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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 (ΔT1/2 up to 30°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.
The macrocyclic scaffold has proven to be highly valuable for the design of efficient and selective G-quadruplex ligands (see the review article in the present issue, “One Ring to Bind Them All”—Part I, by Monchaud et al.) [1–4]. Along these lines, two families of macrocyclic ligands have been particularly and thoroughly studied for their ability to specifically interact with quadruplex-DNA, namely, the families of telomestatin-  and porphyrin (TMPyP4)-related compounds [6–8]. Recently, a third family has emerged, namely the polyammonium cyclophane-type macrocycles, generally referred to as “CBI macrocycles” (for Cyclo-Bis-Intercalators), that adopt a non-planar conformation and therefore are structurally highly different from the two aforementioned classes. The efficiency of this family of ligands with regard to quadruplex DNA recognition is particularly illustrated by the compound BOQ1 (Figure 1(a)) [9–11] as discussed in “One Ring to Bind Them All”—Part I. This compound is comprised of two large aromatic quinacridine units [12–14] able to interact with DNA bases through π-π interactions, and linked together via two short polyamine side chains, which ensure a global tetracationic charge at physiological pH affording both high water solubility and strong electrostatic attraction to DNA.
We have previously reported that BOQ1 exhibits a high affinity for the human telomeric G-quadruplex DNA, along with a poor ability to associate with double-stranded DNA [9–11]. Consequently, this compound not only rivals the performances of telomestatin and TMPyP4 in terms of quadruplex-affinity (Figure 1(c)) , but elicits a quadruplex- over duplex-DNA selectivity comparable to that of telomestatin and highly superior to that of TMPyP4. One key to explain the very good quadruplex-binding properties of CBIs stands in their peculiar structural organization, which represents a non-planar conformation with a significant degree of molecular flexibility , thus clearly distinct from the planar rigid macrocycles telomestatin and TMPyP4. Indeed, previous works (NMR, X-rays and molecular modelling studies, vide infra) have shown that the CBI scaffold exists in a semiclosed conformation (Figure 1(b)) that results both from internal hydrophobic interactions between the two aromatic moieties and from a constrained distance between them imposed by the linkers . This particular spatial organization is an essential determinant that governs the interaction of CBIs with secondary structures of DNA. In the case of quadruplex-DNA, BOQ1 was initially thought to interact in an open-state conformation , but subsequent modelling studies  as well as recent molecular dynamics studies , suggested that the CBI scaffold never adopts the fully open conformation. In fact the semi-closed conformation of BOQ1 has been shown to be not only the most stable conformation after in silico geometry optimization (Figure 1(b)) but also to be the favoured one when interacting with quadruplex structures (see “One Ring to bind them all”—Part I) . It is thus hypothesized that this particular structure is at the origin of the efficient G-quadruplex recognition performed by BOQ1, which is supposed to occur via a mixed binding mode combining quartet and loop interactions. Finally, given that the length of the polyammonium linker does not enable intercalation of both aromatic residues between contiguous base pairs in duplex-DNA due to the neighbour-exclusion principle [19, 20], the macrocyclic scaffold of BOQ1 impedes binding to duplex-DNA, and consequently is responsible for the good selectivity for quadruplex- versus duplex-DNA.
The promising results obtained with BOQ1 in terms of quadruplex recognition and selectivity prompted us to screen a collection of 26 CBI-type macrocycles previously synthesized [17, 18, 21, 22]. The members of this series (represented schematically in Figure 2, in extenso in Figure 3) differ by the nature of the aromatic units, the topology of the macrocyclic framework that contains either two different aromatic units or the same units, but with different connectivities; the nature and the derivatization of the linking chains; and finally the number of the constitutive “intercalator” units (two for CBI, three for CTI (Cyclo-Tris-Intercalators), Figure 2). In detail, the macrocycles are comprised of either polyaromatic cycles (naphthalene, anthracene, and biphenyl moieties), or heterocycles (such as acridine, quinacridine, phenanthroline, phenazine and bipyridine moieties) or even an organometallic unit (ferrocene), along with the possibility of various connectivities that can be present in a given series. Moreover, oxygen or sulphur atoms were introduced in the linking chains in place of the secondary amino groups present in BOQ1 (X = Y = NH, O or S, Figure 2), and in some cases the linkers bear tertiary amino groups due to substitution with pendant arms (X = Y = NR). All these structural variations allow to divide the present collection of macrocycles into four categories (Figure 3): (a) homodimeric macrocycles, containing two identical aromatic units (A = B); (b) heterodimeric macrocycles, containing two different aromatic units (A ≠ B); (c) pendant-arm macrocycles containing a bisnaphthalene scaffold (A = B = 2,6-naphthylene) with one (monobrachial) or two (bibrachial) side-chains with additional functional groups; and (d) homotrimeric macrocycles (cryptand–type) containing three identical aromatic units. With this series in hand, we systematically studied the influence of the various structural components of the macrocyclic scaffold on both the quadruplex-affinity and selectivity.
