Affinity Chromatography-Based Assays for the Screening of Potential Ligands Selective for G-Quadruplex Structures

Abstract

DNA G-quadruplexes (G4s) are key structures for the development of targeted anticancer therapies. In this context, ligands selectively interacting with G4s can represent valuable anticancer drugs. Aiming at speeding up the identification of G4-targeting synthetic or natural compounds, we developed an affinity chromatography-based assay, named G-quadruplex on Oligo Affinity Support (G4-OAS), by synthesizing G4-forming sequences on commercially available polystyrene OAS. Then, due to unspecific binding of several hydrophobic ligands on nude OAS, we moved to Controlled Pore Glass (CPG). We thus conceived an ad hoc functionalized, universal support on which both the on-support elongation and deprotection of the G4-forming oligonucleotides can be performed, along with the successive affinity chromatography-based assay, renamed as G-quadruplex on Controlled Pore Glass (G4-CPG) assay. Here we describe these assays and their applications to the screening of several libraries of chemically different putative G4 ligands. Finally, ongoing studies and outlook of our G4-CPG assay are reported.

Keywords: G-quadruplexes; affinity chromatography; cancer; drug discovery; oligonucleotides.

Figure 1

A) Structure and schematic representation of a G‐quartet. B) Schematic representation of unimolecular, bimolecular and tetramolecular G4s formed by stacking of three G‐quartets. C) Examples of three different topologies of unimolecular G4. D) Types of linking loops in a G4. E) The anti/syn conformations of the 2′‐deoxyguanosine. M⁺ indicates a stabilizing metal cation, for example K⁺ or Na⁺. R=1‐β‐D‐2‐deoxyribofuranosyl group. Green circles indicate the 5’‐end.

Figure 2

A) Schematic representation of the commercially available Oligo Affinity Support (OAS), a polystyrene/polyethylene glycol (PS)‐based resin functionalized with a 4,4′‐dimethoxytriphenylmethyl (DMT)‐protected adenosine derivative. B) Schematic representation of the OAS resin functionalized with the 26‐mer d[(TTAGGG)₄TT] sequence (tel26), a truncation of the human telomeric DNA, folded into a G4 structure in a K⁺‐containing solution. Ac=acetyl. Reproduced with permission from Ref. [37] Copyright 2014, American Chemical Society.

Figure 3

Schematic representation of the affinity‐chromatography‐based G4‐binding assay. r. t.=room temperature.

Figure 4

Chemical structures of some known G4 ligands (TMPyP4, TO, RHPS4, Distamycin and Netropsin) and two molecules unable to bind the G4s (9‐Acr‐COOH and Resveratrol), used as models for the G4‐binding assays.

Figure 5

General scheme for the screening of a mixture of multiple small molecules by the G4‐OAS assay.

Figure 6

Chemical structures of the best G4 ligands from Library I (2, 6, 7) and II (10B, 4D, 7E, 1F, 2F, 7F, 8F).[ ³⁷ , ⁴⁰ ]

Figure 7

A) Commercially available Long Chain AlkylAmine Controlled Pore Glass (LCAA‐CPG) solid support. B) Functionalization of LCAA‐CPG with 5′‐O‐DMT, 3′‐O‐acetyl‐thymidine through the selected hexaethylene glycol spacer. Ac=acetyl; DCC=N,N′‐dicyclohexylcarbodiimide; DEAD=diethyl azodicarboxylate; DMF=N,N‐dimethylformamide; DMT=4,4′‐dimethoxytriphenylmethyl; HOBt=1‐hydroxybenzotriazole; Ph=phenyl; TCA=trichloroacetic acid; THF=tetrahydrofuran. Adapted with permission from Ref. [41] Copyright 2018, Elsevier.

Figure 8

A) Chemical structure of c_ex‐NDI. B) Schematic picture of the fluorescent behaviour of c_ex‐NDI when bound to hybrid G4s, producing a dramatic fluorescence enhancement, and parallel G4s or duplex DNA with a weaker fluorescence emission. C) Representative confocal images of tel26‐, c‐kit1‐ and ds27‐functionalized CPG supports after incubation with the c_ex‐NDI. Merged images of c_ex‐NDI fluorescence and transmitted light. Scale bars correspond to 100 μm. D) Mean fluorescence intensity values (±S.D.) taken from different edge glass beads‐containing regions of each image acquired for tel26‐, c‐kit1‐ and ds27‐functionalized CPG supports. p‐values have been calculated using the Student's t‐test (****p<0.0001). Adapted with permission from Ref. [41] Copyright 2018, Elsevier.

Figure 9

Chemical structures of the best G4 ligand (S4‐5) from Library III and its parent compound (4).

Figure 10

Chemical structures of the best G4 ligands from Library IV–V.[ ⁶⁶ , ⁶⁷ ]

Figure 11

A) Summary of the binding assay data for NDI‐5 on nude and functionalized CPG supports. Bound ligand calculated as a difference from the unbound ligand and expressed as % of the amount initially loaded on the support. The errors associated with the % are within ±2 %. B) Representative merged images of immunofluorescence of BJ‐EHLT cancer cell line treated with NDI‐5; H2AX (DNA damage marker) spots are green, TRF1 (telomeric marker) spots are red and nuclei are blue. Enlarged views of Telomere Induced Foci (TIFs) are reported on the right smaller panels. C) Job plot analysis for the binding of NDI‐5 to tel26 G4. D) CD spectra of tel46 G4 in the absence and presence of increasing amounts of NDI‐5. Black arrows indicate intensity changes of the specific bands upon increasing NDI‐5 concentration. E) ITC binding isotherm for titration of tel46 G4 with NDI‐5. F) Binding mode of NDI‐5 to tel46 G4 obtained by molecular docking. A), B) and C) are adapted with permission from Ref. [66] under CC‐BY 4.0, Copyright 2020 by the authors; D), E) and F) are adapted with permission from Ref. [67] Copyright 2020 Elsevier.

Figure 12

Chemical structures of the best G4 ligands from Library VI.

Figure 13

Chemical structures of Canadine, D‐Glaucine, Dicentrine, Deguelin and Millettone from Library VII, and of J10, the best G4 ligand from Library VIII.[ ³³ , ⁷⁴ ]