Development of novel pseudopeptides targeting the 20s proteasome as anticancer agents

Proteasome is a multicatalytic threonine protease complex responsible for the turnover of cellular proteins including those involved in signal transduction, cell cycle control and apoptosis. Defects in the proteasome activity can lead to anarchic cell proliferation and tumors development. For these reasons, proteasome is a target of great interest in drug discovery for anticancer therapy.(1) Among the three active sites of proteasome, the greatest interest has been focused on the inhibition of β5 subunit (chymotrypsin-like, ChT-L). Starting from the structure of bortezomib as lead compound, we recently synthesized a series of conformationally constrained analogs that showed a promising inhibitory profile by blocking primarily the ChT-L activity of the proteasome with Ki values in submicromolar/micromolar range. The obtained results were rationalized by means of docking experiments that also provided essential insights for the optimization of the inhibitors.(2) On these bases, starting from the most active peptidomimetic boronates 1 and 2 (Ki = 0.098 and 0.17 μM), we designed a novel series of boronic acids 3-4 (Fig. 1). In the optimization of compound 1, the 1Hpyridin- 2-one ring has been kept unchanged in view of its ability to enclose the amide moiety of the pyrazinamide of bortezomib. Also the anilino moiety at position 4 of the pyridone nucleus has been maintained, since docking studies pointed out that this substituent projected towards S3 binding pocket and that NH form a hydrogen bond with the side chain of D114 of β6 subunit.(2) The anilino moiety has been also shifted in position 3 to verify if its ability to interact with β6-D114 is retained. Additional structural modifications were the introduction of β-alanine and phenylalanine residues at P2 position to optimize the interactions with S2 pocket. Regarding compound 2, docking studies clearly indicated that this compound adopted a folded conformation that flip the constrained scaffold to the S2 pocket instead of the originally assumed S3 pocket. Therefore, the replacement of the Gly residue at P2 with β-alanine or rigid synthons should impede the folding and optimize the interactions with the target. Furthermore, we decided to investigate the real contribution of N1 (corresponding to N4 of the pyrazine ring of bortezomib) to the inhibitory activity by replacing the naphthyridinone scaffold with the isosteric isoquinolin-1(2H)-one. The results of the biological evaluation of the new compounds will be reported and discussed