To evaluate the 26 compounds shown in Figure 3, we used the FRET-melting assay, which has been recently developed to a high-throughput screening format and provides reliable information concerning quadruplex-affinity and -selectivity in a straightforward manner . This method is based on monitoring the ligand-induced stabilization of a fluorescently labelled quadruplex-forming structure (F21T, FAM-G3[T2AG3T]3-Tamra). The stabilization, measured via a fluorescence resonance energy transfer (FRET) between the two fluorescent partners (6-carboxyfluorescein, FAM, and 6-carboxytetramehtylrhodamine, Tamra), is expressed as an increase in melting temperature of F21T (ΔT1/2) in the presence of the ligand. This method also enables to gain insight into the quadruplex- over duplex-DNA selectivity if the melting experiments are carried out in the presence of various amounts of a 26-bp double-stranded DNA competitor (ds26, the self-complementary [5′-CA2TCG2ATCGA2T2CGATC2GAT2G-3′], up to 50 equiv.) Last but not least, FRET-melting enables also a direct comparison with recently reported ligands, given that this technique is now widely used by the quadruplex DNA community and often, as it is the case herein, as a test for the primary selection of ligands.
In the first round of experiments, we searched for the ligands with high affinity for the human telomeric quadruplex-DNA. FRET-melting experiments were thus carried out in the standard conditions previously calibrated for selection of high-affinity ligands , that is, with 0.2μM F21T and 1μM ligand. This first round of selection was performed both in sodium (10mM lithium cacodylate, 100mM NaCl) and potassium conditions (10mM lithium cacodylate, 10mM KCl, 90mM LiCl) of identical ionic strength. This systematic comparison was necessary since the telomeric quadruplex is known to fold into different conformations depending on the cation present in the medium, displaying different loop arrangements that might result in differences in ligand binding [23–25]. The two conformations currently admitted to correspond to the predominant species in solution and in each are represented in Figure 1 of “One Ring to Bind Them All”—Part I. The results of the FRET-melting experiments with the 26 macrocycles are presented as a bar graph in Figure 4.
This series of experiment enabled us to classify the compounds in two categories, namely the high-affinity ligands (ΔT1/2 > 10°C) and the ligands of moderate to low affinity (ΔT1/2 < 10°C). The selection criteria, that is, the affinity threshold of 10°C, was motivated by the experience acquired over the past years when developing the FRET-melting assay.
As depicted in Figure 4, ten compounds (corresponding to blue structures in Figure 3) did not reach the affinity threshold (2,7-BisNP-S, 1,5-BisNP, 9,10-BisAN, 1,8-BisAN, 1,8/9,10-BisAN, 4,4′-BisBPy, 2,8-BisPZ, 4,7-BisPN, 2,7-NP/1,1′-FC, and 2,8-TrisPZ). Among the ligands which induce a stabilization by over 10°C, 8 ligands (corresponding to black structures in Figure 3) display a high quadruplex-affinity (10°C < ΔT1/2 < 20°C: 2,7-BisNP, 2,7-BisNP-N, 1,5/2,6-BisNP, 3,3′-BisBP, 4,5-BisA, 2,7-NP/9,10-AN, 2,7-NP/2,7-A and 3,3′-TrisBP), and 8 ligands (corresponding to red structures in Figure 3) elicit a very high affinity (ΔT1/2 > 20°C: 2,6-BisNP, 2,9-BisPN,BisNP-1NH2, BisNP-1PY,BisNP-2PY, BisNP-1FC, BisNP-2FC, and 2,7-TrisNP). It is worth noting that a ΔT1/2 value of 30–35°C as observed for BisNP-2PY represents the upper practical limit of the assay (corresponding to a Tm ≥ 80–85°C for the ligand-DNA complex).
