Optimization of G-Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones)

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules (ligands) and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate ready-to-screen libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.

Automated summary

G-quadruplexes, secondary structures adopted by guanine-rich DNA and RNA sequences, have attracted increasing interest as potential drug targets. In this study, a simple method of parallel synthesis was developed to generate ready-to-screen libraries of cationic acylhydrazones. Using this method, a combinatorial library of 90 bis(acylhydrazones) was synthesized and screened against five different nucleic acid structures. Three novel G4 ligands with promising interactions with G4-DNAs of different topologies were identified and studied in detail by various biophysical techniques.