Binding modes of a new epoxysuccinyl^peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity? Cezary Czaplewski a , Zbigniew Grzonka a , Mariusz Jasko ¨ lski b;c , Franciszek Kasprzykowski a , Maciej Kozak b , Ewa Politowska a , Jerzy Ciarkowski a ; * a Faculty of Chemistry, University of Gdan ¨sk, ul. Sobieskiego 18, 80-952 Gdan ¨sk, Poland b Faculty of Chemistry, A. Mickiewicz University, ul. Grunwaldzka 6, 60-780 Poznan ¨, Poland c Institute of Bioorganic Chemistry, Polish Academy of Sciences, ul. Noskowskiego 12/14, Poznan ¨, Poland Received 3 December 1998; received in revised form 19 February 1999; accepted 24 February 1999 Abstract Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain^ZPACK, papain^E64c and papain^leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop^ prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. ß 1999 Elsevier Science B.V. All rights reserved. Keywords : Thiol protease ; Enzyme selectivity ; Covalent protease inhibitor ; Molecular dynamics ; Constrained simulated annealing 1. Introduction Papain from the latex of Carica papaya is the most common member of the largest family of endopro- teases that depend on the thiol nucleophile of a cys- teine residue. Other families of cysteine proteases are structurally not related to papain [1]. Other members of the papain family, e.g., the mammalian cathepsins 0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII:S0167-4838(99)00053-9 Abbreviations : Z, benzyloxycarbonyl ; E64, 1-[L-N-(trans-ep- oxysuccinyl)leucyl]amino-4-guanidinobutane ; E64c, N-[-N-(L-3- trans-carboxyoxirane 2-carbonyl)-L-leucyl]-isoamylamide; CA028, epoxysuccinyl^L-Ile^L-Pro^OH ; CA030, ethyl ester of CA028 ; CA074, propylamide of CA028; other abbreviations (Nnv, Oxi, CSA, etc.) are de¢ned in the text, where applicable * Corresponding author. Fax: +48-58-341-0357; E-mail : jurek@chemik.chem.univ.gda.pl Biochimica et Biophysica Acta 1431 (1999) 290^305 www.elsevier.com/locate/bba