Synthesis, Characterization, and Optimization for in Vivo Delivery of a Nonselective Isopeptidase Inhibitor as New Antineoplastic Agent

Abstract

Bis-arylidenecycloalkanones structurally related to the nonselective isopeptidase inhibitor G5 were synthesized and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted arylidene groups, confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydroxycyclohexanone scaffold, which favors apoptosis over necrosis, was selected for further development. 2c inhibited representative deubiquitinases with micromolar IC50, and its proapoptotic activity was studied on several cancer cell lines. Inhibitor 2c was conjugated to PEG via dicarbamate and diester linkers. While the dicarbamate was inactive, the diester (2cPE) behaves like a prodrug and is converted into the active species 2c by secreted esterase activities. Finally, 2cPE was also tested in vivo on A549 lung carcinoma xenografts generated in mice. Intravenous treatment with 2cPE led to a significant reduction in primary tumor growth, without appreciable toxicity to mice.

The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells

“…In leukemia cells from patients LLC122, LLC195 and LLC270, resistance is not due to deficit in pro-drug maturation, as recently observed in other cancer cells [20]. Hierarchical gene clustering analysis of the different patient-derived B-CLL cells and the presence of the 11q deletion suggested that ATM could be a candidate for such resistant phenotype.…”

“…The 2cPE pro-drug and its unconjugated version, 2c induce comparable levels of cell death (Figure 3A), thus indicating that this cell line secretes the esterase PLA2G7 [20]. 2c and 2cPE induced cell death was apoptotic, as confirmed by the caspase-dependent proteolytic cleavage of the death substrate GAS2 (Figure 3B) [27].…”

“…This failure could arise from an incapability to process the pro-drug 2cPE in the extracellular environment, as observed in certain cancer cells [20]. mRNA expression levels analysis of PLAG7, the secreted esterase responsible for 2cPE pro-drug maturation, excludes this hypothesis (Supplementary Figure S2).…”

“…We have recently developed a PEG-conjugated P-SII, named 2cPE optimized for the in vivo delivery. 2cPE is a pro-drug version of G5 [11], which can be activated by secreted esterase and exhibits promising anti-neoplastic activities in vivo [20]. In this manuscript, we have investigated the effect of 2cPE against B-CLL cells, in comparison with bortezomib.…”

Novel Linear Bis-Crown Receptors with Cross-Conjugated and Conjugated Central Cores

“…[16][17][18][19][20][21][22] Recently, cross-conjugated dienones with pyridine substituents have been used as ligands to produce discrete and polymeric coordination compounds. [23][24][25][26] Thus, it has been possible to produce discrete dimers based on cyclopentanone-based ligands, [23] one-dimensional coordination polymers, in the case of cyclohexanone-based ligands, [24], [27] and two-dimensional coordination polymers using piperidones-4.…”