A close examination of these results enables us to identify the parameters that influence the efficiency of the quadruplex-recognition by the macrocyclic scaffold, namely, (a) the nature and (b) the connectivity of the intercalator units, (c) the nature of the linker, (d) the global charge of the ligand and (e) the number of intercalator units.
Altogether the results of this primary screen confirm that the association between the macrocycles and the quadruplex-DNA is strongly dependent on both the rigidity and the topology of the macrocyclic framework. Finally, as depicted in Figure 6, the behaviour of the majority of compounds shows a poor dependency on the cation present in the medium, thereby confirming that the quadruplex-recognition is primarily governed by tetrad-recognition.
The first selection based on the amplitude of the F21T stabilisation induced by the ligand resulted in the selection of 16 compounds. For entering the second round of experiments, we further decided to arbitrary remove two candidates (3, 3′-BisBP and 2,7-NP/9,10-AN), since they do not reach the affinity threshold (ΔT1/2 = 10°C) in the two cationic conditions (Figure 4). The 14 remaining compounds were thus subjected to the second selection step that consists in reproducing the melting experiment of F21T in the presence of the nonlabelled duplex-DNA competitor ds26. As shown in previous works, the presence of the duplex-DNA competitor diminishes the quadruplex-binding of nonselective compounds (thus resulting in decreased ΔT1/2 values) and poorly affects the binding of selective compounds (thus resulting in unchanged ΔT1/2 values). The competitive FRET-melting assay enables thus a rapid evaluation of quadruplex- over duplex-DNA selectivity, a criterion that is of utmost importance for further developments of quadruplex ligands in cell-based assays.
Competitive FRET-melting experiments were carried out in the conditions used in the first experiment , that is, with 0.2μM F21T, 1μM ligand without ds26 or with two different concentrations of ds26 (3 and 10μM, i.e., 15- and 50-molar excess relative to F21T). These experiments have been performed with the 14 selected macrocycles both in Na+ and K+ conditions; the results (ΔT1/2 values) are summarized as bar graphs shown in Figure 7.
In this second selection, 8 compounds (whose name is squared in red in Figure 7, see also Table 1) appear to keep on stabilizing efficiently the quadruplex-DNA (ΔT1/2 > 10°C, red dashed line in Figure 7(a), lower red dashed line in Figure 7(b)) in presence of 10μM of duplex-DNA (i.e., 2,9-BisPN, 2,7-NP/2,7-A, BisNP-1PY, BisNP-2PY, BisNP-1FC, BisNP-2FC, 2,7-TrisNP, and 3, 3′-TrisBP); on the other hand, 6 compounds (2,7-BisNP, 2,7-BisNP-N, 2,6-BisNP, 1,5/2,6-BisNP, 4,5-BisA and BisNP-1NH2) are found less selective since their ΔT1/2 value was reduced to <10°C in presence of 10μM of ds26.
To further examine the properties of the eight ligands selected after the second selection step, we quantified the quadruplex- versus duplex-DNA selectivity by the FRETS value , which is defined as FRETS = T1/2 (10μM ds26)/ΔT1/2 (no ds26); consequently, FRETS→ 1 for highly selective ligands. This ratio has the advantage to paramter the ΔT1/2 value in presence of ds26 competitor as a function the ΔT1/2 value without ds26. Moderate (down to 0.36/0.48, Table 1) to excellent FRETS values (up to 1.01/0.98, Table 1) were determined for the eight selected compounds. This values ranking enables us to discard the two ligands showing the lowest selectivity (i.e., FRETS < 0.5), namely BisNP-2PY and 2,7-TrisNP, thereby leaving six promising candidates (2,9-BisPN, 2,7-NP/2,7-A, BisNP-1PY, BisNP-1FC, and BisNP-2FC, 3, 3′-TrisBP) for entering the final round of selection (vide infra).
