PAC-1

First procaspase-activating compound (PAC-1) is a small-molecule activator of procaspase-3 that directly catalyzes the maturation of procaspase-3 to the active caspase-3 by inducing the cleavage of procaspase-3 in a time-dependent manner. As a result of the direct and immediate activation of procaspase-3, PAC-1 potently induces apoptosis in cancer cell lines. The PAC-1 induced apoptosis has been observed to be proportional to the concentrations of procaspase-3 inside the cells of primary colon cancer isolates. Study results have demonstrated that PAC-1 is able to induce cell death in both primary cancerous cells and adjacent normal tissues with 50% inhibition concentration IC₅₀ values ranging from 0.003 to 1.41 μM and 5.02 to 9.98 μM respectively.

Reference

Putt KS, Chen GW, Pearson JM, Sandhorst JS, Hoagland MS, Kwon JT, Hwang SK, Jin H, Churchwell MI, Cho MH, Doerge DR, Helferich WG, Hergenrother PJ. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol. 2006 Oct;2(10):543-50. Epub 2006 Aug 27.

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.[Pubmed:26658105]

Oncotarget. 2016 Jan 19;7(3):3461-76.

The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.

SM-1, a novel PAC-1 derivative, activates procaspase-3 and causes cancer cell apoptosis.[Pubmed:27488460]

Cancer Chemother Pharmacol. 2016 Sep;78(3):643-54.

PURPOSE: To develop more potent procaspase-3 activator, 7 novel derivatives of PAC-1 were synthesized and evaluated. Among them, SM-1 stood out for its promising activity and good pharmacokinetics properties. The purpose of this study is to elucidate the pharmacological mechanism of SM-1 and evaluate its efficacy and toxicity in-depth. METHODS: To reveal the effects of SM-1 on caspase-3 activity, both in vitro activation assay and in cells fluorometric assay were tested. The protein levels and distributions of procaspase-3 and cleaved caspase-3 were also measured by western blot and immunostaining. MTT assay, apoptosis assay and mouse xenograft model were applied to evaluate the efficacy of SM-1. Preliminary safety assessments also tested the acute toxicity and tissue distribution of SM-1. RESULTS: Compared to PAC-1, SM-1 showed higher cytotoxicity in cancer cells. Further investigation demonstrated that SM-1 relieved zinc-mediated inhibition of procaspase-3 and activated the caspase-3 activity both in tube test and in cells. Efficacy evaluation showed SM-1-induced cell apoptosis mainly via activation of caspase-3 and reduced tumor size in mouse xenograft model. Its apoptosis induction efficacy was higher than PAC-1. The preliminary safety assessment demonstrated that the overall LD50 of SM-1 lied between 500 and 1000 mg/kg and the distribution of SM-1 in brain was low. CONCLUSIONS: We identified SM-1 as a promising antitumor candidate, which displayed enhanced procaspase-3 activating activity and potent cytotoxicity for cancer cells but low toxicity for normal cells.

Synergistic antitumor activity of pro-apoptotic agent PAC-1 with cisplatinum by the activation of CASP3 in pulmonary adenocarcinoma cell line H1299.[Pubmed:26620316]

Asia Pac J Clin Oncol. 2016 Mar;12(1):41-51.

AIM: Evasion of apoptosis is a hallmark of human cancer cells. We sought to explore the potential synergistic antitumor activity and underlying mechanisms of the pro-apoptotic agent PAC-1 plus cisplatinum (Cis) in non-small cell lung cancer (NSCLC) cell lines. METHODS: The adenocarcinoma cell lines H1299, A549, PC9, H1650 and H1975 were used as in vitro models. Colorimetric MTT assays, Western blotting and flow cytometry were used to evaluate the anti-growth effects of PAC-1 and/or Cis and apoptosis status. The activated form of CASP3 (C-CASP3) was assessed by immunofluorescent staining. RESULTS: Single-agent Cis and PAC-1 were able to inhibit the cancer cell growth in certain dose ranges, with IC50 values of 1.9-11.7 and 5.6-14.8 muM, respectively. Sequential Cis-->PAC-1 or concurrent Cis + PAC-1, but not PAC-1-->Cis combinations showed synergistic effects on cell growth inhibition in H1299 cells (combination index, CI 1.0). Flow cytometric analysis showed that Cis-->PAC-1 sequential combination showed strong pro-apoptotic effects in H1299 cells. Western blots showed that in H1299, PC9 and H1975 cells, PAC-1 promoted the C-CASP3, but only in H1299 cells was there a synergistic effect with Cis on the CASP3 activation. CONCLUSIONS: PAC-1 showed anti-tumor activity in NSCLCs in vitro and a synergistic effect with cisplatin in EGFR(wt)KRAS(wt) H1299 cells. Our data suggest a potential treatment approach using cisplatin plus a pro-apoptotic agent acting via CASP3 activation for this subgroup of pulmonary adenocarcinomas.

Derivatives of Procaspase-Activating Compound 1 (PAC-1) and their Anticancer Activities.[Pubmed:26630918]

Curr Med Chem. 2016;23(3):201-41.

PAC-1 induces the activation of procaspase-3 in vitro and in cell culture by chelation of inhibitory labile zinc ions via its ortho-hydroxy-N-acylhydrazone moiety. First reported in 2006, PAC-1 has shown promise in cell culture and animal models of cancer, and a Phase I clinical trial in cancer patients began in March 2015 (NCT02355535). Because of the considerable interest in this compound and a well-defined structure-activity relationship, over 1000 PAC-1 derivatives have been synthesized in an effort to vary pharmacological properties such as potency and pharmacokinetics. This article provides a comprehensive examination of all PAC-1 derivatives reported to date. A survey of PAC-1 derivative libraries is provided, with an indepth discussion of four derivatives on which extensive studies have been performed.

Procaspase-3 activation as an anti-cancer strategy: structure-activity relationship of procaspase-activating compound 1 (PAC-1) and its cellular co-localization with caspase-3.[Pubmed:19708658]

J Med Chem. 2009 Sep 24;52(18):5721-31.

A goal of personalized medicine as applied to oncology is to identify compounds that exploit a defined molecular defect in a cancerous cell. A compound called procaspase-activating compound 1 (PAC-1) was reported that enhances the activity of procaspase-3 in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Experimental evidence indicates that PAC-1 activates procaspase-3 in vitro through chelation of inhibitory zinc ions. Described herein is the synthesis and biological activity of a family of PAC-1 derivatives where key functional groups have been systematically altered. Analysis of these compounds reveals a strong correlation between the in vitro procaspase-3 activating effect and their ability to induce death in cancer cells in culture. Importantly, we also show that a fluorescently labeled version of PAC-1 co-localizes with sites of caspase-3 activity in cancer cells. The data presented herein further bolster the hypothesis that PAC-1 induces apoptosis in cancer cells through the direct activation of procaspase-3, has implications for the design and discovery of next-generation procaspase-3 activating compounds, and sheds light on the anti-apoptotic role of cellular zinc.

Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy.[Pubmed:16936720]

Nat Chem Biol. 2006 Oct;2(10):543-50.

Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.