Discovery of novel Atg4B inhibitors with a benzotropolone core structure and therapeutic evaluation in a HT29 colon carcinoma mouse xenograft model

Date: 16 December 2016

Venue: UAntwerp - Campus Drie Eiken - Building R - Auditorium R2 - Universiteitsplein 1 - 2610 Antwerp (Wilrijk) (route: UAntwerpen, Stadscampus)

Time: 4:00 PM - 6:00 PM

PhD candidate: Matthias Cleenewerck

Principal investigator: Koen Augustyns

Co-principal investigator: Pieter Van der Veken

Short description: PhD defence Matthias Cleenwerck - Department of Pharmaceutical Sciences


Autophagy plays an essential role in fundamental life processes and acts in a beneficial manner in a wide range of diseases, including cardiovascular and neurodegenerative disease. In cancerogenesis however, autophagy can either function tumour suppressive, as is observed during initial tumorigenesis, or tumour protective, as seen during times of hypoxia or starvation.

These conditions are often met in fast growing tumours or during application of chemotherapeutics or radiotherapy. In this regard, autophagy targeting is hypothesized as a valuable add-on therapy to existing anticancer therapies. Generally accepted as an attractive target for specific autophagy modulation is the cysteine protease Atg4B/autophagin-1. In this work, we assessed the possibility to inhibit Atg4B with chemical substances and the therapeutic potential of Atg4B inhibition in cancerogenesis.

A gel-based method was developed and validated for Atg4B inhibitor screening. 7 reference Atg4B inhibitors and a 182-member Substrate Activity Screening (SAS) library of fragment-sized N-7-amino-4-methylcoumarines were screened for Atg4B inhibition. Two reference compounds (a benzotropolone and aurintricarboxylic acid) and one fragment were identified as weak Atg4B inhibitors. A library of 36 benzotropolones was synthesized and screened for autophagy/Atg4B inhibition using the gel-based assay and an in cellulo CYTO-ID and luciferase reporter assay. A total of 5 hit structures showed promising activity in all screening assays. Based on in vitro ADME properties, one hit compound was used as a starting point for an in-depth SAR-characterization for Atg4B/autophagy inhibition around the specific substitution pattern of this compound. A total of 19 analogues were synthesized and screened using the gel-based, CYTO-ID and luciferase reporter assay.

None of the derivatives showed promising activity in all screening assays and the parent compound was assessed for autophagy inhibition in a healthy mouse model. A strong autophagy inhibition could be observed in the group treated with the autophagy inhibitor. As a next step, the autophagy inhibitor was evaluated in a tumour xenograft model in mice using a HT29 colon cancer celline using four different testing groups: a control group, a group receiving either the autophagy inhibitor or the chemotherapeutic oxaliplatin and a group receiving both oxaliplatin and the autophagy inhibitor. An intermediate tumour growth inhibition was noted for the autophagy inhibitor in combination therapy with oxaliplatin, while a low inhibition was observed for the oxaliplatin group.