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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
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- Gubat, Johannes, 1986- (author)
- Linköpings universitet,Avdelningen för klinisk kemi och farmakologi,Medicinska fakulteten
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- Selvaraju, Karthik, 1983- (author)
- Linköpings universitet,Avdelningen för klinisk kemi och farmakologi,Medicinska fakulteten
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- Sjöstrand, Linda (author)
- Linköpings universitet,Avdelningen för klinisk kemi och farmakologi,Medicinska fakulteten
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- Kumar Singh, Dhananjay (author)
- Linköpings universitet,Institutionen för biomedicinska och kliniska vetenskaper,Medicinska fakulteten,Department of Pharmacy, Central University of South Bihar, Gaya, India
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- Turkina, Maria V, 1973- (author)
- Linköpings universitet,Avdelningen för cellbiologi,Medicinska fakulteten
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- Schmierer, Bernhard (author)
- Karolinska Institutet,Department of Medical Biochemistry and Biophysics, Division of Chemical Biology, Karolinska Institutet, Stockholm, Sweden
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- Sabatier, Pierre (author)
- Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry I, Karolinska Institutet, Stockholm, Sweden
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- Zubarev, Roman A. (author)
- Karolinska Institutet,Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry I, Karolinska Institutet, Stockholm, Sweden; Department of Pharmacological and Technological Chemistry, I.M. Sechenov First Moscow State Medical University, Moscow, Russia
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- Linder, Stig, 1954- (author)
- Linköpings universitet,Avdelningen för klinisk kemi och farmakologi,Medicinska fakulteten,Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
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- D´arcy, Pádraig, 1978- (author)
- Karolinska Institutet,Linköpings universitet,Avdelningen för klinisk kemi och farmakologi,Medicinska fakulteten
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(creator_code:org_t)
- 2022-04-22
- 2022
- English.
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In: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 12
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Abstract
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- 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.
Subject headings
- MEDICIN OCH HÄLSOVETENSKAP -- Klinisk medicin -- Cancer och onkologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Clinical Medicine -- Cancer and Oncology (hsv//eng)
Keyword
- b-AP15
- proteasome inhibitor
- mitochondrial dysfunction
- dienone
- Michael acceptor
- target screening
Publication and Content Type
- ref (subject category)
- art (subject category)
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- By the author/editor
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Gubat, Johannes, ...
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Selvaraju, Karth ...
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Sjöstrand, Linda
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Kumar Singh, Dha ...
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Turkina, Maria V ...
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Schmierer, Bernh ...
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Sabatier, Pierre
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Zubarev, Roman A ...
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Linder, Stig, 19 ...
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D´arcy, Pádraig, ...
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- About the subject
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- MEDICAL AND HEALTH SCIENCES
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MEDICAL AND HEAL ...
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and Clinical Medicin ...
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and Cancer and Oncol ...
- Articles in the publication
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Frontiers in Onc ...
- By the university
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Linköping University
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Karolinska Institutet