No abstract

The roles of cysteine proteases (CP) as protein degrading and protein processing enzymes both in physiological and pathological processes of mammals are well known. Furthermore, the key roles of CP;s in the life cycles of infectious agents like protozoa and viruses turn them into new important targets for anti-infective drugs. Thus, the effective inhibition of pathologically relevant cysteine proteases has raised increasing interest in drug development. One strategy to create CP inhibitors is the use of electrophilic moieties, which covalently bind to the cysteine residue of the active site of the target protease. In a previous approach we have selected the aziridine-2,3-dicarboxylic acid as weak electrophilic inhibitor fragment. In order to achieve effective enzyme inhibition this electrophile was incorporated into peptidic or peptidomimetic sequences addressing the substrate binding sites of the protease. High selectivity could be obtained with compounds, which bind into both the primed and non-primed substrate binding pockets. In a second approach the alpha,beta-unsaturated ketone of the well-known diuretic drug ethacrynic acid was found to be another appropriate electrophilic moiety. Derivatives thereof turned out to be new non-peptidic CP inhibitors. Results of inhibition assays obtained with these two inhibitor series on various proteases of human, protozoan, and viral origin, theoretical studies to investigate binding modes and inhibition mechanisms, and structure-activity relationships are presented. Furthermore, the results of in vitro assays on respective pathogens as well as the results of first toxicity studies are summarized.

A comprehensive screening of N-acylated aziridine (aziridide) based cysteine protease inhibitors containing either Boc-Leu-Caa (Caa=cyclic amino acid), Boc-Gly-Caa, or Boc-Phe-Ala attached to the aziridine nitrogen atom revealed Boc-(S)-Leu-(S)-Azy-(S,S)-Azi(OBn)(2) (18 a) as a highly potent cathepsin L (CL) inhibitor (K(i)=13 nM) (Azy=aziridine-2-carboxylate, Azi=aziridine-2,3-dicarboxylate). Docking studies, which also accounted for the unusual bonding situations (the flexibility and hybridization of the aziridides) predict that the inhibitor adopts a Y shape and spans across the entire active site cleft, binding into both the nonprimed and primed sites of CL.

Selectin-mediated leukocyte adhesion to endothelia, the crucial first step initiating the pathogenic cascade of inflammation, is an attractive target for specific therapies. The small-molecule macrolide, efomycine M, inhibits selectin-mediated leukocyte adhesion in vitro and in vivo, and effectively alleviates inflammatory disorders in vivo. To define the molecular basis of the therapeutically relevant antiadhesive properties of efomycines, several new species of this family were purified and/or synthesized. Efomycines E and G were isolated from Steptomyces BS1261. Efomycine O was synthesized by Lewis acid-catalyzed acetalization and efomycine M was generated by base-catalyzed deglycosylation. Efomycine S resulted from ester cleavage of the macrolide ring system, and efomycine T represents the peracetylated form of efomycine M. When the functional activity of efomycines on adhesion of leukocytes to vascular endothelium was studied, some remarkable differences between the compounds became apparent, inasmuch as efomycines E, G, M, and O significantly inhibited adhesion of both human and porcine leukocytes to the vascular endothelium, whereas efomycines S and T did not show any biological activity. A novel docking engine (ProPose), generating an improved, fully configurable protein-ligand interaction model, demonstrated that biological activities of efomycines can be predicted in silico, thus highlighting the utility of such combinatorial approaches.