PP3(University of Florida)

Increased mitochondrial Ca 2+ uptake has been implicated in the QT prolongation and lethal arrhythmias associated with nonischemic cardiomyopathy. We attempted to define the role of mitochondria in ischemic arrhythmic risk and to identify upstream regulators.

Myocardial infarction (MI) was induced in wild-type FVB/NJ mice by ligation of the left anterior descending coronary artery. Western blot, immunoprecipitation, ECG telemetry, and patch-clamp techniques were used.

After MI, c-Src (proto-oncogene tyrosine-protein kinase Src) and its active form (phosphorylated Src, p-Src) were increased. The activation of c-Src was associated with increased diastolic Ca 2+ sparks, action potential duration prolongation, and arrhythmia in MI mice. c-Src upregulation and arrhythmia could be reversed by treatment of mice with the Src inhibitor PP1 but not with the inactive analogue PP3. Tyrosine phosphorylated mitochondrial Ca 2+ uniporter (MCU) was upregulated in the heart tissues of MI mice and patients with ischemic cardiomyopathy. In a heterologous expression system, c-Src could bind MCU and phosphorylate MCU tyrosines. Overexpression of wild-type c-Src significantly increased the mitochondrial Ca 2+ transient while overexpression of dominant-negative c-Src significantly decreased the mitochondrial Ca 2+ transient. c-Src inhibition by PP1, MCU inhibition by Ru360, or MCU knockdown could reduce the action potential duration, Ca 2+ sparks, and arrhythmia after MI. The human heart tissue showed that patients with ischemic cardiomyopathy had significantly increased c-Src active form associated with increased MCU tyrosine phosphorylation and ventricular arrhythmia.

MI leads to increased c-Src active form that results in MCU tyrosine phosphorylation, increased mitochondrial Ca 2+ uptake, QT prolongation, and arrhythmia, suggesting c-Src or MCU may represent novel antiarrhythmic targets.

BCL-X_L and BCL-2 are important targets for cancer treatment. BCL-X_L specific proteolysis-targeting chimeras (PROTACs) have been developed to circumvent the on-target platelet toxicity associated with BCL-X_L inhibition. However, they have minimal effects on cancer cells that are dependent on BCL-2 or both BCL-X_L and BCL-2. Here we report a new series of BCL-PROTACs. The lead PZ703b exhibits high potency in inducing BCL-X_L degradation and in inhibiting but not degrading BCL-2, showing a hybrid dual-targeting mechanism of action that is unprecedented in a PROTAC molecule. As a result, PZ703b is highly potent in killing BCL-X_L dependent, BCL-2 dependent, and BCL-X_L/BCL-2 dual-dependent cells in an E3 ligase (VHL)-dependent fashion. We further found that PZ703b forms stable {BCL-2:PROTAC:VCB} ternary complexes in live cells that likely contribute to the enhanced BCL-2 inhibition by PZ703b. With further optimization, analogues of PZ703b could potentially be developed as effective antitumor agents by co-targeting BCL-X_L and BCL-2.