It is worth mentioning here that the selectivity ranking is not globally affected by the nature of the cation (Na+ versus K+, Figures 7(a) and 7(b), resp., Table 1); however, the values are significantly higher in presence of K+, thereby indicating a higher resistance of the ligands to the competition of the duplex-DNA in these conditions. For this reason and also for the sake of clarity, we used only results obtained in K+ (Figure 7(b)) for rationalizing the data: a close examination of the results enables us to identify three structural parameters that influence the selectivity of the quadruplex-recognition by the macrocycles, namely, (a) the presence of pendant arms, (b) the existence of a specific three-dimensional conformation, and (c) the rigidity of the macrocyclic framework.
Thus, the two-step selection process described herein (step 1: affinity, step 2: selectivity) enables the rapid identification of six candidates (2,9-BisPN, 2,7-NP/2,7-A, BisNP-1PY, BisNP-1FC, BisNP-2FC, and 3, 3′-TrisBP) with remarkable quadruplex-interacting properties, that is, high affinity and selectivity. To further increase the selection pressure and select hits for subsequent developments, we decided to focus our attention on compounds that display ΔT1/2 ≥ 15°C in presence of 10μM ds26 (upper red dashed line in Figure 7(b)). Four compounds fulfill this requirement, namely, 2,7-NP/2,7-A, BisNP-1PY, BisNP-1FC, and 3, 3′-TrisBP (Figures (Figures7 and7 and and8,8, it is worth noting that this selection has been extended to 3, 3′-TrisBP, since its ΔT1/2 in presence of 10μM ds26 (14.7°C) is an average value resulting from experiments with ΔT1/2 ≥ 15°C). Amongst these four compounds, two were thus elected as the most promising compounds over the 26 macrocycles screened: (i) BisNP-1PY, since this CBI combines the best affinity and selectivity on the basis of the three values used for the selection procedure (in K+ conditions: ΔT1/2 = 29.7 and 21.5°C in absence and presence of 10μM ds26, resp. FRETS = 0.72; in Na+ conditions: ΔT1/2 = 31.9 and 22.9°C in absence and presence of 10μM ds26, resp., FRETS = 0.71), and (ii)- 3, 3′-TrisBP, since, while displaying a decent affinity, this CTI reaches an unprecedented level of quadruplex- over duplex-DNA selectivity (FRETS = 1.01 and 0.98 in Na+ and K+ conditions, resp.).
The CBIs were initially designed to trap flat aromatic substrates between their aromatic units [22, 26, 32]; however, it turned out very rapidly that these compounds display remarkable binding preference for single-stranded and locally unpaired DNA (abasic , mismatched sites) [17, 18, 21, 34] and various secondary structures (hairpins , trinucleotide repeats , G-quadruplex) [9–11, 37]. We show here that this class of macrocycles offers unique advantages for targeting G-quadruplex-DNA.
The two-step selection procedure followed herein as well as the structural diversity of the collection studied enabled us to identify several structural features that govern the efficiency of the quadruplex-recognition by the CBI ligands. Among these, the presence of a pendant side-arm and the existence of a particular topological arrangement appear to be strong determinants. The selected hits possess sophisticated architectures and are thus likely to create multiple contacts with the quadruplex-DNA structure. This should result from a complex interplay of π-stacking interactions, H-bonding, and electrostatic forces both with the accessible G-tetrad of the quadruplex-DNA and with the quadruplex-elements that surround the G-tetrad (loops and/or grooves). These secondary interactions may be at the origin of the high selectivity observed.
In conclusion, we have performed the first step toward the identification of two molecules that are highly promising ligands of the human telomeric quadruplex in vitro and opened the way towards new molecular designs of high efficiency. We are thus now performing further studies with these ligands to investigate their peculiar binding mode and in vivo properties.
The authors would like to thank sincerely their coworkers, C. Guetta, A. De Cian, E. Largy, and L. Lacroix, for technical assistance and stimulating scientific discussions related to G-quadruplex DNA and ligands. A special acknowledgment is due to J. R. R. Tolkien for the title inspiration.