Synthesis of Peptidyl Ene Diones: Selective Inactivators of the Cysteine Proteinases

Abstract

A series of synthetic peptides in which the C-terminal carboxyl grouping (-CO(2)H) of each has been chemically converted into a variety of ene dione derivatives (-CO-CH=CH-CO-X; X = -H, -Me, -OBut, -OEt, -OMe, -CO-OMe), have been prepared and tested as inactivators against typical members of the serine and cysteine protease families. For example, the sequences Cbz-Pro-Phe-CH=CH-CO-OEt (I) which fulfils the known primary and secondary specificity requirements of the serine protease chymotrypsin, and Cbz-Phe-Ala-CH=CH-CO-OEt (II) which represents a general recognition sequence for cysteine proteases such as cathepsins B, L and S, have been tested as putative irreversible inactivators of their respective target proteases. It was found that, whereas II, for example, functioned as a time-dependent, irreversible inactivator of each of the cysteine proteases, I behaved only as a modest competitive reversible inhibitor of chymotrypsin. Within the simple ester sequences Cbz-Phe-Ala-CH=CH-CO-R, the rank order of inhibitor effectiveness decreases in the order R = -OMe > -OEt >> -OBut. It was also found that the presence of both an unsaturated double bond and an ester (or alpha-keto ester) moiety were indispensable for obtaining irreversible inactivators. Of the irreversible inactivators synthesized, Cbz-Phe-Ala-CH=CH-CO-CO-OEt (which contains a highly electrophilic alpha-keto ester grouping) was found to be the most effective exhibiting, for example, second-order rate constants of approximately 1.7 x 10(6)M(-1)min(-1) and approximately 4.9 x 10(4)M(-1)min(-1) against recombinant human cathepsin S and human spleenic cathepsin B, respectively. This initial study thus holds out the promise that this class of inactivator may well be specific for the cysteine protease subclass.

Derivatives of aryl amines containing the cytotoxic 1,4-dioxo-2-butenyl pharmacophore

“…A number of studies report that protein thiolation plays an important role in biochemical processes. Some representative examples of this phenomenon include inhibition of human cytomegalovirus protease, 16 inhibition of Ref-1, a therapeutic target for asthma, 17 inactivation of cysteine proteases, 18 blockade of platelet-derived growth factor BB-stimulated Akt phosphorylation, 19 catalytic inhibition of human topoisomerase-2 alpha, an established target for the development of anticancer agents, 20,21 etc. This encouraged us the study the reactivity of representative compounds in each of the series 1 – 5 and 9 towards a model thiol, benzyl mercaptan, under simulated physiological conditions These experiments were undertaken in order to evaluate whether the compounds in these series are in fact thiol alkylators and, if so, to determine the locus of thiolation.…”

Dipeptide-derived nitriles containing additional electrophilic sites: Potentially irreversible inhibitors of cysteine proteases

“…Irreversible inhibitors are therefore attractive candidates for treating microbial infections and valuable tools for elucidating the in vivo functions of the enzyme. Compounds of different classes have been reported as irreversible inactivators of cysteine proteases 3 , including peptide-derived epoxides 2,4 and related aziridines 5 , diazoketones 6 , and Michael acceptors such as vinyl sulfones and peptidyl ene diones [7][8][9] . The inherent chemical reactivity of these agents limits their applicability due to unspecific reactions with other biomolecules.…”

On-Bead Screening of a Combinatorial Fumaric Acid Derived Peptide Library Yields Antiplasmodial Cysteine Protease Inhibitors with Unusual Peptide Sequences

“…Several efficient irreversible inhibitors of proteases based on activated olefins are known. For example, the fumaric acid derivative of the well-known epoxysuccinyl peptide ( S , S )-epoxysuccinyl-( S )-leucylamido(4-guanidino)butane (E-64), the papain inhibitor DC-11, has been shown to irreversibly block CAC1 cysteine proteases, and recently related peptidyl ene diones and azapeptide Michael acceptors have been identified as potent inactivators of cysteine proteases. Other fumaric acid derivatives have also been tested against these proteases. , In order to further explore the suitability of fumaric acid derivatives, we established a synthetic strategy for a fumaric acid diamide library and developed on-bead screening assays for falcipain-2 and rhodesain.…”

“…Several efficient irreversible inhibitors of proteases based on activated olefins are known. For example, the fumaric acid derivative of the well-known epoxysuccinyl peptide (S,S)epoxysuccinyl-(S)-leucylamido(4-guanidino)butane (E-64), the papain inhibitor DC-11, 19 has been shown to irreversibly block CAC1 cysteine proteases, and recently related peptidyl ene diones 20 and azapeptide Michael acceptors 21 have been identified as potent inactivators of cysteine proteases. Other fumaric acid derivatives have also been tested against these proteases.…”