Inactivation of Papain by Epoxysuccinyl Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 17 3365
and Osteoarthritis. Clin. Chem. 1992, 38, 1694-1697. (b) van
Noorden, C. J . F.; Vogels, I. M. C. Cathepsin B Activity in
Unfixed and Unrecalcified Cryostat Sections of Normal and
Arthritic Mouse Knee J oints. Adv. Biosci. 1987, 65, 27-31.
(8) (a) Rosenthal, P. J ., McKerrow, J . H., Aikawa, M., Nagasawa,
H., Leech, J . J . A Malarial Cysteine Proteinase is Necessary for
Hemoglobin Degradation by Plasmodium falciparum. J . Clin.
Invest. 1988, 82, 1560-1566. (b) Ashall, F. Cancer Cells and
Parasites: Two of a Kind. Trends Biochem. Sci. 1986, 11, 518-
520.
(9) Kra¨usslich, H.-G.; Wimmer, E. Viral Proteinases. Annu. Rev.
Biochem. 1988, 57, 701-754.
(10) Musil, D.; Zucic, D.; Turk, D.; Engh, R. A.; Mayr, I.; Huber, R.;
Popovic, T.; Turk, V.; Towatari, T.; Katunuma, N.; Bode, W. The
Refined 2.15 Å X-ray Crystal Structure of Human Liver Cathe-
psin B: The Structural Basis for its Specificity. EMBO J . 1991,
10, 2321-2330.
(11) Kamphuis, I.; Drenth, J .; Baker, I. Thiol Proteases: Comparative
Studies Based on the High-resolution Structures of Papain and
Actinidin, and on Amino Acid Sequence Information for Cathe-
psins B and H, and Stem Bromelain. J . Mol. Biol. 1985, 182,
317-329.
(12) (a) Hanada, K.; Tamai, M.; Yamagishi, M.; Ohmura, S.; Sawada,
J .; Tanaka, I. Isolation and Characterization of E-64, a New
Thiol Protease Inhibitor. Agric. Biol. Chem. 1978, 42, 523-528.
(b) Hanada, K.; Tamai, M.; Ohmura, S.; Sawada, J .; Seki, T.;
Tanaka, I. Structure and Synthesis of E-64, a New Thiol
Protease Inhibitor. Agric. Biol. Chem. 1978, 42, 529-536.
(13) Tamai, M.; Hanada, K.; Adachi, T.; Oguma, K.; Kashiwagi, K.;
Omura, S.; Ohzeki, M. Papain Inhibitions by Optically Active
E-64 Analogs. J . Biochem. 1981, 90, 255-257.
(14) Barrett, A. J .; Kembhavi, A. A.; Brown, M. A.; Kirschke, H.;
Knight, C. G.; Tamai, M.; Hanada, K. L-trans-Epoxysuccinyl-
leucylamido(4-guanidino)butane (E-64) and its Analogues as
Inhibitors of Cysteine Proteinases Cathepsins B, H and L.
Biochem. J . 1982, 201, 189-198.
(15) Yabe, Y.; Guillaume, D.; Rich, D. H. Irreversible Inhibition of
Papain by Epoxysuccinyl Peptides. 13C NMR Characterization
of the Site of Alkylation. J . Am. Chem. Soc. 1988, 110, 4043-
4044.
(25) Mori, K.; Iwasawa, H. Stereoselective Synthesis of Optically
Active Forms of δ-Multistriatin, the Attractant for European
Populations of the Smaller European Elm Bark Beetle. Tetra-
hedron 1980, 36, 87-90.
(26) Tamai, M.; Yokoo, C.; Murata, M.; Oguma, K.; Sota, K.; Sato,
E.; Kanaoka, Y. Efficient Synthetic Method for Ethyl (+)-(2S,3S)-
3-[(S)-3-Methyl-1-(3-methylbutylcarbamoyl)butylcarbamoyl]-2-
oxiranecarboxylate (EST), a New Inhibitor of Cysteine Protein-
ases. Chem. Pharm. Bull. 1987, 35, 1098-1104.
(27) However, Bihovsky et al.21 report results comparable to ours
using DCC/HOBt with the potassium salt of (S,S)-methyl-1,2-
dicarboxylate oxirane in the absence of an additional base.
(28) Korn, A.; Rudolph-Bohner, S.; Moroder, L. A Convenient Syn-
thesis of Optically Pure (2R,3R)-2,3-Epoxysuccinyl-Dipeptides.
Tetrahedron 1994, 50, 8381-8392.
(29) J orgenson, M. J . Preparation of Ketones from the Reactions of
Organolithium Reagents with Carboxylic Acids. Org. React.
1970, 18, 1-97.
