An Autophagy-Disrupting Small Molecule Promotes Cancer Cell Death via Caspase Activation.

A novel autophagy inhibitor, autophazole (Atz), which promoted cancer cell death via caspase activation, is described. This compound was identified from cell-based high-content screening of an imidazole library. The results showed that Atz was internalized into lysosomes of cells where it induced lysosomal membrane permeabilization (LMP). This process generated nonfunctional autolysosomes, thereby inhibiting autophagy. In addition, Atz was found to promote LMP-mediated apoptosis. Specifically, LMP induced by Atz caused release of cathepsins from lysosomes into the cytosol. Cathepsins in the cytosol cleaved Bid to generate tBid, which subsequently activated Bax to induce mitochondrial outer membrane permeabilization (MOMP). This event led to cancer cell death via caspase activation. Overall, the findings suggest that Atz will serve as a new chemical probe in efforts aimed at gaining a better understanding of the autophagic process.

Mitochondrial Cl--Selective Fluorescent Probe for Biological Applications.

Herein we describe the development of the first mitochondrial Cl--selective fluorescent probe, Mito-MQAE, and its applications in biological systems. Fluorescence of Mito-MQAE is insensitive to pH over the physiological pH range and is quenched by Cl- with a Stern-Volmer quenching constant of 201 M-1 at pH 7.0. The results of cell studies using Mito-MQAE show that substances with the ability to disrupt mitochondrial membranes cause increases in the mitochondrial Cl- concentration.

An Inhibitor of the Interaction of Survivin with Smac in Mitochondria Promotes Apoptosis.

Herein we report the first small molecule that disrupts the survivin-Smac interaction taking place in mitochondria. The inhibitor, PZ-6-QN, was identified by initially screening a phenothiazine library using a fluorescence anisotropy assay and then conducting a structure-activity relationship study. Mutagenesis and molecular docking studies suggest that PZ-6-QN binds to survivin similarly to the known Smac peptide, AVPI. The results of the effort also show that PZ-6-QN exhibits good anticancer activity against various cancer cells. Moreover, cell-based mechanistic studies provide evidence for the proposal that PZ-6-QN enters mitochondria to inhibit the survivin-Smac interaction and promotes release of Smac and cytochrome c from mitochondria into the cytosol, a process that induces apoptosis in cancer cells. Overall, the present study suggests that PZ-6-QN can serve as a novel chemical probe for study of processes associated with the mitochondrial survivin-Smac interaction and it will aid the discovery of novel anticancer agents.

Subcellular Hsp70 Inhibitors Promote Cancer Cell Death via Different Mechanisms.

Mechanisms underlying cancer cell death caused by inhibitors of subcellular Hsp70 proteins have been elucidated. An inhibitor of Hsp70, apoptozole (Az), is mainly translocated into lysosomes of cancer cells where it induces lysosomal membrane permeabilization, thereby promoting lysosome-mediated apoptosis. Additionally, Az impairs autophagy in cancer cells owing to its ability to disrupt the lysosomal function. However, the Az-triphenylphosphonium conjugate, Az-TPP-O3, localizes mainly to mitochondria of cancer cells where it inhibits the mortalin-p53 interaction and induces mitochondrial outer membrane permeabilization, consequently leading to mitochondria-mediated apoptosis. Unlike Az, Az-TPP-O3 does not have an effect on autophagy in cancer cells. Collectively, the findings indicate that inhibitors of lysosomal Hsp70 and mitochondrial mortalin enhance cancer cell death via distinctively different mechanisms. Additionally, the findings arising from this effort demonstrate that studies aimed at determining subcellular locations and functions of small-molecule modulators provide a deeper understanding of their modes of action in cells.