A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids

Abstract

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, β-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.

Keywords: Activity-based protein profiling; Beta-lactones; Inhibitor; Serum stability; Structure-activity relationship studies.

Figure 1

Representative structures of human or yeast 20S proteasome and fatty acid synthase inhibitors.

Figure 2

Representative Initial dual inhibitors 8a–c displaying inhibitory activity toward both FAS-TE and the proteasome and design process based on structures of orlistat (4) and the belactosin C derivative 7.

Figure 3

A) In situ ABPP labeling of MCF-7 and HeLa cells with increasing concentrations of alkyne ABPP probe 28. B) Identification of FAS and proteasome beta-5 by Western Blotting using specific anti-FAS and anti-PSB-5 antibodies. C) Competitive labeling with inhibitors 21, 25 and ABPP probe 28. (PSB-5: proteasome subunit beta type, FAS: Fatty acid synthase)

Scheme 1

Synthesis of disubstituted α-lactone acids 12a and 12a’ and α-methyl-β-lactone acids 12b and 12b’ employing a chiral auxiliary-based, tandem Mukaiyama aldol-β-lactonization. (a) ZnCl₂, CH₂Cl₂, 23 °C, 30 min (R = H) or 15 h (R = Me); (b) H₂, Pd/C (10% w/w), THF.

Scheme 2

Representative coupling strategy for synthesis of dual inhibitors as shown for dipeptide β-lactone 17.