(30) Pojer, P. M.; Ritchie, E.; Taylor, W. C. The Preparation of Some
Aryl Methyl Ketones. Austr. J . Chem. 1968, 21, 1375-1378.
(31) Scholtz, J . M.; Bartlett, P. A. A Convenient Differential Protec-
tion Strategy for Functional Group Manipulation of Aspartic and
Glutamic Acids. Synthesis 1989, 542-544.
(32) Me´nard, R.; Khouri, H. E.; Plouffe, C.; Dupras; Ripoll, D.; Vernet,
T.; Tessier, D. C.; Laliberte´, F.; Thomas, D. Y.; Storer, A. C. A
Protein Dengineering Study of the Role of Aspartate 158 in the
Catalytic Mechanism of Papain. Biochemistry 1990, 29, 6706-
6713.
(33) (a) Dixon, M. The Effect of pH on the Affinities of Enzymes for
Substrates and Inhibitors. Biochem. J . 1953, 55, 161-170. (b)
Cleland, W. W. The Use of pH Studies to Determine Chemical
Mechanisms of Enzyme-Catalyzed Reactions. Methods Enzymol.
1982, 87, 390-405.
(34) Serjeant, E. P.; Dempsey, B. Ionisation constants of organic acids
in aqueous solution; Pergamon Press: Oxford, 1979.
(35) Although it is possible that the 1-2% of ionized hydroxamic acid
binds especially well to the enzyme and leads to a higher
apparent Ki, the experimental differences between inhibitors 2a
and 9 are small, and the data do not allow one to distinguish
among these possibilities.
(16) Varughese, K. I.; Ahmed, F. R.; Carey, P. R.; Hasnain, S.; Huber,
C. P.; Storer, A. C. Crystal Structure of a Papain-E-64 Complex.
Biochemistry 1989, 28, 1330-1332.
(36) Turk, D.; Podobnik, M.; Popovic, T.; Katunuma, N.; Bode, W.;
Huber, R.; Turk, V. Crystal Structure of Cathepsin B Inhibited
with CA030 at 2.0-Å Resolution: A Basis for the Design of
Specific Epoxysuccinyl Inhibitors. Biochemistry 1995, 34, 4791-
4797.
(37) Bihovsky, R. Reactions of R,â-Epoxy Carbonyl Compounds with
Methanethiolate: Regioselectivity and Rate. J . Org. Chem. 1992,
57, 1029-1031.
(38) (a) Chaiken, I. M.; Smith, E. L. Reaction of Chloroacetamide with
the Sulfhydryl Group of Papain. J . Biol. Chem. 1969, 244, 5087-
5094. (b) Chaiken, I. M.; Smith, E. L. Reaction of the Sulfhydryl
Group of Papain with Chloroacetic acid. J . Biol. Chem. 1969,
244, 5095-5099. (c) At neutral pH in solution, simple model
thiols react much faster with chloroacetamide than chloroac-
etate: Webb, J . L. Enzyme and Metabolic Inhibitors, Vol. III;
Academic Press: New York, 1966.
(17) (a) Matsumoto, K.; Yamamoto, D.; Ohishi, H.; Tomoo, K.; Ishida,
T.; Inoue, M.; Sadatome, T.; Kitamura, K.; Mizuno, H. Mode of
Binding of E-64-c, a Potent Thiol Protease Inhibitor, to Papain
as Determined by X-ray Crystal Analysis of the Complex. FEBS
Lett. 1989, 245, 177-180. (b) Yamamoto, D.; Ishida, T.; Inoue,
M. A Comparison Between the Binding Modes of a Substrate
and Inhibitor to Papain as Observed in Complex Crystal
Structures. Biochem. Biophys. Res. Commun. 1990, 171, 711-
716. (c) Yamamoto, D.; Matsmoto, K.; Ohishi, H.; Ishida, T.;
Inoue, M.; Kitamura, K.; Mizuno, H. Refined X-ray Structure of
Papain‚E-64-c Complex at 2.1 Å Resolution. J . Biol. Chem. 1991,
266, 14771-14777. (d) Kim, M.-J .; Yamamoto, D.; Matsmoto,
K.; Inoue, M.; Ishida, T.; Mizuno, H.; Sumiya, S.; Kitamura, K.
Crystal Structure of Papain-E64-c Complex. Binding Diversity
of E64-c to Papain S2 and S3 Subsites. Biochem. J . 1992, 287,
797-803.
(39) Liu, S.; Hanzlik, R. P. Structure-Activity Relationships for
Inhibition of Papain by Peptide Michael Acceptors. J . Med.
Chem. 1992, 35, 1067-1075.
