Identification of benzo[cd]indol-2(1H)-ones as novel Atg4B inhibitors via a structure-based virtual screening and a novel AlphaScreen assay

Targeting autophagy is a promising therapeutic strategy for cancer treatment. As a result, the identification of novel autophagy inhibitors is an emerging field of research. Herein, we report the development of a novel AlphaScreen HTS assay that combined with a MS-based assay and a structure-based high-throughput virtual screening have enabled the identification of benzo[cd]indol-2(1H)-one as a novel scaffold that targets Atg4B. Thus, an initial screening campaign led to the identification of NSC126353 and NSC611216 bearing a chlorohydrin moiety. Structural-activity relationship analysis of the initial hits provided an optimized lead, compound 33, bearing a 7-aminobenzo[cd]indol-2-[1H]-one scaffold and a propyl group replacing the chlorine. Inhibition of autophagy was also investigated in cells by measuring LC3-II and p62 protein levels. Moreover, the synergistic effect of 33 combined with oxaliplatin resulted in an enhanced cell death in the human colorectal adenocarcinoma cell line HT-29. We are convinced that the developed AlphaScreen and MS-based assays can be key tools enabling the high-throughput identification of novel Atg4B inhibitors. Moreover, the aminobenzo[cd]indol-2-[1H]-one scaffold represents a novel chemotype for the further development of small molecule inhibitors of Atg4B. © 2019 Elsevier Masson SASWe would like to thank Karel Hernandez and Roman Bonet for his support in the expression and purification of recombinant proteins and Kristina Lang for her help in the preparation of N-(His) 6 -LC3B-L123C. We are grateful to the Ministerio de Economia and Competitividad for supporting this work with a research grant CTQ2013-44334-P ) and FPI fellowship to A.B.G ( BES-2014-070026 ). Appendix APeer reviewe