Unfolding rates of 1:1 and 2:1 complex of CX-5461 and c-MYC promoter G-quadruplexes revealed by single-molecule force spectroscopy

Abstract

CX-5461, also known as pidnarulex, is a strong G4 stabilizer and has received FDA fast-track designation for BRCA1- and BRCA2- mutated cancers. However, quantitative measurements of the unfolding rates of CX-5461-G4 complexes which are important for the regulation function of G4s, remain lacking. Here, we employ single-molecule magnetic tweezers to measure the unfolding force distributions of c-MYC G4s in the presence of different concentrations of CX-5461. The unfolding force distributions exhibit three discrete levels of unfolding force peaks, corresponding to three binding modes. In combination with a fluorescent quenching assay and molecular docking to previously reported ligand-c-MYC G4 structure, we assigned the ~69 pN peak corresponding to the 1:1 (ligand:G4) complex where CX-5461 binds at the G4’s 5'-end. The ~84 pN peak is attributed to the 2:1 complex where CX-5461 occupies both the 5' and 3'. Furthermore, using the Bell-Arrhenius model to fit the unfolding force distributions, we determined the zero-force unfolding rates of 1:1, and 2:1 complexes to be (2.4 ± 0.9) × 10⁻⁸ s⁻¹ and (1.4 ± 1.0) × 10⁻⁹ s⁻¹ respectively. These findings provide valuable insights for the development of G4-targeted ligands to combat c-MYC-driven cancers.

Graphical abstract

Keywords

Single-molecule / G-quadruplex / Magnetic tweezers / Stoichiometry / Force spectroscopy / Pidnarulex

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Conflict of interestHui Peng, Yashuo Zhang, Qun Luo, Xinyu Wang and Huijuan You declare that they have no conflict of interest. Human and animal rights and informed consent This article does not contain any studies with human or animal subjects performed by any of the authors. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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2024 The Author(s) 2024. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)