Tripeptide mimetics inhibit the 20 S proteasome by covalent bonding to the active threonines

Proteasomes play an important role in protein turnover in living cells. The inhibition of proteasomes affects cell cycle processes and induces apoptosis. Thus, 20 S proteasomal inhibitors are potential tools for the modulation of neoplastic growth. Based on MG132, a potent but nonspecific 20 S proteasome inhibitor, we designed and synthesized 22 compounds and evaluated them for the inhibition of proteasomes. The majority of the synthesized compounds reduced the hydrolysis of LLVY-7-aminomethyl-coumarin peptide substrate in cell lysates, some of them drastically. Several compounds displayed inhibitory effects when tested in vitro on isolated 20 S proteasomes, with lowest IC₅₀ values of 58 nM (chymotrypsin-like activity), 53 nM (trypsin-like activity), and 100 nM (caspase-like activity). Compounds 16, 21, 22, and 28 affected the chymotrypsin-like activity of the β5 subunit exclusively, whereas compounds 7 and 8 inhibited the β2 trypsin-like active site selectively. Compounds 13 and 15 inhibited all three proteolytic activities. Compound 15 was shown to interact with the active site by x-ray crystallography. The potential of these novel inhibitors was assessed by cellular tolerance and biological response. HeLa cells tolerated up to 1 μM concentrations of all substances. Intracellular reduction of proteasomal activity and accumulation of polyubiquitinated proteins were observed for compounds 7, 13, 15, 22, 25, 26, 27, and 28 on HeLa cells. Four of these compounds (7, 15, 26, and 28) induced apoptosis in HeLa cells and thus are considered as promising leads for antitumor drug development.