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

Abstract

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 (P), peptidic core, (P and P), and end-cap (P) of our scaffold. The cystargolide derivative 5k, structurally unique at both P and P, exhibited the most promising inhibitory activity for the β5 subunit of human proteasomes (IC = 3.1 nM) and significant cytotoxicity towards MCF-7 (IC = 416 nM), MDA-MB-231 (IC = 74 nM) and RPMI 8226 (IC = 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 IC measurements and it is cytotoxic against multiple cancer cell lines at levels approaching those of carfilzomib.

Cystargolide-based amide and ester Pz analogues as proteasome inhibitors and anti-cancer agents

“…In order to improve the pharmacokinetic profiles of our proteasome inhibitors, we hypothesized that increasing the number of nitrogen atoms in the structure could deliver additional analogues with improved aqueous solubility and activity. According to our previous observations [ 27 ], the end-cap (Pz) portion of the cystargolide scaffold ( figure 1 ) seemed to better tolerate structural changes while preserving desirable inhibitory activity and cytotoxicity profiles. For this reason, we synthesized and evaluated cystargolide-based Pz analogues incorporating ester or amide linkages, and pyridine or pyrazine aromatic moieties.…”

“…The construction of our nitrogenated Pz analogues was based on methodology used in our previous work on the synthesis of cystargolide derivatives [ 27 ]. Known β-lactone 2 [ 17 , 27 , 28 ] can be efficiently prepared from L-isoleucine by a sequence of reactions involving deamination, asymmetric alkylation and one-pot chlorination/cyclization ( scheme 1 ). Coupling of 2 with the TFA salt derived from known N-Boc O-benzyl L-valine 3 [ 29 ] followed by hydrogenolysis yielded common intermediate 5 in good yield ( scheme 1 ).…”

“…Based on our previously reported procedure [ 27 ], the degree of peptide cleavage in a crude Jurkat cell lysate was measured to determine proteasome activity. The fluorescent substrate Suc-LLVY-AMC (Enzo Life Sciences) was used to determine chymotrypsin-like ( β 5) activity of the proteasomes.…”

“…Analogue 1 emerged as a potent inhibitor of the β 5 subunit of human proteasomes (IC 50 = 3.1 nM) which promotes significant cytotoxicity toward MCF-7 (IC 50 = 416 nM), MDA-MB-231 (IC 50 = 74 nM) and RPMI 8226 (IC 50 = 41 nM) cancer cell lines. The efficacy of 1 toward multiple myeloma cells approaches that of carfilzomib; however, comparison of the proteasome inhibition in cell lysate and in whole cells indicated that cellular infiltration remained the primary barrier for further optimization of 1 and related PIs [ 27 ].…”

Natural product scaffolds as inspiration for the design and synthesis of 20S human proteasome inhibitors

“…177 Subsequent optimization of the cystargolide scaffold was achieved in 2018: benzyl ether 70 inhibits the hb5 subunit of Jurkat cell lysate with an IC 50 value of 3.1 AE 0.2 nM as compared to cystargolide B (IC 50 : 0.90 AE 0.11 mM), and is also cytotoxic against MCF-7 and RPMI-8226 cells. 179…”