Aza-Peptide Michael Acceptors: A New Class of Inhibitors Specific for Caspases and Other Clan CD Cysteine Proteases O ¨ zlem Dog ˇan Ekici, † Marion G. Go ¨tz, † Karen Ellis James, † Zhao Zhao Li, † Brian J. Rukamp, † Juliana L. Asgian, † Conor R. Caffrey, ‡ Elizabeth Hansell, ‡ Jan Dvor ˇa ´k, § James H. McKerrow, ‡ Jan Potempa, |,⊥ James Travis, ⊥ Jowita Mikolajczyk, X Guy S. Salvesen, X and James C. Powers* ,† School of Chemistry and Biochemistry and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, Sandler Center for Basic Research in Parasitic Diseases, University of California-San Francisco, California 94143, Department of Parasitology, Faculty of Science, Charles University, Vinie ` na ´ 7, CZ-12844 Prague 2, Czech Republic, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602, and Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037 Received January 21, 2004 Abstract: Aza-peptide Michael acceptors are a new class of irreversible inhibitors that are highly potent and specific for clan CD cysteine proteases. The aza-Asp derivatives were specific for caspases, while aza-Asn derivatives were effective legumain inhibitors. Aza-Lys and aza-Orn derivatives were potent inhibitors of gingipain K and clostripain. Aza-peptide Michael acceptors showed no cross reactivity toward papain, cathepsin B, and calpain. The cysteine proteases caspases, legumain, clostri- pain, gingipains, and separase, belong to the protease clan CD, which is a small but important group of proteolytic enzymes with a unique R/ fold. 1 Clan CD cysteine proteases are important targets for drug de- velopment. Caspases, cysteine aspartate specific pro- teases, are a family with >15 members, 11 of which are found in humans. Some caspases are important media- tors of inflammation, whereas others are involved in apoptosis. 2 Excessive neuronal apoptosis leads to a variety of diseases such as stroke, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotropic lateral sclerosis (ALS), multiple sclerosis (MS), and spinal muscular atrophy. 3 Caspases are recognized as novel therapeutic targets for central nervous diseases in which cell death occurs mainly by an apoptosis mechanism. Legumain, originally identified in legumi- nous plants 4 and the parasitic blood fluke Schistosoma mansoni, 5 has recently been discovered in mammals 6 and is associated with bacterial antigen processing and immune disorders. 7 Gingipains, from Porphyromonas gingivalis, cause tissue damage in periodontal disease, 8 while clostripain is involved in bacterial infections. 9 Thus, potent and specific inhibitors of clan CD proteases could lead to the development of potential new drugs. A number of different classes of inhibitors have been developed for cysteine proteases including reversible transition state inhibitors and a variety of irreversible inhibitors. 10 Relatively few inhibitors such as peptide aldehydes, halomethyl ketones, and acyloxymethyl ke- tones have been tried with clan CD cysteine proteases, mainly with the caspases. 11 The major disadvantage of peptide aldehydes, halomethyl ketones, and acyloxym- ethyl ketones is their lack of selectivity. It has recently been shown that these inhibitors are not as specific as once claimed. 12 Thus, fluoromethyl and chloromethyl ketones with caspase specific sequences potently inhibit several lysosomal cysteine proteases including cathep- sins B, L, and S. Our laboratory has recently reported aza-peptide epoxide inhibitors (1, Figure 1) that are highly specific for clan CD cysteine proteases. 13 Aza-peptide epoxides with caspase specific sequences showed no cross reactiv- ity toward serine proteases or clan CA cysteine pro- teases such as papain, cathepsin B, and calpain and toward serine proteases. On the basis of our results with the aza-peptide epoxides, we designed a new class of aza-peptide inhibi- tors containing Michael acceptors. We refer to this new class of inhibitors as aza-peptide Michael acceptors (2, Figure 1). A variety of other inhibitors with electrophilic warheads have been reported as irreversible inhibitors effective for clan CA cysteine proteases. One of the first Michael acceptors described in the literature is the fumarate derivative of E-64c (3, Figure 1). This inhibitor contains an R,-unsaturated carbonyl moiety and was found to be an irreversible inhibitor of cathepsins B, H, and L. 14 Vinyl sulfones and R,-unsaturated carbonyl derivatives (4, Figure 1) have been developed as highly potent inhibitors for exopeptidases such as dipeptidyl peptidase I (DPPI) and many clan CA cysteine endopep- tidases including papain, cathepsins B, L, S, and K, calpains, and cruzain. 15,16 The design of vinyl sulfones and R,-unsaturated carbonyl derivatives was based on the optimal peptide sequence of the target enzyme, where the carbonyl of the scissile bond was replaced by the double bond moiety. In contrast, our aza-peptide Michael acceptor structure resembles the substrate * To whom correspondence should be addressed. E-mail: james. powers@chemistry.gatech.edu. † Georgia Institute of Technology. ‡ University of CaliforniasSan Francisco. § Charles University. | Jagiellonian University. ⊥ University of Georgia. X The Burnham Institute. Figure 1. Aza-Peptide Michael acceptor design. 1889 J. Med. Chem. 2004, 47, 1889-1892 10.1021/jm049938j CCC: $27.50 © 2004 American Chemical Society Published on Web 03/11/2004