Article

A Modified HSP70 Inhibitor Shows Broad Activity as an Anticancer Agent

The Wistar Institute.
Molecular Cancer Research (Impact Factor: 4.35). 01/2013; 11(3). DOI: 10.1158/1541-7786.MCR-12-0547-T
Source: PubMed

ABSTRACT The stress-induced heat shock protein 70 (HSP70) is an ATP-dependent molecular chaperone that plays a key role in refolding misfolded proteins and promoting cell survival following stress. HSP70 is marginally expressed in non-transformed cells, but is greatly overexpressed in tumor cells. Silencing HSP70 is uniformly cytotoxic to tumor but not normal cells; therefore, there has been great interest in the development of HSP70 inhibitors for cancer therapy. Here we report that the HSP70 inhibitor 2-phenylethynesulfonamide (PES) binds to the substrate-binding domain of HSP70, and requires the C-terminal helical 'lid' of this protein (amino acids 573-616) in order to bind. Using molecular modeling and in silico docking, we have identified a candidate binding site for PES in this region of HSP70, and we identify point mutants that fail to interact with PES. A preliminary structure-activity relationship analysis has revealed a derivative of PES, 2-(3-chlorophenyl) ethynesulfonamide (PES-Cl), which shows increased cytotoxicity and ability to inhibit autophagy, along with significantly improved ability to extend the life of mice with pre-B cell lymphoma, compared to the parent compound (p=0.015). Interestingly, we also show that these HSP70 inhibitors impair the activity of the Anaphase Promoting Complex/Cyclosome (APC/C) in cell-free extracts, and induce G2/M arrest and genomic instability in cancer cells. PES-Cl is thus a promising new anti-cancer compound with several notable mechanisms of action.

2 Bookmarks
 · 
237 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 2-phenylethynesulfonamide (PES) or pifithrin-μ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by Necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with L-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis.
    Biochemical Pharmacology 10/2014; DOI:10.1016/j.bcp.2014.08.005 · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ric-8A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary time window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because the G protein α subunits are mis-folded and degraded rapidly. Ric-8 proteins also act as Gα subunit guanine nucleotide exchange factors (GEFs) in vitro. It is not clear whether Ric-8 GEF-mediated activation of G proteins produces Gα-GTP in cells to regulate G protein-dependent effector enzymes. Ric-8 GEF activity could be a restricted in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action, but obligate for receptors. It remains an open question of whether Ric-8 has dual function in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 proteins do have profound positive influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets, in which pharmacological inhibition of the Ric-8:Gα subunit interface could serve to attenuate the effects of disease causing G proteins (constitutively active mutants) and/or hyperactive GPCR signaling. This mini-review will chronicle understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for therapeutic development.
    Molecular pharmacology 10/2014; 87(1). DOI:10.1124/mol.114.094664 · 4.12 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PES (2-phenylethynesulfonamide) was initially identified as an inhibitor of p53 translocation to mitochondria and named Pifithrin-µ. Further studies showed that PES selectively killed tumour cells and was thus a promising anticancer agent. PES-induced cell death was characterised by a non-apoptotic, autophagosome-rich phenotype. We observed this phenotype via electron microscopy in wild type (wt) and double Bax-/- Bak-/- (DKO) mouse embryonic fibroblasts (MEFs) treated with PES. We excluded the involvement of effector caspases, BAX and BAK, in causing PES-triggered cell death. Therefore, apoptosis was ruled out as the lethal mode of action of PES. Surprisingly, MEFs containing BAX were significantly protected from PES treatments. BAX overexpression in Bax-/- MEFs confirmed this pro-survival effect. Moreover, this protective effect required the ability of BAX to localise to mitochondrial membranes. Conversely, mitochondrial fusion induced by treatment with Mdivi-1 conferred increased resistance to MEFs subjected to PES treatment. The involvement of BAX in the regulation of mitochondrial dynamics has been reported. We propose the promotion of mitochondrial fusion by BAX to be the pro-survival function attributed to PES.
    Cancer Letters 08/2014; 354(1). DOI:10.1016/j.canlet.2014.07.037 · 5.02 Impact Factor

Full-text

Download
34 Downloads
Available from
May 31, 2014