Towards Profiling of the G-Quadruplex Targeting Drugs in the Living Human Cells Using NMR Spectroscopy.

Silvie Foldynová-Trantírková,Eva Ištvánková,Pavlína Víšková,Daniel Krafčík,Šimon Džatko,Lukáš Trantírek

doi:10.3390/ijms22116042

Abstract

Recently, the 1H-detected in-cell NMR spectroscopy has emerged as a unique tool allowing the characterization of interactions between nucleic acid-based targets and drug-like molecules in living human cells. Here, we assess the application potential of 1H and 19F-detected in-cell NMR spectroscopy to profile drugs/ligands targeting DNA G-quadruplexes, arguably the most studied class of anti-cancer drugs targeting nucleic acids. We show that the extension of the original in-cell NMR approach is not straightforward. The severe signal broadening and overlap of 1H in-cell NMR spectra of polymorphic G-quadruplexes and their complexes complicate their quantitative interpretation. Nevertheless, the 1H in-cell NMR can be used to identify drugs that, despite strong interaction in vitro, lose their ability to bind G-quadruplexes in the native environment. The in-cell NMR approach is adjusted to a recently developed 3,5-bis(trifluoromethyl)phenyl probe to monitor the intracellular interaction with ligands using 19F-detected in-cell NMR. The probe allows dissecting polymorphic mixture in terms of number and relative populations of individual G-quadruplex species, including ligand-bound and unbound forms in vitro and in cellulo. Despite the probe’s discussed limitations, the 19F-detected in-cell NMR appears to be a promising strategy to profile G-quadruplex–ligand interactions in the complex environment of living cells.

Highlights

Nucleic acids (NAs), in particular non-B DNA structures, are an important class of drug targets [1,2]
The ligand-binding capacities determined under non-native conditions do not, in many cases, reflect the efficacy that the ligands display in vivo, where intracellular factors might drastically affect the structure of the target [8]
We show that the use of G4 constructs modified with 3,5-bis(trifluoromethyl)phenyl tag, recently developed by Xu et al [37,38,60], combined with 19F-detected in-cell nuclear magnetic resonance (NMR), can help overcome the problems of 1H-detected in-cell NMR

Summary

Introduction

Nucleic acids (NAs), in particular non-B DNA structures, are an important class of drug targets [1,2]. The most commonly used approaches have relied on using formulated buffer solutions to recapitulate non-specific properties of the cellular environment These factors might include: the composition mimicking the simultaneous presence of various ions (K+, Na+, Mg2+, Ca2+); synthetic additives (polyethylene glycol, Ficoll) emulating the molecular crowding and reduced dielectric permittivity of the cellular interior; micelles and nanopores simulating cellular confinements; the genomic DNA isolated from cells imitating the presence of genomic off-targets [10]. The environmentally promoted polymorphism, the lack of information on the active biological structure of G4, hampers the attempts for rational drug design and virtual screening applications Disregarding both the polymorphism and cellular context in HTS, ligand off-targeting in vivo, results in a high failure rate during the development of specific G4-binding drugs. Our data show 19F in-cell NMR can become a promising strategy to profile G4–ligand interactions in the complex environment of living cells

Towards Characterization of G4–Ligand Interactions In Situ Using In-Cell NMR

Materials and Methods

Conclusions and Perspectives