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<p>Trabectedin is a marine-derived antitumor agent from the ecteinascidin family that is effective in treating Ewing sarcoma. It features a carbinolamine that forms reversible covalent bonds with N2 of guanine, distinguishing it from other structurally similar duplex DNA alkylating agents. Importantly, the ecteinascidins are structurally distinct from other carbinolamine alkylating drugs by inclusion of a C-subunit that widens the DNA minor groove and bends DNA into the major groove. Taking into account the sensitivity of soft tissue sarcomas to Trabectedin and the role of the Ewing sarcoma protein (EWS) in binding to G-quadruplexes (G4s), this research proposes a refined understanding of Trabectedin’s molecular target by exploring its potential interaction with G4 structures. Building on these insights, we designed experiments using Trabectedin in combination with G4s, including the wild-type MYCN G4s, to assess whether Trabectedin could form stable covalent bonds to a consensus sequence within a duplex stem loop potentially influenced by its association with a G4 structure. Using a combination of circular dichroism, quantitative Förster resonance energy transfer (FRET) melting assays, and LC-MS, we demonstrate that Trabectedin can engage and stabilize composite G4–duplex architectures, provided a guanine-containing covalent bonding site is present within the associated hairpin loop. However, an LC-MS affinity assay revealed that Trabectedin still retains a preference for duplex DNA over the G4-containing construct. Finally, we observed that Trabectedin treatment of cells significantly increased G4 frequency, suggesting a modulation of G4 dynamics. We propose a context-dependent recognition model wherein Trabectedin’s dominant reactivity still lies with duplex DNA, but its structural compatibility allows engagement with reactive motifs embedded within G4-associated stem-loop architectures, potentially contributing to its unique downstream biological effects. To explain why Trabectedin is more potent and clinically efficacious than other carbinolamine-containing antitumor antibiotics such as Saframycin A, we propose a unique “search and lock” mechanism in which Trabectedin preferentially covalently bonds to G4-associated stem loops due to context-dependent enhanced stability of its covalent adduct.</p>

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Investigating the role of G-quadruplex-associated duplex stem loops in Trabectedin’s mechanism of action

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月1日

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  • Alena Safarova,
  • Raquel Altares,
  • Marta Martinez-Diez,
  • Ismael Fernández-Miranda,
  • Pablo Aviles,
  • Marcelo L. Ribeiro,
  • Carmen Cuevas,
  • Daniel Von Hoff,
  • Laurence H. Hurley

摘要

Trabectedin is a marine-derived antitumor agent from the ecteinascidin family that is effective in treating Ewing sarcoma. It features a carbinolamine that forms reversible covalent bonds with N2 of guanine, distinguishing it from other structurally similar duplex DNA alkylating agents. Importantly, the ecteinascidins are structurally distinct from other carbinolamine alkylating drugs by inclusion of a C-subunit that widens the DNA minor groove and bends DNA into the major groove. Taking into account the sensitivity of soft tissue sarcomas to Trabectedin and the role of the Ewing sarcoma protein (EWS) in binding to G-quadruplexes (G4s), this research proposes a refined understanding of Trabectedin’s molecular target by exploring its potential interaction with G4 structures. Building on these insights, we designed experiments using Trabectedin in combination with G4s, including the wild-type MYCN G4s, to assess whether Trabectedin could form stable covalent bonds to a consensus sequence within a duplex stem loop potentially influenced by its association with a G4 structure. Using a combination of circular dichroism, quantitative Förster resonance energy transfer (FRET) melting assays, and LC-MS, we demonstrate that Trabectedin can engage and stabilize composite G4–duplex architectures, provided a guanine-containing covalent bonding site is present within the associated hairpin loop. However, an LC-MS affinity assay revealed that Trabectedin still retains a preference for duplex DNA over the G4-containing construct. Finally, we observed that Trabectedin treatment of cells significantly increased G4 frequency, suggesting a modulation of G4 dynamics. We propose a context-dependent recognition model wherein Trabectedin’s dominant reactivity still lies with duplex DNA, but its structural compatibility allows engagement with reactive motifs embedded within G4-associated stem-loop architectures, potentially contributing to its unique downstream biological effects. To explain why Trabectedin is more potent and clinically efficacious than other carbinolamine-containing antitumor antibiotics such as Saframycin A, we propose a unique “search and lock” mechanism in which Trabectedin preferentially covalently bonds to G4-associated stem loops due to context-dependent enhanced stability of its covalent adduct.