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

Abstract

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 ~300K 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 to 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.

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“…[51][52][53] Free Bcl-2 has a BH3 domain that can bind to Beclin-1, a specific marker of autophagy, thereby affecting the occurrence of autophagy. 54 Similarly, our present results showed that Bax, Bcelin-1 and LC3-II were increased significantly in liver IR while Bcl-2 was decreased. However, after drug treatment, these indices were reversed in a dose-dependent manner.…”

Therapeutic modulation of autophagy: which disease comes first?

“…Prominent pharmacological targets for autophagy inhibition include proteases (such as the ATG4 isoenzymes required for the proteolytic maturation of LC3 family proteins), lipid kinases (in particular, phosphatidylinositol 3-kinase catalytic subunit type 3, PIK3C3, which acts within the Beclin 1, BECN1, complex) and protein kinases (especially, Unc-51like autophagy activating kinase 1, ULK1) [19,24]. Druggable targets to enhance autophagic flux include a series of negative regulators of autophagy such as multidomain proteins of the BCL-2 family (which can be targeted by socalled BH-3 mimetics and perhaps other agents that disrupt their inhibitory interaction with the Beclin 1/PIK3C3 complex) [25][26][27][28], the acetyltransferase E1A binding protein P300 (EP300, which is already known as the target of aspirin and spermidine) [20,29], the mammalian target of rapamycin complex 1 (MTORC1, which is inhibited by rapamycin and rapalogs), as well as kinases acting upstream of MTORC1 such as the members of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and protein kinase B (PKB) families [19,30]. However, this list is not exhaustive and other potential targets for autophagy modulation must be considered.…”

Targeting autophagy in thyroid cancers

“…Moreover, assessing the redifferentiative effect of these clinically available agents in a clinical scenario is needed to validate preclinical observations. Apart from these clinically available agents, novel chemical compounds (Galluzzi et al 2017b, Chiang et al 2018, or autophagy-inducing peptides (Peraro et al 2017), could also be used to pharmacologically stimulate autophagy. Since BRAF and MEK inhibitors have been validated as effective in restoring radioiodine incorporation in clinical trials (Ho et al 2013, Rothenberg et al 2015, Huillard et al 2017, Dunn et al 2018, future studies may also explore if there is a synergistic effect in redifferentiating RR-DTC when a combination of molecularly targeted agent with autophagy activator is applied.…”