ATG4B inhibitors with a benzotropolone core structure block autophagy and augment efficiency of chemotherapy in mice

Biochem Pharmacol. 2017 Aug 15;138:150-162. doi: 10.1016/j.bcp.2017.06.119. Epub 2017 Jun 19.


Autophagy is a cell survival mechanism hijacked by advanced tumors to endure a rough microenvironment. Late autophagy inhibitors such as (hydroxy)chloroquine have been used clinically to halt tumor progression with modest success. However, given the toxic nature of these compounds and their lack of specificity, novel targets should be considered. We recently identified a benzotropolone derivative that significantly inhibited the essential autophagy protein ATG4B. Therefore, we synthesized and tested additional benzotropolone compounds to identify a promising ATG4B inhibitor that impairs autophagy both in vitro and in vivo. A compound library containing 27 molecules with a benzotropolone backbone was synthesized and screened for inhibition of recombinant ATG4B. Depending on the benzotropolone compound, inhibition of recombinant ATG4B ranged from 3 to 82%. Active compounds were evaluated in cellular assays to confirm inhibition of ATG4B and suppression of autophagy. Seven compounds inhibited processing of the autophagy protein LC3 and autophagosome formation. Compound UAMC-2526 was selected for further in vivo use because of its fair plasma stability. This compound abolished autophagy both in nutrient-deprived GFP-LC3 mice and in CD1-/- Foxn1nu mice bearing HT29 colorectal tumor xenografts. Moreover, addition of UAMC-2526 to the chemotherapy drug oxaliplatin significantly improved inhibition of tumor growth. Our data indicate that suppression of autophagy via ATG4B inhibition is a feasible strategy to augment existing chemotherapy efficacy and to halt tumor progression.

Keywords: 3-Methyladenine (PubChem CID: 1673); ATG4B; Autophagy; Bafilomycin A1 (PubChem CID: 6436223); Benzotropolone; Chloroquine (PubChem CID: 2719); Colorectal tumor; DMSO (PubChem CID: 679); Ethanol (PubChem CID: 702); Everolimus (PubChem CID: 6442177); Osmotic minipump; Oxaliplatin (PubChem CID: 5310940); Propylene glycol (PubChem CID: 1030); Sevoflurane (PubChem CID: 5206); Tween 20 (PubChem CID: 443314).

MeSH terms

  • Adenocarcinoma / drug therapy*
  • Adenocarcinoma / pathology
  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use*
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Autophagy / drug effects*
  • Autophagy-Related Proteins / antagonists & inhibitors*
  • Autophagy-Related Proteins / genetics
  • Autophagy-Related Proteins / metabolism
  • Cell Proliferation / drug effects
  • Colonic Neoplasms / drug therapy*
  • Colonic Neoplasms / pathology
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • Cysteine Proteinase Inhibitors / chemistry
  • Cysteine Proteinase Inhibitors / pharmacology
  • Cysteine Proteinase Inhibitors / therapeutic use*
  • Drug Design*
  • Drug Stability
  • Forkhead Transcription Factors / genetics
  • Forkhead Transcription Factors / metabolism
  • HEK293 Cells
  • HT29 Cells
  • Humans
  • Jurkat Cells
  • Mice, Knockout
  • Mice, Transgenic
  • Microtubule-Associated Proteins / antagonists & inhibitors
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Organoplatinum Compounds / therapeutic use
  • Oxaliplatin
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Tropolone / analogs & derivatives*
  • Tropolone / chemistry
  • Tropolone / pharmacology
  • Tropolone / therapeutic use
  • Xenograft Model Antitumor Assays


  • Antineoplastic Agents
  • Autophagy-Related Proteins
  • Cysteine Proteinase Inhibitors
  • Forkhead Transcription Factors
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Organoplatinum Compounds
  • Recombinant Proteins
  • UAMC-2526
  • Whn protein
  • Oxaliplatin
  • Tropolone
  • ATG4B protein, human
  • Cysteine Endopeptidases