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 ( up to ) 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.

1. Introduction

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.) [14]. 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- [5] and porphyrin (TMPyP4)-related compounds [68]. 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)) [911] as discussed in “One Ring to Bind Them All”—Part I. This compound is comprised of two large aromatic quinacridine units [1214] able to interact with DNA bases through - 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 exhibits a high affinity for the human telomeric G-quadruplex DNA, along with a poor ability to associate with double-stranded DNA [911]. Consequently, this compound not only rivals the performances of telomestatin and TMPyP4 in terms of quadruplex-affinity (Figure 1(c)) [16], 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 [11], 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 [17]. 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, was initially thought to interact in an open-state conformation [9], but subsequent modelling studies [11] as well as recent molecular dynamics studies [18], suggested that the CBI scaffold never adopts the fully open conformation. In fact the semi-closed conformation of 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) [11]. It is thus hypothesized that this particular structure is at the origin of the efficient G-quadruplex recognition performed by , 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 impedes binding to duplex-DNA, and consequently is responsible for the good selectivity for quadruplex- versus duplex-DNA.

The promising results obtained with 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 ( or , Figure 2), and in some cases the linkers bear tertiary amino groups due to substitution with pendant arms ( ). 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 ( ); (b) heterodimeric macrocycles, containing two different aromatic units ( ); (c) pendant-arm macrocycles containing a bisnaphthalene scaffold ( -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.

2. Results and Discussion

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 [15]. This method is based on monitoring the ligand-induced stabilization of a fluorescently labelled quadruplex-forming structure ( - -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 ( ) 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 [ - ], 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.

3. Selection of the High-Affinity 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 [15], that is, with 0.2  M F21T and 1 M ligand. This first round of selection was performed both in sodium (10 mM lithium cacodylate, 100 mM NaCl) and potassium conditions (10 mM lithium cacodylate, 10 mM KCl, 90 mM 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 [2325]. 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 ( ) and the ligands of moderate to low affinity ( ). The selection criteria, that is, the affinity threshold of , 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- - , 1,5- , 9,10- , 1,8- , 1,8/9,10- , 4, - , 2,8- , 4,7- , 2,7- /1, -FC, and 2,8- ). Among the ligands which induce a stabilization by over , 8 ligands (corresponding to black structures in Figure 3) display a high quadruplex-affinity ( : 2,7- , 2,7- - , 1,5/2,6- , 3, - , 4,5- , 2,7- /9,10- , 2,7- /2,7- and 3, - ), and 8 ligands (corresponding to red structures in Figure 3) elicit a very high affinity ( : 2,6- , 2,9- , - , - , - , - , - , and 2,7- ). It is worth noting that a value of C as observed for - represents the upper practical limit of the assay (corresponding to a 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.(a)The nature of the intercalator unit: (i) rigid fused aromatic units lead to better results than the unfused flexible ones. Indeed, the two CBIs containing biphenyl or bipyridine units 3, - and 4, - induce a lower stabilization ( / = 8.2/10.4 and 1.3/ C, resp.) than the corresponding naphthalene analogue 2,7- ( / = 9.1/ C). (ii)- The size of the aromatic units appears less important than their relative arrangement, since the four ligands comprising an anthracene unit (i.e., the largest intercalator used herein), namely, 1,8-, 9,10-, 1,8/9,10- and the anthracene-naphthalene hybrid 2,7- /9,10- , give results either below (the three former) or just reaching (the latter) the affinity threshold, whereas most naphthalene derivatives strongly stabilize the quadruplex-DNA. (iii)- Finally, the presence of two flat aromatic units in the typical CBI arrangement is an absolute requirement, as shown by the poor quadruplex-stabilizing properties of the unsymmetrical macrocycle 2,7-NP/1,1 -FC containing a bulky ferrocene unit ( C).(b)The connectivity of the intercalator units: the connectivity of the two aromatic units determines the conformation and the internal molecular dynamics of the macrocycle. Consequently, it must have a profound influence on the DNA binding ability. This is fully confirmed herein by the striking differences observed within a given series: for example, in the naphthalene series, the 1,5- does not induce notable stabilization ( C) while 2,6- and 2,7- are above the affinity threshold ( / = 19.6/18.3 and 9.1/12. C, resp.). Similar conclusions can be drawn for the phenanthroline series, since 2,9-BisPN is much more efficient than its analogue 4,7- ( = 13.8/16.8 and 2.5/9. C, resp.).(c)The nature of the linker: interestingly, the replacement of the central oxygen atom by a sulfur atom (2,7- versus 2,7- - ) significantly reduces the stabilisation ( / = 9.1/12.2 and 6.3/9. C, resp.), an observation that can be attributed to the larger steric hindrance of the sulfur atom. On the other hand, the replacement of the central oxygen atom by a nitrogen atom improves the stabilization (2,7- versus 2,7- - , / = 9.1/12.2 and 13.3/16. C, resp.), suggesting a possible involvement of the group in -bond interactions with the quadruplex target.(d)The global charge of the ligand: previous studies have shown that only the benzylic nitrogens of the linkers are protonated at the experimental (7.2) both in the and series [2628]. Thus, it can be assumed that the global cationic charge of the whole series varies from 4 (for ) to 5 (for monosubstituted ) up to 6+ (for disubstituted and ). The data collected (Figure 4) show that there is no obvious dependence on the global charge of the ligand given that compounds (4+) globally induce better stabilizations than compounds (6+) and that the monosubstituted (5+) are more efficient than their disubstituted analogues (6+). We can conclude that the quadruplex-recognition is not dominated only by electrostatic parameters, although this interaction may contribute strongly to the stabilisation effect.(e)The number of intercalative units: the number of intercalative units appears to play a crucial role since, in the biphenyl series, the bismacrocycle , - is less efficient than the corresponding trismacrocyclic compound - ( / = 8.2/ C and 13.0/14. C). Although this difference could be explained by the higher cationic charge of the trimer (6+ for the versus 4+ for the ), the poor activity of the trisphenazine (2,8- ) that is also hexacationic argues against a dominant electrostatic effect (see also (d)). Thus the large difference between the two compounds is more likely attributable to the higher rigidity of the as compared to that of the corresponding . To support this hypothesis, molecular dynamic simulation in a water box has been performed with the two compounds: the results presented in Figure 5 show that - has an extremely high degree of flexibility and adopts a largely open conformation whilst - is strongly conformationally restrained [29].

