title: Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.
abstract: Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.
authors.affiliation: Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA. dnomura@berkeley.edu.
authors.forename: L A
authors.initials: LA
authors.lastname: Bateman
authors.forename: T B
authors.initials: TB
authors.lastname: Nguyen
authors.forename: A M
authors.initials: AM
authors.lastname: Roberts
authors.forename: D K
authors.initials: DK
authors.lastname: Miyamoto
authors.forename: W-M
authors.initials: WM
authors.lastname: Ku
authors.forename: T R
authors.initials: TR
authors.lastname: Huffman
authors.forename: Y
authors.initials: Y
authors.lastname: Petri
authors.forename: M J
authors.initials: MJ
authors.lastname: Heslin
authors.forename: C M
authors.initials: CM
authors.lastname: Contreras
authors.forename: C F
authors.initials: CF
authors.lastname: Skibola
authors.forename: J A
authors.initials: JA
authors.lastname: Olzmann
authors.forename: D K
authors.initials: DK
authors.lastname: Nomura
mesh_terms: D020106:Acrylamide / Q000737:chemistry / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D003545:Cysteine / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D000070798:Nogo Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism; D040901:Proteomics
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D000070798:Nogo Proteins; C000603199:RTN4 protein, human; D020106:Acrylamide; D003545:Cysteine