Eltrombopag directly activates BAK and induces apoptosis
Meng Chen1,2,3, Lei Hu4, Xuyuan Bao5
1Anhui Province Key Laboratory of Medical Physics and Technology, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
Abstract:
Small molecule direct BAK activators can potentially be used for the development of anti-cancer drugs or as tools to study BAK activation. The thrombopoietin receptor agonist eltrombopag (Eltro) inhibits BAX activation and BAX-mediated apoptosis. Here we report that, in contrast to its function as a BAX inhibitor, Eltro directly binds BAK but induces its activation in vitro. Moreover, Eltro induces or sensitizes BAK-dependent cell death in mouse embryonic fibroblasts (MEFs) and Jurkat cells. Chemical shift perturbation analysis by NMR indicates that Eltro binds to the BAK α4/α6/α7 groove to initiate BAK activation. Further molecular docking by HADDOCK suggests that several BAK residues, including R156, F157, and H164, play an important role in the interaction with Eltro. The introduction of an R156E mutation in the BAK α4/α6/α7 groove not only decreases Eltro binding and Eltro-induced BAK activation in vitro but also diminishes Eltro-induced apoptosis. Thus, our data suggest that Eltro directly induces BAK activation and BAK-dependent apoptosis, providing a starting point for the future development of more potent and selective direct BAK activators.
Insights
Eltrombopag (Eltro) directly activates BAK, a protein involved in cell death, contrary to its known role as a BAX inhibitor. This discovery offers potential for developing new anti-cancer drugs targeting BAK activation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Small molecules that directly activate BCL2-associated X protein (BAX) or BCL2-antagonist/killer (BAK) are valuable for cancer therapy and biological research.
- Eltrombopag (Eltro), a thrombopoietin receptor agonist, is known to inhibit BAX activation and BAX-mediated apoptosis.
Purpose of the Study:
- To investigate the direct interaction of eltrombopag (Eltro) with BAK.
- To determine if Eltro can induce BAK activation and subsequent cell death.
- To elucidate the molecular mechanism of Eltro-induced BAK activation.
Main Methods:
- In vitro binding assays to assess Eltro's interaction with BAK.
- Cell death assays in mouse embryonic fibroblasts (MEFs) and Jurkat cells to evaluate Eltro's effect on BAK-dependent apoptosis.
- Nuclear Magnetic Resonance (NMR) chemical shift perturbation analysis to map Eltro's binding site on BAK.
- Molecular docking using HADDOCK to identify key residues involved in Eltro-BAK interaction.
- Site-directed mutagenesis (R156E) to validate the role of specific residues.
Main Results:
- Eltro directly binds to BAK and induces its activation in vitro, contrasting with its BAX inhibitory function.
- Eltro treatment leads to increased or sensitized BAK-dependent cell death in MEFs and Jurkat cells.
- NMR and molecular docking studies identified the BAK α4/α6/α7 groove as the binding site for Eltro, with residues R156, F157, and H164 being crucial for interaction.
- A mutation at R156 significantly reduced Eltro binding, BAK activation, and Eltro-induced apoptosis.
Conclusions:
- Eltro directly activates BAK, inducing BAK-dependent apoptosis.
- This finding provides a novel mechanism for Eltro and a foundation for developing targeted BAK activators for cancer therapy.
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