Biochem. Soc. Symp. 70, 15–30 (Printed in Great Britain) © 2003 Biochemical Society 15 2 Papain-like lysosomal cysteine proteases and their inhibitors: drug discovery targets? D˘ usan Turk 1 , Boris Turk and Vito Turk Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, Ljubljana, 1000 Slovenia Abstract Papain-like lysosomal cysteine proteases are processive and digestive enzymes that are expressed in organisms from bacteria to humans. Increasing knowledge about the physiological and pathological roles of cysteine proteases is bringing them into the focus of drug discovery research. These proteases have rather short active-site clefts, comprising three well defined substrate- binding subsites (S2, S1 and S1') and additional broad binding areas (S4, S3, S2' and S3'). The geometry of the active site distinguishes cysteine proteases from other protease classes, such as serine and aspartic proteases, which have six and eight substrate-binding sites respectively. Exopeptidases (cathepsins B, C, H and X), in contrast with endopeptidases (such as cathepsins L, S, V and F), pos- sess structural features that facilitate the binding of N- and C-terminal groups of substrates into the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from the specific exclusion of substrate types. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assem- bly of inhibitor fragments will bring the desired result. Introduction The physiological roles of lysosomal cysteine proteases now emerging from research are bringing these proteases increasingly into focus as drug tar- gets for a wide range of diseases, such as cancer, rheumatoid arthritis and osteoarthritis, multiple sclerosis and muscular dystrophy (reviewed in [1]). For many diseases resulting from excess proteolysis, no inhibitors have yet been 1 To whom correspondence should be addressed (e-mail Dusan.Turk@ijs.si).