Cargando…

Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action

Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we ext...

Descripción completa

Detalles Bibliográficos
Autores principales: Gubat, Johannes, Selvaraju, Karthik, Sjöstrand, Linda, Kumar Singh, Dhananjay, Turkina, Maria V., Schmierer, Bernhard, Sabatier, Pierre, Zubarev, Roman A., Linder, Stig, D’Arcy, Pádraig
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9076133/
https://www.ncbi.nlm.nih.gov/pubmed/35530310
http://dx.doi.org/10.3389/fonc.2022.852980
_version_ 1784701845576351744
author Gubat, Johannes
Selvaraju, Karthik
Sjöstrand, Linda
Kumar Singh, Dhananjay
Turkina, Maria V.
Schmierer, Bernhard
Sabatier, Pierre
Zubarev, Roman A.
Linder, Stig
D’Arcy, Pádraig
author_facet Gubat, Johannes
Selvaraju, Karthik
Sjöstrand, Linda
Kumar Singh, Dhananjay
Turkina, Maria V.
Schmierer, Bernhard
Sabatier, Pierre
Zubarev, Roman A.
Linder, Stig
D’Arcy, Pádraig
author_sort Gubat, Johannes
collection PubMed
description Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level.
format Online
Article
Text
id pubmed-9076133
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-90761332022-05-07 Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action Gubat, Johannes Selvaraju, Karthik Sjöstrand, Linda Kumar Singh, Dhananjay Turkina, Maria V. Schmierer, Bernhard Sabatier, Pierre Zubarev, Roman A. Linder, Stig D’Arcy, Pádraig Front Oncol Oncology Dienone compounds have been demonstrated to display tumor-selective anti-cancer activity independently of the mutational status of TP53. Previous studies have shown that cell death elicited by this class of compounds is associated with inhibition of the ubiquitin-proteasome system (UPS). Here we extend previous findings by showing that the dienone compound b-AP15 inhibits proteasomal degradation of long-lived proteins. We show that exposure to b-AP15 results in increased association of the chaperones VCP/p97/Cdc48 and BAG6 with proteasomes. Comparisons between the gene expression profile generated by b-AP15 to those elicited by siRNA showed that knock-down of the proteasome-associated deubiquitinase (DUB) USP14 is the closest related to drug response. USP14 is a validated target for b-AP15 and we show that b-AP15 binds covalently to two cysteines, Cys203 and Cys257, in the ubiquitin-binding pocket of the enzyme. Consistent with this, deletion of USP14 resulted in decreased sensitivity to b-AP15. Targeting of USP14 was, however, found to not fully account for the observed proteasome inhibition. In search for additional targets, we utilized genome-wide CRISPR/Cas9 library screening and Proteome Integral Solubility Alteration (PISA) to identify mechanistically essential genes and b-AP15 interacting proteins respectively. Deletion of genes encoding mitochondrial proteins decreased the sensitivity to b-AP15, suggesting that mitochondrial dysfunction is coupled to cell death induced by b-AP15. Enzymes known to be involved in Phase II detoxification such as aldo-ketoreductases and glutathione-S-transferases were identified as b-AP15-targets using PISA. The finding that different exploratory approaches yielded different results may be explained in terms of a “target” not necessarily connected to the “mechanism of action” thus highlighting the importance of a holistic approach in the identification of drug targets. We conclude that b-AP15, and likely also other dienone compounds of the same class, affect protein degradation and proteasome function at more than one level. Frontiers Media S.A. 2022-04-22 /pmc/articles/PMC9076133/ /pubmed/35530310 http://dx.doi.org/10.3389/fonc.2022.852980 Text en Copyright © 2022 Gubat, Selvaraju, Sjöstrand, Kumar Singh, Turkina, Schmierer, Sabatier, Zubarev, Linder and D’Arcy https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Oncology
Gubat, Johannes
Selvaraju, Karthik
Sjöstrand, Linda
Kumar Singh, Dhananjay
Turkina, Maria V.
Schmierer, Bernhard
Sabatier, Pierre
Zubarev, Roman A.
Linder, Stig
D’Arcy, Pádraig
Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title_full Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title_fullStr Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title_full_unstemmed Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title_short Comprehensive Target Screening and Cellular Profiling of the Cancer-Active Compound b-AP15 Indicate Abrogation of Protein Homeostasis and Organelle Dysfunction as the Primary Mechanism of Action
title_sort comprehensive target screening and cellular profiling of the cancer-active compound b-ap15 indicate abrogation of protein homeostasis and organelle dysfunction as the primary mechanism of action
topic Oncology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9076133/
https://www.ncbi.nlm.nih.gov/pubmed/35530310
http://dx.doi.org/10.3389/fonc.2022.852980
work_keys_str_mv AT gubatjohannes comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT selvarajukarthik comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT sjostrandlinda comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT kumarsinghdhananjay comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT turkinamariav comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT schmiererbernhard comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT sabatierpierre comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT zubarevromana comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT linderstig comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction
AT darcypadraig comprehensivetargetscreeningandcellularprofilingofthecanceractivecompoundbap15indicateabrogationofproteinhomeostasisandorganelledysfunctionastheprimarymechanismofaction