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. 2023 Feb 14;18(4):e202200549.
doi: 10.1002/cmdc.202200549. Epub 2023 Jan 23.

Peptides Targeting RNA m6 A Methylations Influence the Viability of Cancer Cells

Rushdhi Rauff  1 , Sudeshi M Abedeera  1 , Stefani Schmocker  1 , Jiale Xie  1 , Sanjaya C Abeysirigunawardena  1
Affiliations

Peptides Targeting RNA m6 A Methylations Influence the Viability of Cancer Cells

Rushdhi Rauff et al. ChemMedChem. .

Abstract

N6-methyladenosine (m6 A) is the most abundant nucleotide modification observed in eukaryotic mRNA. Changes in m6 A levels in transcriptome are tightly correlated to expression levels of m6 A methyltransferases and demethylases. Abnormal expression levels of methyltransferases and demethylases are observed in various diseases and health conditions such as cancer, male infertility, and obesity. This research explores the efficacy of m6 A-modified RNA as an anticancer drug target. We discovered a 12-mer peptide that binds specifically to m6 A-modified RNA using phage display experiments. Our fluorescence-based assays illustrate the selected peptide binds to methylated RNA with lower micromolar affinity and inhibit the binding of protein FTO, a demethylase enzyme specific to m6 A modification. When cancer cell lines were treated with mtp1, it led to an increase in m6 A levels and a decrease in cell viability. Hence our results illustrate the potential of mtp1 to be developed as a drug for cancer.

Keywords: FTO; epitranscriptomics; m6A; peptide inhibitors; phage display.

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Figures

Figure 1.
Figure 1.
The most enriched peptide in phage display against m6A-modified RNA target, mtp1 binds tightly to RNA. a) Percentage occurrence of peptides after four cycles of biopanning is shown. b) sequence logo for all the selected peptides illustrates high sequences conservation among the enriched peptides. c) The structure of the most abundant peptide (mtp1) predicted by the PEPFOLD3 algorithm. The mtp1 peptide is composed of positively charged (red), negatively charged (blue), and aromatic (lilac) amino acid residues. d) RNA titration curve (0-10 μM) constructed using tryptophan fluorescence change for mtp1 peptide. e) Peptide titration curve (0-12 μM). The fraction bound was calculated using circular dichroism change of RNA (276-280 nm). All titrations were performed in triplicate to ensure reproducibility.
Figure 2.
Figure 2.
The mtp1 peptide inhibits the binding and demethylase activity of FTO. a) Schematic representation of FTO binding inhibition assay. b) FRET between cyanine dyes on RNA and FTO plotted against FTO concentrations. FTO titration curves were fitted quadratic equation to determine the Kd for the FTO-RNA complex. c) Changes in FRET for FTO-RNA complexes with increasing mtp1 concentration are shown. d) m6A levels in total RNA increase in the presence of mtp1. Error bars represent the standard deviation of biological triplicates.
Figure 3.
Figure 3.
The mtp1 peptide decreases cancer cell viability. a) The decrease in viability of various cancer cells upon transfection of mtp1 is shown. The standard deviation of three biological replicates is shown as error bars. b) The IC50 of mtp1 for MDA-MB 231 cells. c) Viability of MDA-MB cells upon transfection of a control peptide is shown.
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