Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore.

Bisindolylpyrrole at 0.1 µM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 µM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.

The yeast mitochondrial permeability transition is regulated by reactive oxygen species, endogenous Ca2+ and Cpr3, mediating cell death.

We investigated the properties of the permeability transition pore (PTP) in Saccharomyces cerevisiae in agar-embedded mitochondria (AEM) and agar-embedded cells (AEC) and its role in yeast death. In AEM, ethanol-induced pore opening, as indicated by the release of calcein and mitochondrial membrane depolarization, can be inhibited by CsA, by Cpr3 deficiency, and by the antioxidant glutathione. Notably, the pore opening is inhibited, when mitochondria are preloaded by EGTA or Fluo3 to chelate matrix Ca2+, or are pretreated with 4-Br A23187 to extract matrix Ca2+, prior to agar-embedding, or when pore opening is induced in the presence of EGTA; opened pores are re-closed by sequential treatment with CsA, 4-Br A23187 plus EGTA and NADH, indicating endogenous matrix Ca2+ involvement. CsA also inhibits the pore opening with low conductance triggered by exogenous Ca2+ transport with ETH129. In AEC, the treatment of tert-butylhydroperoxide, a pro-oxidant that triggers transient pore opening in high conductance in AEM, induces yeast death, which is also dependent on CsA and Cpr3. Furthermore, AEMs from mutants lacking three ADP/ATP carrier (AAC) isoforms and with defective ATP synthase dimerization exhibit high and low conductance pore openings with CsA sensitivity, respectively. Collectively, these data show that the yeast PTP is regulated by Cpr3, endogenous matrix Ca2+, and reactive oxygen species, and that it is involved in yeast death; furthermore, ATP synthase dimers play a key role in CsA-sensitive pore formation, while AACs are dispensable.

A practical method for oxazole synthesis by cycloisomerization of propargyl amides.

[reaction: see text] 2,5-disubstituted and 2,4,5-trisubstituted oxazol-5-yl carbonyl compounds were prepared in good yields by a mild SiO2-mediated cycloisomerization of propargyl amides.

Bisindolylmaleimide I and V inhibit necrosis induced by oxidative stress in a variety of cells including neurons.

Although free radical-mediated necrosis is implicated in many diseases such as neurodegeneration, potent anti-necrotic drugs have not yet been exploited. We found that bisindolylmaleimide I (BMI or GF 109203X), a PKC inhibitor, protected a variety of cells, including neurons, from oxidant-induced necrosis, although calphostin C, another type of PKC inhibitor, and staurosporine, a broad kinase inhibitor, had no such effect. BMI was significantly protective in neuronal cells whereas chronic application of BMI induced neurotoxicity. BMV, a BMI-derivative devoid of PKC inhibition activity, exhibited cytoprotective effects similar to those of BMI but had no neurotoxic effects. Oxidation treatment of BMI and BMV did not impact their cytoprotective effects. These findings suggest that the cytoprotective mechanisms are independent of the inhibition of PKC and are not attributable to a direct free radical-scavenging effect. Moreover, the BM compounds did not affect classic, caspase-dependent apoptosis. These data suggest that BMV could act as a tool for elucidating necrotic mechanisms and as a lead for exploiting drugs to treat necrosis-involved diseases.