"One Ring to Bind Them All"-Part II: Identification of Promising G-Quadruplex Ligands by Screening of Cyclophane-Type Macrocycles

Abstract

A collection of 26 polyammonium cyclophane-type macrocycles with a large structural diversity has been screened for G-quadruplex recognition. A two-step selection procedure based on the FRET-melting assay was carried out enabling identification of macrocycles of high affinity (DeltaT(1/2) up to 30 degrees C) and high selectivity for the human telomeric G-quadruplex. The four selected hits possess sophisticated architectures, more particularly the presence of a pendant side-arm as well as the existence of a particular topological arrangement appear to be strong determinants of quadruplex binding. These compounds are thus likely to create multiple contacts with the target that may be at the origin of their high selectivity, thereby suggesting that this class of macrocycles offers unique advantages for targeting G-quadruplex-DNA.

Figure 1

(a) Structure of BOQ₁. (b) Side-view of the lowest-energy conformation of BOQ₁ during the molecular dynamic simulation. (c) FRET-melting results obtained with telomestatin, TMPyP4, and BOQ₁ (1 μM), for experiments carried out with 0.2 μM F21T in lithium cacodylate buffer (10 mM, pH 7.2) with NaCl (100 mM), in the absence (black bars) or in the presence of 3 (dark green bars) or 10 μM (light green bars) of double-stranded competitor (ds26). (It is worth noting that the FRET-melting values obtained herein for BOQ₁ differ from that of the initial publication (see [9]). This discrepancy originates in the different experimental conditions used, given that FRET-melting is performed herein as an HTS unlike the initial report (single-cell experiment); the disparities between the two methods are extensively detailed in [15] (among which can be cited the final volume (25 versus 600 μL), the buffer conditions (10 mM lithium cacodylate + 100 mM NaCl versus 10 mM sodium cacodylate, 100 mM LiCl), etc.).)

Figure 2

General representation of CBI (a) and CTI (b) macrocycles studied in this work. A, B: (hetero)aromatic residues; X, Y: O, NH, S, or pendant side arms.

Figure 3

Structures of the 26 studied CBIs; see text below for the explanations of the color codes related to affinity and selectivity of the ligands.

Figure 4

Stabilisation of F21T oligonucleotide (ΔT _1/2, °C) induced by CBI macrocycles (1 μM) in Na⁺- (black bars) or K⁺-rich conditions (green bars); see main text for experimental details.

Figure 5

Lowest energy conformations of (a) 3, 3′-BisBP and (b) 3, 3′-TrisBP (front and side-view) during a molecular dynamic simulation in a water box [29].

Figure 6

Plot of stabilization (ΔT _1/2) measured in K⁺- versus Na⁺-rich conditions for the CBI macrocycles.

Figure 7

Stabilisation of F21T oligonucleotide (ΔT _1/2, °C) induced by CBI and CTI macrocycles (1 μM) in (a) Na⁺- and (b) K⁺-rich conditions, for experiments carried out in the absence (black bars) or in the presence of 3 (dark green bars) or 10 μM (light green bars) of the duplex competitor (ds26).

Figure 8

Structures of the four most promising members of the macrocyclic series.