title: An ultrasensitive assay format for detecting ULK1 inhibition by monitoring the phosphorylation status of Atg13.
abstract: A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer(318)) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer(318)-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer(318)-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer(318)-Atg13 upon ULK1 inhibition.
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Zhong-Hua
authors.initials: ZH
authors.lastname: Yan
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Wenhai
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Rollins
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Tayber
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Jiejin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Dongyun
authors.initials: D
authors.lastname: Wu
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Pam
authors.initials: P
authors.lastname: Brauer
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Johara
authors.initials: J
authors.lastname: Chouitar
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Bosse
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Yu
authors.affiliation: Takeda Pharmaceuticals International Co., Cambridge, MA, USA. Electronic address: michael.bembenek@takeda.com.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bembenek
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D000071189:Autophagy-Related Protein-1 Homolog / Q000378:metabolism*; D000071183:Autophagy-Related Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D064593:Heterografts; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*
chemical_list: C534291:ATG13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000071189:Autophagy-Related Protein-1 Homolog; C484754:ULK1 protein, human
keywords: Endogenous Atg13; Immunoassay; Quanterix; Sandwich ELISA; SiMoA; ULK1 inhibition