Development and characterization of proteasome inhibitors

Abstract

Although many proteasome inhibitors have been either synthesized or identified from natural sources, the development of more sophisticated, selective proteasome inhibitors is important for a detailed understanding of proteasome function. We have found that antitumor natural product epoxomicin and eponemycin, both of which are linear peptides containing a alpha,beta-epoxyketone pharmacophore, target proteasome for their antitumor activity. Structural studies of the proteasome-epoxomicin complex revealed that the unique specificity of the natural product toward proteasome is due to the alpha,beta-epoxyketone pharmacophore, which forms an unusual six-membered morpholino ring with the amino terminal catalytic Thr-1 of the 20S proteasome. Thus, we believe that a facile synthetic approach for alpha,beta-epoxyketone linear peptides provides a unique opportunity to develop proteasome inhibitors with novel activities. In this chapter, we discuss the detailed synthetic procedure of the alpha',beta'-epoxyketone natural product epoxomicin and its derivatives.

FIG. 1

Proteasome inhibitors derived from other known peptide-based protease inhibitors possessing common pharmacophores.

FIG. 2

α′,β′-Epoxyketone-containing proteasome inhibitors from natural sources.

FIG. 3

The mechanism of proteasome inhibition by epoxomicin is proposed on the basis of the x-ray structure of yeast 20S proteasome–epoxomicin complex. It is postulated that the unique specificity of epoxomicin is due to the formation of an unusual six-membered morpholino ring between Thr-1 of the catalytic subunit of 20S proteasome and the α′,β′-epoxyketone pharmacophore of epoxomicin.

FIG. 4

Synthetic α′,β′-epoxyketone proteasome inhibitors designed to target a certain proteasomal proteolytic activity/subunit with a high degree of specificity. YU101 is a chymotrypsin-like activity (CT-L)-selective inhibitor, whereas YU102 is shown to be specific for caspase-like activity.

FIG. 5

A convergent approach for the total synthesis of epoxomicin.

FIG. 6

Two potential strategies for the introduction of a α′,β′-epoxyketone group.

FIG. 7

Synthesis of the right-hand fragment (leucine epoxyketone).

FIG. 8

Preparation of the tripeptide left-hand fragment.

FIG. 9

Schematic of hydrogenation reaction, in which hydrogen gas is provided to the reaction mixture through a stream of hydrogen gas.

FIG. 10

The final assembly of epoxomicin peptide backbone and preparation of epoxomicin.

FIG. 11

Epoxomicin/eponemycin chimerae were prepared by a random combination of left- and right-hand and central fragments.

FIG. 12

Preparation of the epoxyketone residue of eponemycin.

FIG. 13

The final assembly of eponemycin peptide backbone, which yielded dihydroeponemycin by TBDPS deprotection from serine side chain.