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NAIA for cysteine profiling in HepG2 cells, protein target identification of covalent ligand and studying cysteine oxidative modifications

ABSTRACT: New cysteine probes, N-acryloylindole-alkynes (NAIAs), have been developed. We have applied NAIA-5 for cysteine profiling in HepG2 cell lysates, and the results have been compared to the conventional cysteine probe iodoacetamide-alkyne (IAA). NAIA-5 allows identification of more ligandable Cys than IAA in our experiment, and more interesting, a unique pool of Cys which have not been profiled previously by any reported cysteine probe. We have further utilized NAIA-5 for covalent ligand screening experiment. This allows us to discover hit compounds targeting the newly identified cysteines and proteins. One of the hit compounds, CL1, demonstrated good bioactivity by arresting cell cycle at G1 phase through targeting proteins associated with cell cycle and regulations. In addition, NAIA-5 has been successfully coupled with NAI compound for studying cysteine oxidative modifications in HepG2 cells by MS experiments.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte

SUBMITTER: Clive Yik-Sham CHUNG

LAB HEAD: Clive Yik-Sham CHUNG

PROVIDER: PXD041264 | Pride | 2023-06-14

REPOSITORIES: Pride

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Project description:The application of toxicogenomics as a predictive tool for chemical risk assessment has been under evaluation by the toxicology community for more than a decade. However, toxicogenomics predominately remains a tool for investigative research rather than for regulatory risk assessment. In this study, we aimed to determine whether the current generation of microarray technology in combination with an in vitro experimental design was capable of generating robust, reproducible data of sufficient quality to show promise as a tool for regulatory risk assessment. To this end, we designed a prospective collaborative study to determine the level of inter- and intra-laboratory reproducibility between three independent laboratories. All test sites adopted the same protocols for all aspects of the toxicogenomic experiment including cell culture, chemical exposure, RNA extraction, microarray data generation and analysis. As a case study, the genotoxic carcinogen B[a]P and the human hepatocyte cell line HepG2 were used to generate three comparable toxicogenomic data sets. High levels of technical reproducibility were demonstrated using a widely employed gene expression microarray platform. While differences at the global transcriptome level were still observed between the test sites, a common subset of B[a]P responsive genes was identified across all test sites which included both genes previously reported in the literature as B[a]P responsive in addition to the same most highly up and down regulated genes. Pending further analysis, these preliminary data show promise that the current generation of microarray technology in combination with a standard in vitro experimental design can produce robust data that can be reproducibly generated in independent laboratories. Future work will need to determine whether in vitro model(s) can be not only reproducible but also predictive for a range of toxic chemicals with different mechanisms of action. Such an approach may potentially be part of future in vitro testing regimes for regulatory risk assessment. The aim of this study was to evaluate the inter- and intra-laboratory reproducibility of toxicogenomics data for toxicity testing in a regulatory setting. 3 Test Centres performed the same experimental protocol treating HepG2 cells with 3 mM Benzo[a]pyrene (B[a]P) (in 0.5% DMSO) for 24 hours prior to RNA extraction and microarray analysis. Three biological replicates of B[a]P and DMSO treatments were analysed by microarray analysis in each Test Centre. Two Test Centres analysed one sample in triplicate to determine technical reproducibility of the chosen array platform. One Centre analysed two samples in duplicate. 24 samples in total were used in this study

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NAIA for cysteine profiling in HepG2 cells, protein target identification of covalent ligand and studying cysteine oxidative modifications

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