Mechanism of Action of Prethioviridamide, an Anticancer Ribosomally Synthesized and Post-Translationally Modified Peptide with a Polythioamide Structure

Abstract

Thioviridamide, prethioviridamide, and JBIR-140, which are ribosomally synthesized and post-translationally modified peptides (RiPPs) possessing five thioamide bonds, induce selective apoptosis in various cancer cells, especially those expressing the adenovirus oncogene E1A. However, the target protein of this unique family of bioactive compounds was previously unknown. To investigate the mechanism of action, we adopted a combined approach of genome-wide shRNA library screening, transcriptome profiling, and biochemical identification of prethioviridamide-binding proteins. An shRNA screen identified 63 genes involved in cell sensitivity to prethioviridamide, which included translation initiation factors, aminoacyl tRNA synthetases, and mitochondrial proteins. Transcriptome profiling and subsequent analysis revealed that prethioviridamide induces the integrated stress response (ISR) through the GCN2-ATF4 pathway, which is likely to cause cell death. Furthermore, we found that prethioviridamide binds and inhibits respiratory chain complex V (F1Fo-ATP synthase) in mitochondria, suggesting that inhibition of complex V leads to activation of the GCN2-ATF4 pathway. These results imply that the members of a unique family of RiPPs with polythioamide structure target mitochondria to induce the ISR.

The Utilization of Lanthipeptide Synthetases Is a General Strategy for the Biosynthesis of 2‐Aminovinyl‐Cysteine Motifs in Thioamitides**

“…[1] Thioamitides are as ubgroup of RiPPs that contain thioamides in place of amides in peptide backbones as their class-defining feature. [2] As the first example of this group of compounds,a poptosis inducer thioviridamide contains anumber of unnatural amino acids,i ncluding backbone thioamides,ab-hydroxy-N1,N3dimethylhistidinium (hdmHis) residue,aN-terminal d-hydroxy-d-methyl-4-oxopentanoyl group and aC -terminal 2aminovinyl-cysteine (AviCys) motif ( Figure 1A). [3] Posttranslational modification enzymes are identified in the tva gene cluster( Figure S1A), includingT vaH/TvaI as ap airo f homologs to YcaO/TfuAp roteinsf or thet hioamidation of peptidebackboneand TvaG as aputativemethyltransferase for thedoublemethylation of His (12) residue.…”

Targeting the Mitochondrial Metabolic Network: A Promising Strategy in Cancer Treatment

“…Various natural products have also been shown to have an inhibitory activity against F 1 F O -ATPase, exerting a significant antiproliferative/cytotoxic effect. These compounds include resveratrol and related polyphenols [ 63 ], aurovertin B [ 64 ], and thioviridamide-like molecules (TLMs), which have been shown to alter metabolism in cancer cells, to induce oxidative stress, and to inhibit tumor proliferation in vitro and in preliminary in vivo studies [ 65 , 66 , 67 ].…”

Lanthipeptide Synthetases Participate the Biosynthesis of 2-Aminovinyl-Cysteine Motifs in Thioamitides

“…1 Thioamitides 2 are a subgroup of RiPPs that contain thioamides in place of amides in peptide backbones as their class-defining feature. [3][4][5][6][7] As the first example of this group of compounds, apoptosis inducer thi-2 oviridamide contains a number of unnatural amino acids, including backbone thioamides, a β-hydroxy-N1,N3dimethylhistidinium (hdmHis) residue, a N-terminal δ-hydroxy-δ-methyl-4-oxopentanoyl group and a C-terminal 2aminovinyl-cysteine (AviCys) motif ( Fig. 1A).…”