"Protease Day" Meeting, Istituto di Ricerche di Biologia Molecolare (IRBM), Pomezia, February 24.

PEPTIDYL PHENOXYMETHYL KETONES AS ACTIVE-SITE DIRECTED INHIBITORS OF CYSTEINE PROTEASES
Morea, V. (1), Calabretta, R. (2), Giordano, C. (2),Consalvi, V. (3), Scandurra, R. (3) and Gallina, C.(1)

(1) Universita' Gabriele d'Annunzio, Chieti, Italy (2) Centro di Studio per la Chimica del Farmaco (CNR) (3) Dipartimento di Scienze Biochimiche, Universita' La Sapienza, Rome, Italy

Cysteine proteases play important roles in a number of pathological conditions including inflammation, tumour metastasis, myocardial tissue damage, osteoclastic bone resorption 
and muscular distrophy (1).

Potent and selective low molecular weight synthetic inhibitors of these enzymes are thus of interest both for biochemical in vitro and in vivo investigations and, more importantly, 
as lead compounds for the development of therapeutic agents (2,3). We present here novel examples of substrate-based peptidyl methylketones cysteine protease inhibitors (4) 
containing phenylalanine at the P2 position (5), glycine at P1 and a phenoxy group with electron-withdrawing substituents (F, Cl, NO2) at the methyl adjacent to the ketone group.

These compounds were synthesized and tested as inhibitors of papain, which they inactivated over a concentration range 10-6-10-9 M, with a reversible or irreversible mechanism 
depending on the nature of the substituents on the phenoxy ring. The kinetic inhibition constants (6) Ki measured for the reversible inhibitors were in the range of 0.8-1.1 uM; 
the k2/KI values for the irreversible ones were in the range of 20,000-300,000 M-1s-1. Molecular modeling studies were also performed, in order to understand the structural basis 
of their activity toward papain.

The most active irreversible papain inhibitors also inactivated irreversibly bovine spleen cathepsin B with k2/KI constants from 40,000 to 2,500,000 M-1s-1. On the basis of the high 
sequence homology between bovine and human cathepsin B and the conservation of the active site residues, the active site structure of these enzymes should be the same; 
therefore, peptidyl phenoxymethylketones can be considered as valuable models for the development of therapeutic agents against cysteine protease dependent pathologies.

(1) Willenbrock, F.; Salih, E.; Brocklehurst, K. Cysteine proteinases In Hydrolytic Enzymes; Neuberger, A. and Brocklehurst, K., Ed.; Elsevier: Amsterdam, 1987; pp 39-200.
(2) Rich, D. H. Inhibitors of cysteine proteinases In Proteinase Inhibitors; Barrett, A. J. and Salvesen, G., Ed.; Elsevier: Amsterdam, 1986; pp 153-178.
(3) Shaw, E. Cysteinyl proteinases and their selective inactivation. Adv. Enzymol. Relat. Areas Mol. Biol. 1990, 63, 271-347.
(4) Calabretta, R., Giordano, C., Gallina, C., Morea, V., Consalvi, V. e Scandurra, R.: "Peptidyl and azapeptidylmethylketones as substrate analog inhibitors of papaina and cathepsin B", 
Eur. J. Med. Chem. (1995) 30, 931-934.
(5) Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Comm. 1967, 27, 157-162.
(6) Knight, G. C. Characterization of enzyme inhibition In Proteinase Inhibitors; Barrett, A. J. and Salvesen, G., Ed.; Elsevier: Amsterdam, 1986; pp 23-51.