Uhlmann et al., 2000). All members are characterised by a catalytic dyad which is present in a His-Gly-spacer-Ala- Cys motif. Comparison of the crystal structures of cas- pase 1 and 3 with gingipain revealed a very similar active centre topology where the active site residues occupy identical positions. Intriguingly, the two active site residues are located on opposite sides of the scissile bond. A striking characteristic of the clan CD cysteine peptidases is the lack of irreversible inhibition by E64, of- ten considered to be a general inhibitor of cysteine en- dopeptidases. So far, synthetic inhibitors against clan CD peptidases of the caspase, gingipain, and clostripain families have been reported. The benzoyloxymethyl ketone inhibitor Cbz-L-Phe-L-Lys-CH 2 O-CO-2,4,6-Me 3 -Ph specifically inhibits gingipain K. The chloromethylketones (CMK) H- D-Phe-L-Pro-L-Arg-CH 2 Cl and H-D-Phe-L-Phe-L-Arg- CH 2 Cl react irreversibly with Arg-specific gingipain and clostripain, allowing active site titration (Potempa et al., 1997). Chloromethylketones with the core-structure H-L- Phe-L-Ala-L-Lys-L-Arg having an ethyl residue and methyl residue were shown to be potent irreversible in- hibitors of clostripain (Wikstrom et al., 1989). For the cas- pases, several different classes of inhibitors have been described, including aldehydes (Chapman, 1992), non- peptidic pyridone aldehydes (Golec et al., 1997), pheny- lalkyl ketones (Mjalli et al., 1993), acyloxymethyl ketones (Revesz et al., 1994; Thornberry et al., 1994), 1-phenyl-3- (trifluoromethyl)-pyrazol-5-yl-oxymethyl ketones (Dolle et al., 1994), fluoromethylketones (Revesz et al., 1994; Hara et al., 1997), and chloromethylketones (Schulz et al., 1996). Legumain was first discovered in plants and the inver- tebrate animal, Schistosoma, but was found in human placenta by Chen et al. (1997). From all sources, legu- main shows strict specificity for cleavage after as- paragine residues, which suggests that the physiological function may involve limited proteolysis. So far, there is strong evidence in plants that legumain might be in- volved in protein splicing (Min and Jones, 1994). An acti- vation of precursor proteins has been reported in plants as well (Hara-Nishimura et al., 1995). The mammalian lysosomal enzyme was reported to play a key role in the processing of bacterial antigen for presentation in the MHC II class system (Manoury et al., 1998; Antoniou et al., 2000). In addition, by screening an osteoclast cDNA expression library, legumain has been identified in- hibiting osteoclast formation and bone resorption (Choi et al., 1999). The regulation of legumain activity is still un- clear, but its inhibition by some cystatins, the general ex- tracellular cysteine peptidase inhibitors, was reported Biol. Chem., Vol. 383, pp. 1205 – 1214, July / August 2002 · Copyright © by Walter de Gruyter · Berlin · New York André J. Niestroj 1 , Kirstin Feußner 1 , Ulrich Heiser 1 , Pam M. Dando 2 , Alan Barrett 2 , Bernd Gerhartz 1 and Hans-Ulrich Demuth 1, * 1 Probiodrug AG, Weinbergweg 22 – Biocenter, D-06120 Halle (Saale), Germany 2 MRC Molecular Enzymology Laboratory, Babraham Institute, Cambridge CB2 4AT, UK * Corresponding author Legumain is a lysosomal cysteine peptidase specific for an asparagine residue in the P 1 -position. It has been classified as a member of clan CD peptidases due to predicted structural similarities to caspases and gingipains. So far, inhibition studies on legumain are limited by the use of endogenous inhibitors such as cystatin C. A series of Michael acceptor inhibitors based on the backbone Cbz-L-Ala-L-Ala-L-Asn (Cbz= benzyloxycarbonyl) has been prepared and resulted in an irreversible inhibition of porcine legumain. Vari- ation of the molecular size within the ‘war head’ re- vealed the best inhibition for the compound contain- ing the allyl ester (k obs /I=766 M –1 s –1 ). To overcome cyclisation between the amide moiety of the Asn residue and the ‘war head’, several asparagine ana- logues have been synthesised. Integrated in halo- methylketone inhibitors, azaasparagine is accepted by legumain in the P 1 -position. The most potent inhibitor of this series, Cbz-L-Ala-L-Ala-AzaAsn- chloromethylketone, displays a k obs /I value of 139 000 M –1 s –1 . Other cysteine peptidases, such as papain and cathepsin B, are not inhibited by this compound at concentrations up to 100 μM. The synthetic in- hibitors described here represent useful tools for the investigation of the structural and physiological prop- erties of this unique asparagine-specific peptidase. Key words: α,β-Unsaturated esters / Azaasparagine / Benzoyloxymethylketone / Cysteine protease / Halomethylketone / Legumain / Michael acceptors. Introduction Cysteine peptidases grouped into the clan CD represent a new emerging class of enzymes displaying a novel cat- alytic mechanism different to classical papain-like pepti- dases of clan CA. So far, five different families have been placed in the clan CD, namely family C11 clostripain, family C13 legumain, family C14 caspases, family C25 gingipain, and family C50 separin (Chen et al., 1998a; Inhibition of Mammalian Legumain by Michael Acceptors and AzaAsn-Halomethylketones Bereitgestellt von | SUB Göttingen Angemeldet Heruntergeladen am | 22.01.15 10:25