosing to rats. The most effective inhibitor, Z-Phe-Lys-CH2OCO-(2,4,6-Me3)Ph (8), was found to give ED50 values of 18 mg/kg po (orally) and 5.0 mg/kg ip (intraperitoneally) at 4-5 h postdose, and 2.4 mg/kg sc (subcutaneously) at 24 h postdose, for liver cathepsin B inhibition (measured ex vivo). The subcutaneous route of administration of (acyloxy)methyl ketone 8 also provided potent cathepsin B inhibition in certain peripheral tissues (e.g., ED50 1.0 mg/kg for skeletal muscle, 0.1 mg/kg for heart). These investigations demonstrate that peptidyl (acyloxy)methyl ketones such as 8 have promise as tools for the characterization of in vivo biochemical processes and as therapeutic agents.

Potent inactivation of cathepsins S and L by peptidyl (acyloxy)methyl ketones.

Peptidyl (acyloxy)methyl ketones (Z-Aa-Aa-CH2-O-CO-R), a new class of irreversible inhibitors whose chemical reactivity can be modulated by varying the substitution pattern of the carboxylate leaving group, are shown to be extremely potent inactivators of the lysosomal cysteine proteinases cathepsin L and cathepsin S. The highest k2/Ki values measured were found to exceed 10(6) M-1s-1 for both cathepsin L and cathepsin S. The rate of inactivation can be controlled by varying the dipeptidyl moiety or the carboxylate leaving group, with the second-order rate constants for both enzymes found to be strongly dependent on the pKa values of the leaving group. The specificities of the cathepsins S and L reveal a different selectivity towards the nature of substitution of the aryl P' leaving group of the inhibitor. This new inhibitor class opens the possibility of the design of selective and specific inhibitors for lysosomal cysteine proteinases.

Comparative behaviour of calpain and cathepsin B toward peptidyl acyloxymethyl ketones, sulphonium methyl ketones and other potential inhibitors of cysteine proteinases.

Peptidyl acyloxymethyl ketones, previously established as potent inactivators of the lysosomal cysteine proteinase cathepsin B, were evaluated against smooth-muscle calpain, a member of the family of Ca(2+)-dependent cysteine proteinases. Only modest rates of time-dependent inhibition could be achieved, even with peptidyl affinity groups optimized for calpain and linked to a carboxylate leaving group of very low pKa [2,6-(CF3)2PhCOO-, pKa 0.58]. Selective inactivation of cathespin B versus calpain was consistently observed with this type of inhibitor. Examination of other potential inhibitors revealed a rank order of potency against calpain to be: peptidyl sulphonium methyl ketones > fluoromethyl ketones, diazomethyl ketones >> acyloxymethyl ketones, an order which differs sharply from that found for cathespin B.

Peptidyl (acyloxy)methyl ketones and the quiescent affinity label concept: the departing group as a variable structural element in the design of inactivators of cysteine proteinases.

(Acyloxy)methyl ketones, of general structure Z-[AA2]-[AA1]-CH2OCOAr, are potent inactivators of the cysteine proteinase cathepsin B. These reagents have been designed as affinity labels in which the dipeptidyl moiety serves as an affinity group (complementary to the S1 and S2 sites of the enzyme), while the (acyloxy)methyl ketone unit (-COCH2OCOR), containing a weak leaving group in the form of a carboxylate nucleofuge, functions as the potentially reactive entity that labels the enzyme. The inhibition is time dependent, active site directed, and irreversible. The apparent second-order rate constant kinact/Kinact, which characterizes the inhibition of cathepsin B by this series, spans several orders of magnitude and in certain cases exceeds 10(6) M-1 s-1. The activity of this series of inhibitors was found to be exquisitely sensitive to the nature of the carboxylate leaving group as well as the affinity group. A strong dependence of second-order inactivation rate on leaving group pKa was uncovered for Z-Phe-Ala (acyloxy)methyl ketones [log(k/K) = 1.1 (+/- 0.1) X pKa + 7.2 (+/- 0.4); r2 = 0.82, n = 26]. Heretofore in constructing affinity labels the choice of leaving group was quite restricted. The aryl carboxylate group thus offers considerable variation as a design element in that both its binding affinity and reactivity can be controlled by substituent effects. Specific peptidyl (acyloxy)methyl ketones thus represent prime examples of highly potent, chemically stable enzyme inhibitors with variable structural elements in both the affinity and departing groups.

Peptidyl O-acyl hydroxamates: potent new inactivators of cathepsin B.

Peptidyl O-acyl hydroxamates having appropriate active-site recognition features are very potent time-dependent inhibitors of the cysteine proteinase cathepsin B. The inhibition is irreversible, and the inactivation rate is strongly dependent on peptide structure and correct positioning of the P1 amino acid carbonyl group. Lipophilic O-acyl groups provide the most rapid inactivators, as exemplified by the inhibitor O-mesitoyl N-benzyloxycarbonyl-L-phenylalanyl-L-alanine hydroxamate (kmax/Ki = 640,000 M-1s-1).