Identifying G-Quadruplex-DNA-Disrupting Small Molecules

Abstract

The quest for small molecules that strongly bind to Gquadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV−vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

Modulation of the Conformational Space of SARS‐CoV‐2 RNA Quadruplex RG‐1 by Cellular Components and the Amyloidogenic Peptides α‐Synuclein and hIAPP

“…G4Qs have therefore received considerable attention over the last twenty years due to their involvement in the regulation of cellular processes including replication, transcription and translation. [ 10 , 11 , 12 , 13 , 14 , 15 ] Dysregulation of G4Q formation and their binding proteins, which assist them in regulating the equilibrium between their structured and unstructured/unfolded forms, due to mutations or through the alteration of their stability by environmental factors (for example, by changes in intracellular solution conditions or by G4Q‐stabilization induced by a ligand), have been found to contribute to many human pathologies, including neurodegenerative diseases, cancer, and microbial infections. [ 4 , 5 , 6 , 7 ]…”

Ligand-induced unfolding mechanism of an RNA G-quadruplex

“…32 It is only recently (while this manuscript was under review) that Monchaud and co-workers have published a G4-helicase based destabilization assay. 44 Nevertheless, there are small molecules that have been reported to destabilize G4s. For example, TMPyP4 disrupted the G4 structure in the Fragile X FMR1 gene, 42 and in the MT3 endopeptidase mRNA sequence; 45 a triarylpyridine derivative disrupted G4 in the cKit-1 and 2 sequences; 46 an anthrathiophenedione, 47 and a stiffstilbene derivative was shown to unfold a sodium form of telomeric G4.…”

Major Achievements in the Design of Quadruplex-Interactive Small Molecules

“…Moreover, molecular dynamics and NMR studies suggested that STI binds to G4 grooves and intercalates between G-quartets, which may lead to disruption of the G-quartets H-bond network [327]. Very recently, the ability of a set of chemically diverse G4 ligands to disrupt G4 structures was investigated using several biophysical and biochemical methods [329]. The authors concluded that the effect of G4 ligands on the G4 structure is strongly dependent on the technique and concentration of the ligand, in agreement with a previous study [330].…”

“…Very recently, the ability of a set of chemically diverse G4 ligands to disrupt G4 structures was investigated using several biophysical and biochemical methods [ 329 ]. The authors concluded that the effect of G4 ligands on the G4 structure is strongly dependent on the technique and concentration of the ligand, in agreement with a previous study [ 330 ].…”

“…The authors concluded that the effect of G4 ligands on the G4 structure is strongly dependent on the technique and concentration of the ligand, in agreement with a previous study [ 330 ]. Moreover, the study revealed a new G4 disrupting small molecule, the G-clamp analogue PhpC ( Figure 20 ), whose ability to efficiently disrupt G4 structures facilitated G4-helicase activity [ 329 ].…”