PAC-1 Activates Procaspase-3 in Vitro through Relief of Zinc-Mediated Inhibition

Abstract

SUMMARYThe direct induction of apoptosis has emerged as a powerful anti-cancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key "executioner" caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3, and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small molecule mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.

Synergistic antitumor activity of pro‐apoptotic agent PAC‐1 with cisplatinum by the activation of CASP3 in pulmonary adenocarcinoma cell line H1299

“…40 Mechanistically, PAC-1 may bind Zn ions competitively and promote cleavage of CASP3 into C-CASP. 14,41 PAC-1-mediated C-CASP3 activation was also confirmed by confocal microscopy in our study. In EGFR wt KRAS wt ALK wt H1299 cells, PAC-1 induced greater levels of C-CASP3 compared with EGFR or KRAS mutant cells, suggesting that in H1299 cells, CASP3 might be activated more readily by PAC-1, consistent with the flow cytometry results, which showed sequential treatment of Cis followed by PAC-1 could cause a high rate of apoptosis in H1299 cells.…”

Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation

“…9). These results in addition to previous reports would suggest that the proenzyme can auto-activate (20,21,(33)(34)(35); however, the above proteolytic susceptibility assay shows that intermolecular processing between two procaspase-3 molecules is restricted. Therefore, we assessed if intramolecular processing of procaspase-3 can generate a trace amount of mature caspase-3, which can then rapidly feedback to promote autocatalytic maturation of the proenzyme (Fig.…”

“…C, procaspase-3 activation may proceed through an initiation event that forms the first mature caspase-3 molecule, which can rapidly feedback to process additional procaspase-3 molecules in a propagation phase. (33,34)-Uncleavable procaspase-3 (D9A/D28A/D175A) or mature caspase-3 was agitated at 37°C with the catalytically inactive procaspase-3 (C163A) in a caspase activity buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 0.1 mM EDTA, 10 mM DTT, and 0.1% CHAPS) in the presence or absence of 1541. The irreversible inhibitor, Ac-DEVD-cmk, was added under the designated conditions to inhibit any cleaved caspase-3 impurity present in the uncleavable procaspase-3 expression.…”

Molecular Basis of Drug Interactions of Methotrexate, Cyclophosphamide and 5-Fluorouracil as Chemotherapeutic Agents in Cancer

“…Activated CAD/DFF40 then results in the degradation of genomic DNA into nucleosomal fragments ( Fig. 1) (Cao et al, 2001;Janicke et al, 1998;McIlroy et al, 2000;O'Donovan et al, 2003;Peterson et al, 2009). …”