A Proteomic Platform to Identify Off-Target Proteins Associated with Therapeutic Modalities that Induce Protein Degradation or Gene Silencing

ABSTRACT: Novel modalities such as Proteolysis Targeting Chimera (PROTAC) and RNA interference (RNAi) have the ability to inadvertently alter the abundance of endogenous proteins. Currently available in vitro secondary pharmacology assays, which evaluate off-target binding or activity of small molecules, do not fully assess the off-target effects of PROTAC and are not applicable to RNAi. To address this gap, we developed a proteomics-based platform to comprehensively evaluate abundance of off-target proteins. The first part of the study involves selecting a panel of off-target proteins and a platform of cell lines using evidence from genetics and pharmacology. This process yielded 2,813 proteins, forming the basis of a panel that we refer to as the “selected off-target proteome” (SOTP). An iterative algorithm was then used to identify appropriate cell lines. Four human cell lines out of 932 were selected that, collectively, expressed ~ 80% of the SOTP based on transcriptome data. Second, we used mass spectrometry to quantify the intracellular and extracellular proteins of interest in the 4 selected cell lines. Among over 10,000 quantifiable proteins identified, 1,828 were part of the predefined SOTP. The SOTP was designed to be easily modified or expanded, owing rationale selection process developed and the label free LC-MS/MS approach chosen. This versatility inherent to our platform is essential to design fit-for-purpose studies that can address the dynamic questions faced in investigative toxicology.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ye Zhang  

LAB HEAD: Fan Fan

PROVIDER: PXD024312 | Pride | 2021-09-10

REPOSITORIES: pride