Design, synthesis, and evaluation of cystargolide-based β-lactones as potent proteasome inhibitors

The peptidic β-lactone proteasome inhibitors (PIs) cystargolides A and B were used to conduct structure-activity relationship (SAR) studies in order to assess their anticancer potential. A total of 24 different analogs were designed, synthesized and evaluated for proteasome inhibition, for cytotoxicity towards several cancer cell lines, and for their ability to enter intact cells. X-ray crystallographic analysis and subunit selectivity was used to determine the specific subunit binding associated with the structural modification of the β-lactone (P₁), peptidic core, (P_x and P_y), and end-cap (P_z) of our scaffold. The cystargolide derivative 5k, structurally unique at both P_y and P₁, exhibited the most promising inhibitory activity for the β5 subunit of human proteasomes (IC₅₀ = 3.1 nM) and significant cytotoxicity towards MCF-7 (IC₅₀ = 416 nM), MDA-MB-231 (IC₅₀ = 74 nM) and RPMI 8226 (IC₅₀ = 41 nM) cancer cell lines. Cellular infiltration assays revealed that minor structural modifications have significant effects on the ability of our PIs to inhibit intracellular proteasomes, and we identified 5k as a promising candidate for continued therapeutic studies. Our novel drug lead 5k is a more potent proteasome inhibitor than carfilzomib with mid-to-low nanomolar IC₅₀ measurements and it is cytotoxic against multiple cancer cell lines at levels approaching those of carfilzomib.