High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl‑2 Binding

Autophagy,
a lysosomal degradation pathway, plays a crucial role
in cellular homeostasis, development, immunity, tumor suppression,
metabolism, prevention of neurodegeneration, and lifespan extension.
Thus, pharmacological stimulation of autophagy may be an effective
approach for preventing or treating certain human diseases and/or
aging. We sought to establish a method for developing new chemical
compounds that specifically induce autophagy. To do this, we developed
two assays to identify compounds that target a key regulatory node
of autophagy inductionspecifically, the binding of Bcl-2 (a
negative regulator of autophagy) to Beclin 1 (an allosteric modulator
of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy
initiation). These assays use either a split-luciferase assay to measure
Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly
measure direct Beclin 1/Bcl-2 binding in vitro. We
screened two different chemical compound libraries, comprising ∼300
K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2
binding and induce autophagy. Three novel compounds were identified
that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds
do not demonstrate significant cytotoxicity, and they exert selectivity
for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared
with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax
and Bim. Thus, we have identified candidate molecules that serve as
lead templates for developing potent and selective Beclin 1/Bcl-2
inhibitors that may be clinically useful as autophagy-inducing agents.