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.

4. Selection of Highly Selective Ligands

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 ( -BisBP and 2,7- /9,10- ), since they do not reach the affinity threshold ( 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 values) and poorly affects the binding of selective compounds (thus resulting in unchanged 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 [15], that is, with 0.2 M F21T, 1 M ligand without ds26 or with two different concentrations of ds26 (3 and 10 M, i.e., 15- and 50-molar excess relative to F21T). These experiments have been performed with the 14 selected macrocycles both in and conditions; the results ( 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 ( , red dashed line in Figure 7(a), lower red dashed line in Figure 7(b)) in presence of 10 M of duplex-DNA (i.e., 2,9- , 2,7- /2,7- , -1 , -2 , -1 , -2 , 2,7- , and - ); on the other hand, 6 compounds (2,7- , 2,7- - , 2,6- , 1,5/2,6- , 4,5- and -1 ) are found less selective since their value was reduced to C in presence of 10 M 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 value [30], which is defined as (10 M ds26)/ (no ds26); consequently, → 1 for highly selective ligands. This ratio has the advantage to paramter the value in presence of ds26 competitor as a function the value without ds26. Moderate (down to 0.36/0.48, Table 1) to excellent 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., ), namely -2 and 2,7- , thereby leaving six promising candidates (2,9- , 2,7- /2,7- , -1 , -1 , and -2 , - ) 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 ( versus , Figures 7(a) and 7(b), resp., Table 1); however, the values are significantly higher in presence of , 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 (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.(a)The presence of pendant side-arm(s): this parameter contributes strongly to the quadruplex-selectivity, as demonstrated by the much higher values obtained in presence of 10 M ds26 for the monobrachial and bibrachial compounds ( = 21.5, 17.2, 12.6 and 11. C for -1 , -1 , -2 and -2 , resp.) as compared with their nonsubstituted analogue 2,6- ( = 5. C). Remarkably, pendant side-arms terminated by a functional group (pyrene (PY) or ferrocene (FC)) lead to ligands that exhibit higher quadruplex-affinity and -selectivity than their amino-terminated counterpart (i.e., -1 , Figure 7 and Table 1; e.g., in presence of 10 M ds26, = 29.7 versus 20. C and = 0.72 versus 0.38 for -1 and -1 , resp.). This clearly indicates that these functional groups are responsible for additional interactions with quadruplex-DNA, eventually with loops and/or grooves, which may result in the multiple anchorage of the ligand in its binding site. Finally, the better performances of monobrachial compounds as compared to bibrachial ones, both in terms of affinity (in presence of 10 M ds26, = 29.7/25. C for -1 / -1 versus 25.9/13. C for -2 / -2 ) and selectivity ( = 0.72/0.66 for -1 / -1 versus 0.48/0.84 for -2 / -2 ) suggest that one pendant side-arm is enough to create optimized interactions with the quadruplex-DNA. The second pendant side-arm may impair the quadruplex-binding due to steric hindrance and also may participate to create nonspecific interactions since it increases the global charge of the molecule.