John F. Lynas

Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN(2) (I) and Cbz-Leu-Pro-Ala-Thr-CH(2)Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtA(DeltaN)). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k(i)/K(i)) of 5.3x10(4) M(-1).min(-1), approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN(2) (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.

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.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

ABSTRACT Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (Ki = 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

Inhibition of escherichia coli glucosamine synthetase by novel electrophilic analogues of glutamine—comparison with 6-diazo-5-oxo-norleucine

A series of electrophilic glutamine analogues based on 6-diazo-5-oxo-norleucine has been prepared, using novel synthetic routes, and evaluated as inhibitors of Escherichia coli glucosamine synthetase. The gamma-dimethylsulphonium salt analogue of glutamine was found to be one of the most potent inactivators of this enzyme yet reported, with an apparent second order rate constant (k2/Ki) of 3.5 x 10(5) M(-1) min(-1).

Development of peptidyl α-keto-β-aldehydes as new inhibitors of cathepsin L — comparisons of potency and selectivity profiles with cathepsin B

Abstract We have utilized previously known substrate and inhibitor specificity profiles for the lysosomal cysteine protease, cathepsin L, to design a new series of putative inhibitors of this enzyme, based on di- and tri-peptidyl α-keto-β-aldehydes. Kinetic evaluation of these compounds revealed Z-Phe-Tyr(OBut)-COCHO, with a K i =0.6 nM, to be the most potent, synthetic reversible inhibitor of cathepsin L reported to date.

Synthesis and kinetic evaluation of peptide α-keto-β-aldehyde-based inhibitors of trypsin-like serine proteases

New, synthetic peptide analogues bearing a C‐terminal basic α‐keto‐β‐aldehyde moiety were prepared as novel inhibitors of the trypsin‐like serine proteases. The compounds, Ac‐Leu‐Leu‐Arg‐COCHO, Ac‐Arg‐Gln‐Arg‐COCHO and Boc‐Val‐Leu‐Lys‐COCHO were evaluated kinetically against trypsin and three other trypsin‐like serine proteases, tryptase, plasmin and thrombin, all of which are implicated as mediators of important disease processes. Results illustrate that α‐keto‐β‐aldehydes are potent inhibitors, with similar potency to comparable peptide aldehydes, and intriguingly, appear to act, in some instances, by a novel mechanism of action. Ac‐Leu‐Leu‐Arg‐COCHO, an analogue of the natural product leupeptin, is a potent, tight‐binding inhibitor of trypsin (Ki(final) = 1.9 μm), plasmin (Ki(final) = 4.9 μm) and tryptase (Ki(final) = 1.2 μm) and an irreversible inactivator of thrombin (k2nd 4500 m−1. min−1). Boc‐Val‐Leu‐Lys‐COCHO was found to be a tight‐binding inhibitor of its target protease plasmin (Ki(final) = 3.1 μm) and was inactive against thrombin. Ac‐Arg‐Gln‐Arg‐COCHO was a slow‐binding inhibitor of tryptase (Ki(final) = 1.6 μm) and also irreversibly inactivated trypsin (k2nd = 8920 m−1 min−1). Peptides or peptidomimetics with a C‐terminal basic α‐keto‐β‐aldehyde function thus provide a useful new molecular template for the development of new therapeutic agents against a wide range of disorders, such as coagulopathies and asthma, which may be mediated by the aberrant activity of trypsin‐like serine proteases.

Peptide argininol “inverse substrates” of anisic acid: Novel inhibitors of the trypsin-like serine proteinases

Abstract Peptides containing a C-terminal argininol residue linked, via an ester bond, to anisic acid have been synthesized as putative inhibitors of trypsin-like serine proteinases. The most potent analogue, Boc-Ile-Glu-Gly-Arg-ψ-(CH2O)COC6H4-OMe, that was modelled on a known recognition sequence for the clotting enzyme factor Xa, was found to inactivate the protease with a second-order rate constant of ∼4.5 × 105 M−1.min−1.

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).

Potent new leucine aminopeptidase inhibitor of novel structure synthesised by a modified Wadsworth-Emmons (Horner) Wittig procedure.

The use of a leucine-derived alpha-keto-beta-aldehyde (glyoxal) as a substrate in the Horner-Emmons (Wadsworth) Wittig reaction has enabled the synthesis of (Z)-7-methyl-5(S)-amino-4-oxo-methyl-oct-2-eneoate. This novel compound is a potent inhibitor (Ki = 76 nM) of leucine aminopeptidase and provides an interesting new template for the development of metallopeptidase inhibitors.