(18) (a) Murata, M.; Miyashita, S.; Yokoo, C.; Tamai, M.; Hanada,
K.; Hatayama, K.; Towatari, T.; Nikawa, T.; Katunuma, N. Novel
Epoxysuccinyl Peptides. Selective Inhibitors of Cathepsin B, in
vitro. FEBS Lett. 1991, 280, 307-310. (b) Towatari, T.; Nikawa,
T.; Murata, M.; Yokoo, C.; Tamai, M.; Hanada, K.; Katunuma,
N. Novel Epoxysuccinyl Peptides. Selective Inhibitors of Cathe-
psin B, in vivo. FEBS Lett. 1991, 280, 311-315.
(40) Tamai, M.; Adachi, T.; Oguma, K.; Morimoto, S.; Hanada, K.;
Ohmura, S.; Ohzeki, M. Relationship Between Structure and
Papain Inbhibitory Activity of Epoxy succinyl Amino Acid
Derivatives. Agric. Biol. Chem. 1981, 675-679.
(41) Glazer, A. N.; Smith, E. L. Papain and Other Plant Sulfhydryl
Proteolytic Enzymes. In The Enzymes, Vol. 3; Boyer, P. D., Ed.;
Academic Press: New York, 1971; pp 519-523.
(19) Gour-Salin, B. J .; Lachance, P.; Plouffe, C.; Storer, A. C.; Me´nard,
R. Epoxysuccinyl Dipeptides as Selective Inhibitors of Cathepsin
B. J . Med. Chem. 1993, 36, 720-725.
(20) Tamai, M.; Adachi, T.; Oguma, K.; Morimoto, S.; Hanada, K.;
Ohmura, S.; Ohzeki, M. Relationship Between Structure and
Papain Inhibitory Activity of Epoxysuccinyl Amino Acid Deriva-
tives. Agric. Biol. Chem. 1981, 45, 675-79.
(21) Their results are essentially in agreement with ours except that
they report a ki′ for compound 10 (as a mixture of diastereomers
at the epoxide) of only 160 M-1 s-1. Bihovsky, R.; Powers, J . C.;
Kam, C.-M.; Walton, R.; Loew, R. C. Further Evidence for the
Importance of Free Carboxylate In Epoxysuccinate Inhibitors
of Thiol Proteases. J . Enzyme Inhib. 1993, 7, 15-25.
(22) Meara, J . P.; Rich, D. H. Measurement of Individual Rate
Constants of Irreversible Inhibition of a Cysteine Proteinase by
an Epoxysuccinyl Inhibitor Bioorg. Med. Chem. Lett. 1995, 5,
2277-2282.
(42) Lewis, S. D.; J ohnson, F. A.; Ohno, A. K.; Shafer, J . A.
Dependence of the Catalytic Activity of Papain on the Ionization
of Two Acidic Groups. J . Biol. Chem. 1978, 253, 5080-5086.
(43) (a) Lewis, S. D.; J ohnson, F. A.; Shafer, J . A. Effect of Cysteine-
25 on the Ionization of Histidine-159 in Papain as Determined
by Proton Nuclear Magnetic Resonance Spectroscopy. Evidence
for a His-159-Cys-25 Ion Pair and Its Possible Role in Catalysis.
Biochemistry 1981, 20, 48-51. (b) J ohnson, F. A.; Lewis, S. D.;
Shafer, J . A. Determination of a Low pK for Histidine-159 in
the S-Methylthio Derivative of Papain by Proton Nuclear
Magnetic Resonance Spectroscopy. Biochemistry 1981, 20, 44-
48.
(44) J ohnson, F. A.; Lewis, S. D.; Shafer, J . A. Perturbations in the
Free Energy and Enthalpy of Ionization of Histidine-159 at the
Active Site of Papain as Determined by Fluorescence Spectros-
copy. Biochemistry 1981, 20, 52-58.
(45) (a) Brocklehurst, K.; Little, G. Reactions of Papain and of Low-
Molecular-Weight Thiols with some Aromatic Disulphides. Bio-
chem. J . 1973, 133, 67-80. (b) J olley, C. J .; Yankeelov, J . A.,
J r. Reaction of Papain with R-Bromo-â-(5-imidazolyl)propionic
Acid. Biochemistry 1972, 11, 164-169. (c) Wallenfels, K.; Eisele,
B. Stereospecific Alkylation with Asymmetric Reagents. Eur. J .
Biochem. 1968, 3, 267-275.
(23) Rich, D. H. Inhibitors of Cysteine Proteinases. In Proteinase
Inhibitors; Barrett, A. J ., Salvesen, G. S., Eds.; Elsevier Science
Publishers B. V.: Amsterdam, 1986; pp 153-178.
(24) Frankfater, A.; Kuppy, T. Mechanism of Association of N-Acetyl-
L-phenylalanyl-glycinal to Papain. Biochemistry 1981, 20, 5517-
5524.