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Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

ABSTRACT: Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays. Biophysical analyses find no direct evidence for interaction of remodelin with the NAT10 acetyltransferase active site. Cellular studies verify that N4-acetylcytidine (ac4C) is a nonredundant target of NAT10 activity in human cell lines and find that this RNA modification is not affected by remodelin treatment in several orthogonal assays. These studies display the potential for remodelin's chemotype to interact with multiple protein targets in cells and indicate remodelin should not be applied as a specific chemical inhibitor of NAT10-catalyzed RNA acetylation.

SUBMITTER: Shrimp JH

PROVIDER: S-EPMC8201477 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

Shrimp Jonathan H JH Jing Yihang Y Gamage Supuni Thalalla ST Nelson Kathryn M KM Han Joseph J Bryson Keri M KM Montgomery David C DC Thomas Justin M JM Nance Kellie D KD Sharma Sunny S Fox Stephen D SD Andressen Thorkell T Sinclair Wilson R WR Wu Hong H Allali-Hassani Abdellah A Senisterra Guillermo G Vedadi Masoud M Lafontaine Denis D Dahlin Jayme L JL Marmorstein Ronen R Walters Michael A MA Meier Jordan L JL

ACS medicinal chemistry letters 20200727 6

Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in AL ...[more]

PMID: 34141066

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Project description:BackgroundN-4 cytidine acetylation (ac4C) is an epitranscriptomics modification catalyzed by N-acetyltransferase 10 (NAT10); important for cellular mRNA stability, rRNA biogenesis, cell proliferation and epithelial to mesenchymal transition (EMT). However, whether other crucial pathways are regulated by NAT10-dependent ac4C modification in cancer cells remains unclear. Therefore, in this study, we explored the impact of NAT10 depletion in cancer cells using unbiased RNA-seq.MethodsHigh-throughput sequencing of knockdown NAT10 in cancer cells was conducted to identify enriched pathways. Acetylated RNA immunoprecipitation-seq (acRIP-seq) and RIP-PCR were used to map and determine ac4C levels of RNA. Exogenous palmitate uptake assay was conducted to assess NAT10 knockdown cancer cells using Oil Red O staining and lipid content analysis. Gas-chromatography-tandem mass spectroscopy (GC/MS) was used to perform untargeted lipidomics.ResultsHigh-throughput sequencing of NAT10 knockdown in cancer cells revealed fatty acid (FA) metabolism as the top enriched pathway through the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in differentially downregulated genes. FA metabolic genes such as ELOLV6, ACSL1, ACSL3, ACSL4, ACADSB and ACAT1 were shown to be stabilised via NAT10-dependent ac4C RNA acetylation. Additionally, NAT10 depletion was shown to significantly reduce the levels of overall lipid content, triglycerides and total cholesterol. Further, NAT10 depletion in palmitate-loaded cancer cells showed decrease in ac4C levels across the RNA transcripts of FA metabolic genes. In untargeted lipidomics, 496 out of 2 279 lipids were statistically significant in NAT10 depleted cancer cells, of which pathways associated with FA metabolism are the most enriched.ConclusionsConclusively, our results provide novel insights into the impact of NAT10-mediated ac4C modification as a crucial regulatory factor during FA metabolism and showed the benefit of targeting NAT10 for cancer treatment.

Dataset Information

Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

Publications

Remodelin Is a Cryptic Assay Interference Chemotype That Does Not Inhibit NAT10-Dependent Cytidine Acetylation.

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