March 26, 2021

Oksana Reznichenko (2021)

Combinatorial chemistry approaches for the development of G-quadruplex DNA and RNA ligands

Abstract

G-quadruplexes (G4s) are four-stranded structures of nucleic acids (DNA or RNA) that consist of at least two coplanar guanine quartets. An important feature of G4s is their ability to form stable complexes with exogenous small molecules (ligands) and thus influence biological processes in which they are involved. G4 targeting is often associated with oncology, where G4 ligands may suppress the expression of oncogenes, inhibit telomerase, or induce DNA damage in cancer cells. This work aims to develop methodologies for rapid and simple synthesis and screening of compounds, in order to identify selective and highly affine ligands of given non-canonical structures of nucleic acids, in particular G4s. Specifically, this works exploits the chemistry of reversible synthesis of acylhydrazones, which has been barely applied for the development of DNA or RNA ligands before. First, a small library of 20 cationic bis(acylhydrazones), analogues of the previously reported G4-ligands PDC (360A) and PhenDC3, was obtained by preparative synthesis. Through fluorescence melting experiments it is demonstrated that some of compounds indeed have high affinity to G4-DNA, validating the suitability of the acylhydrazone motif as a scaffold for the development of G4 ligands. Next, a method of dynamic combinatorial chemistry (DCC), which consists in simultaneous one-pot generation of libraries of up to 20 compounds with consecutive pull-down of most affine ligands by bead-immobilized targets (i.e., G4-DNA), was developed. By using this method, a non-symmetrical bis(acylhydrazone) was identified as a promising ligand of a parallel G4-DNA Pu24T. However, biophysical experiments with its close structural analogues did not confirm their preferential binding in comparison with the symmetrically substituted compound. It is proposed that the outcome of DCC experiments may be biased by non-specific interactions of ligands with magnetic beads, leading to false-positive results. In order to improve the analysis of dynamic combinatorial libraries, a novel method based on solid-phase extraction of the G4-ligand complex was developed and applied to two libraries of non-symmetric acylhydrazones. In a few rounds of selection, 13 hits were obtained out of 70 in situ generated compounds. Three of them were selected for preparative synthesis and detailed study of interaction with G4-DNA. In parallel, a classical combinatorial chemistry approach was developed, resulting in generation of a combinatorial library of 90 individual bis(acylhydrazone) derivatives in the form of ready-to-use 2 mM solutions in DMSO, with an average purity of 87%. These samples were directly used for biophysical screening experiments towards four G4-DNA targets of three different topologies. Three most active compounds were obtained in preparative manner and their interaction with the mentioned biological targets was studied in detail by several biophysical methods, including native mass spectrometry experiments. This way, at least one derivative with a G4-DNA affinity superior to that of PhenDC3 and unprecedented selectivity towards anti-parallel G4-DNA could be identified. Finally, in the framework of a collaborative project (M. Blondel, University of Western Brittany) the ligands synthesized in this work were studied with respect to their capacity to act as modulators of the immune evasion of Epstein–Barr virus (EBV). Specifically, it was shown that several bis(acylhydrazones) bind in vitro to G4-RNA structures formed by the guanine-rich repeat sequence of mRNA encoding for the glycine-alanine rich (GAr) domain of viral genome maintenance protein EBNA1. Moreover, two derivatives were found to displace the host cell factor nucleolin from EBNA1 mRNA, leading to overexpression of EBNA1 protein and a concomitant increase of antigen presentation in EBV-infected cell cultures. This effect represents an interesting therapeutic opportunity for treatment of EBV-related cancers. (Defended on March 26, 2021)