Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction

Gal Chaim Nuta, Yuval Gilad, Nadav Goldberg, Sara Meril, Marcela Bahlsen, Silvia Carvalho, Noga Kozer, Haim Barr, Yael Fridmann Sirkis, Kamil Hercík, Petra Břehová, Radim Nencka, Shani Bialik, Miriam Eisenstein, Adi Kimchi

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Macroautophagy/autophagy is a catabolic process by which cytosolic content is engulfed, degraded and recycled. It has been implicated as a critical pathway in advanced stages of cancer, as it maintains tumor cell homeostasis and continuous growth by nourishing hypoxic or nutrient-starved tumors. Autophagy also supports alternative cellular trafficking pathways, providing a mechanism of non-canonical secretion of inflammatory cytokines. This opens a significant therapeutic opportunity for using autophagy inhibitors in cancer and acute inflammatory responses. Here we developed a high throughput compound screen to identify inhibitors of protein-protein interaction (PPI) in autophagy, based on the protein-fragment complementation assay (PCA). We chose to target the ATG12-ATG3 PPI, as this interaction is indispensable for autophagosome formation, and the analyzed structure of the interaction interface predicts that it may be amenable to inhibition by small molecules. We screened 41,161 compounds yielding 17 compounds that effectively inhibit the ATG12-ATG3 interaction in the PCA platform, and which were subsequently filtered by their ability to inhibit autophagosome formation in viable cells. We describe a lead compound (#189) that inhibited GFP-fused MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) puncta formation in cells with IC50 value corresponding to 9.3 μM. This compound displayed a selective inhibitory effect on the growth of autophagy addicted tumor cells and inhibited secretion of IL1B/IL-1β (interleukin 1 beta) by macrophage-like cells. Compound 189 has the potential to be developed into a therapeutic drug and its discovery documents the power of targeting PPIs for acquiring specific and selective compound inhibitors of autophagy. Abbreviations: ANOVA: analysis of variance; ATG: autophagy related; CQ: chloroquine; GFP: green fluorescent protein; GLuc: Gaussia Luciferase; HEK: human embryonic kidney; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; PCA: protein-fragment complementation assay; PDAC: pancreatic ductal adenocarcinoma; PMA: phorbol 12-myristate 13-acetate; PPI: protein-protein interaction. VCL: vinculin.

Original languageEnglish
Pages (from-to)2372-2385
Number of pages14
JournalAutophagy
Volume19
Issue number8
Early online date22 Feb 2023
DOIs
StatePublished - Aug 2023
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

Funding

This work was supported by funding from Yeda Research and Development, Ltd., Grant #3924. We would like to thank Yehuda Matan Danino for critical assistance with the PANC1 RFP-GFP-LC3B imaging and analysis. We thank Dor Perets, Meital Yona and Tamar Unger for lysate productions. We would also like to thank Galit Cohen and Khriesto Shurrush for compound management and triage.

FundersFunder number
Galit Cohen and Khriesto Shurrush
Yeda Research and Development, Ltd.3924

    Keywords

    • Autophagy inhibition
    • LC3B
    • cancer
    • drug screen
    • pancreatic cancer
    • protein-fragment complementation assay
    • small molecules

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