Adrienne Healy

Inhibitors of the chymotrypsin-like activity of proteasome based on di- and tri-peptidyl α-keto aldehydes (glyoxals)

A series of peptidyl alpha-keto aldehydes (glyoxals) have been synthesised as putative inhibitors of the chymotryptic-like activity of proteasome. The most potent peptides, Cbz-Leu-Leu-Tyr-COCHO and Bz-Leu-Leu-Leu-COCHO, function as slow-binding reversible inhibitors, exhibiting final Ki values of approximately 3.0 nM. These are among the lowest values so far reported for (tri)peptide-based aldehyde-related inhibitors.

Comparative analysis of synovial fluid and plasma proteomes in juvenile arthritis--proteomic patterns of joint inflammation in early stage disease.

Abstract Synovial fluid is a potential source of novel biomarkers for many arthritic disorders involving joint inflammation, including juvenile idiopathic arthritis. We first compared the distinctive protein ‘fingerprints’ of local inflammation in synovial fluid with systemic profiles within matched plasma samples. The synovial fluid proteome at the time of joint inflammation was then evaluated across clinical subgroups to identify early disease associated proteins. We measured the synovial fluid and plasma proteomes using the two-dimensional fluorescence difference gel electrophoresis approach. Image analysis software was used to highlight the expression levels of joint and subgroup associated proteins across the study cohort (n =32). A defined subset of 30 proteins had statistically significant differences (p <0.05) between sample types such that synovial fluid could be differentiated from plasma. Furthermore distinctive synovial proteome expression patterns segregate patient subgroups. Protein expression patterns localized in the chronically inflamed joint therefore have the potential to identify patients more likely to suffer disease which will spread from a single joint to multiple joints. The proteins identified could act as criteria to prevent disease extension by more aggressive therapeutic intervention directed at an earlier stage than is currently possible.

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

A series of synthetic peptides in which the C‐terminal carboxyl grouping (‐CO2H) 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 α‐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 α‐keto ester grouping) was found to be the most effective exhibiting, for example, second‐order rate constants of approximately 1.7 × 106m−1min−1 and approximately 4.9 × 104m−1min−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.

Kinetic Analysis of the Cleavage of Human Protease-Activated Receptor-1 / 2 / 3 and 4 Using Quenched-Fluorescent Peptide Substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat) / K(m) values were determined.

Peptides containing Acylated C‐terminal Gem diamines: Novel Irreversible Inactivators of the Cysteine and Serine Proteinases†

This study reports on the synthesis of peptides containing C‐terminal acylated gem‐diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl‐Val‐Pro‐g‐Val‐CO‐O‐C6H4‐NO2 of neutrophil and pancreatic elastases that functions in a time‐dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long‐lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem‐diamine, Cbz‐Phe‐g‐Ala‐CO‐CH2Hal (Hal = Br, Cl).