(b)The existence of a specific 3D conformation: in the naphthalene (NP) series, the replacement of an NP unit in 2,7- by an acridine unit (i.e., 2,7- /2,7- ) increases the internal stacking interactions between the aromatic units, which in turn results in a more stable semi-closed conformation; given that 2,7- /2,7- displays a better binding properties than 2,7- , both in terms of affinity (in presence of 10 M ds26, = 20.1 versus 12. C) and selectivity ( = 0.79 versus 0.45), we can conclude that a particular topology could be highly favorable for increasing the efficiency of the ligand/quadruplex-DNA interactions. The same trend is observed when the NP units are replaced by the surface-extended phenanthroline (PN) units; the resulting compound, 2,9- , displays improved performances as compared to 2,7- (in presence of 10 M ds26, = 16. C, and = 0.71). These two cases indicate that subtle modifications of the aromatic units that compose the CBI scaffold significantly increase the selectivity; these modifications may contribute to determine a particular topology of the CBI, thus disfavoring the association with duplex-DNA. Interestingly, the crystal structure of 2,9- has recently described [31] evidence of a “horse-shoe” conformation that may be prone to fit into the quadruplex-DNA bonding site. The importance of the 3D topology suggested here is in line with the role of the connectivity observed during the primary screen (vide supra).(c)Finally, in the NP series, the formal introduction of a third NP branch in 2,7- scaffold significantly improves the binding properties of the resulting compounds (i.e., 2,7- , in presence of 10 M ds26, = 22.7, and = 0.57); we can thus conclude that, given that 2,7- is structurally more constrained than 2,7- , the rigidity of the ligand is a valuable parameter to control when searching to improve the quality of the recognition of the DNA target. The same trend is observed in the bipyridine (BP) series, since - displays a higher quadruplex-affinity than - ( = 14.9 versus 10. C); interestingly, - shows a quite exceptional selectivity (with 0.98), although its stabilizing effect is modest as compared to 2,7- . These two examples demonstrate the interest of the design for improving the quadruplex-selectivity, supporting the observation that the rigidity of the is not only favorable for the quadruplex-binding but also for decreasing the duplex-binding. Last but not least, these results also indicate that electrostatic interactions are not critically determinant for the binding of to both quadruplex- and duplex-DNA.

Thus, the two-step selection process described herein (step 1: affinity, step 2: selectivity) enables the rapid identification of six candidates (2,9- , 2,7- /2,7- , -1 , -1 , - and - ) 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 C in presence of 10 M ds26 (upper red dashed line in Figure 7(b)). Four compounds fulfill this requirement, namely, 2,7- /2,7- , -1 , -1 , and - (Figures 7 and 8, it is worth noting that this selection has been extended to - , since its in presence of 10 M ds26 (14. C) is an average value resulting from experiments with C). Amongst these four compounds, two were thus elected as the most promising compounds over the 26 macrocycles screened: (i) -1 , since this combines the best affinity and selectivity on the basis of the three values used for the selection procedure (in conditions: = 29.7 and 21. C in absence and presence of 10 M ds26, resp. = 0.72; in conditions: = 31.9 and 22. C in absence and presence of 10 M ds26, resp., = 0.71), and (ii)- - , since, while displaying a decent affinity, this reaches an unprecedented level of quadruplex- over duplex-DNA selectivity ( = 1.01 and 0.98 in and conditions, resp.).

5. Conclusion

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 [33], mismatched sites) [17, 18, 21, 34] and various secondary structures (hairpins [35], trinucleotide repeats [36], G-quadruplex) [911, 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, -bonding, and electrostatic forces both with the accessible -tetrad of the quadruplex-DNA and with the quadruplex-elements that surround the -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.