Pyridine Derivative of the Natural Alkaloid Berberine as Human Telomeric G₄-DNA Binder: A Solution and Solid-State Study

Abstract

Telomerase is an enzyme deputed to the maintenance of eukaryotic chromosomes; however, its overexpression is a recognized hallmark of many cancer forms. A viable route for the inhibition of telomerase in malignant cells is the stabilization of G-quadruplex structures (G₄) at the 3′ overhang of telomeres. Berberine has shown in this regard valuable G₄ binding properties together with a significant anticancer activity and telomerase inhibition effects. Here, we focused on a berberine derivative featuring a pyridine containing side group at the 13th position. Such modification actually improves the binding toward telomeric G-quadruplexes and establishes a degree of selectivity in the interaction with different sequences. Moreover, the X-ray crystal structure obtained for the complex formed by the ligand and a bimolecular human telomeric quadruplex affords a better understanding of the 13-berberine derivatives behavior with telomeric G₄ and allows to draw useful insights for the future design of derivatives with remarkable anticancer properties.

Eukaryotic telomeres [(TTAGGG)_n] are known to contribute in maintaining chromosome length and integrity.^1,2 G-quadruplexes (G₄’s) formed at the 3′ terminal overhang of telomeres have been shown to directly inhibit the enzyme telomerase, whose overexpression is greatly involved in the limitless replicative potential in about 85% of cancer forms.³⁻⁶ Thus, the stabilization of G₄’s induced by low molecular weight ligands has been envisaged as a promising anticancer strategy.^7,8

G₄’s are four-stranded, intra- or intermolecular architectures, characterized by planes of four hydrogen bonded guanines which stack on each other giving rise to a variety of foldings.⁹⁻¹¹ Consequently, the most important structural features for G₄ ligands are a large aromatic core for π–π stacking and positively charged groups for electrostatic interactions.^12,13 The natural isoquinoline alkaloid berberine (BER) (Scheme 1) matches these requirements and acts as an interesting G₄ stabilizing ligand with a significant G₄ over duplex DNA selectivity.¹⁴⁻¹⁷ These features are possibly connected to the anticancer activity and the telomerase inhibitory effects displayed by the alkaloid.¹⁸⁻²⁰ Not surprisingly, many studies on berberine derivatives are currently ongoing in several laboratories,²¹⁻²⁹ and some of us explored the chemical space around the C-13³⁰ adding hydrocarbon linkers of variable length bearing (hetero)aryl groups to enhance the propensity for additional stacking.^31,32 Most of these 13-substituted analogues showed a higher binding affinity to ds-DNA with respect to parent berberine, with an effect depending on both the chain length and the aromatic grouping.^26−28,33 They were found to exhibit superior in vitro and in vivo antiproliferative potencies in relevant tumor cell lines and models than BER.^{22,28,34−39} Some derivatives have been reported also to bind telomeric G-quadruplex structures,⁴⁰⁻⁴⁴ and Scheme 1 shows some compounds from our laboratories with relevant anticancer activity. The crystal structure of 4 in adduct with a bimolecular G₄ has also been reported.⁴⁵

Scheme 1. Chemical Structures of Berberine (BER) and Berberine Derivatives 1–4 and L.

Here we report an in-depth study on the G₄ binding behavior of ligand L (Scheme 1) that is derivatized by a C₃-alkyl appendage bearing a pyridine group potentially acting as polyfunctional anchorage via stacking and H-bond.

The G₄–L interaction has been analyzed, through thermal and spectroscopic solution characterization and solid state (single crystal XRD) studies. The possible interaction between ligand L and the bimolecular and monomolecular quadruplex structures given by the Tel12 and Tel23 sequences was initially investigated by circular dichroism experiments (see details in the SI).

Upon titration of both sequences folded into G-quadruplex structures with L (Figure 1), the intensity of the positive peaks is found to decrease gradually, evidencing a strong G₄-L stacking interaction.

Figure 1.

Circular dichroism spectra for the interaction of L with (A) Tel12 (6 μM) with 0.0, 3.0, 6.0, 9.0, 15.0, 21.0, 27.0, and 39.0 μM (curves 1–8) and (B) Tel23 (6 μM) with 0.0, 3.0, 6.0, 9.0, 15.0, 21.0, 33.0, and 45.0 μM (curves 1–8).

The spectroscopic titration of the alkaloid with increasing concentration of quadruplex DNA (both Tel12 and Tel23; Figure 2A,B) caused significant decrease of the absorption maxima at 338 and 413 nm, a spectral region where the absorbance of DNA is negligible. Sharp isosbestic points were observed for L at 351 and 433 nm with Tel12 and at 350, 375, and 430 nm with Tel23, respectively. The hypochromicity in the absorption spectra indicates strong intermolecular association between the π-electron cloud of the interacting ligand and the G-quartets.⁴⁶

Figure 2.

Absorption spectral titration of L (3 μM) treated with (A) 0.0, 0.6, 1.2, 2.4, 3.6, and 4.2 μM Tel12 quadruplex DNA (curves 1–6) and (B) treated with 0.0, 0.6, 1.8, 3.0, 4.2, and 5.4 μM Tel23 quadruplex DNA (curves 1–6).

The alkaloid showed a weak intrinsic fluorescence spectrum when excited at 340 nm with a maximum at 507 nm. Upon spectrofluorimetric titration of 1 μM of L with increasing concentration of both forms of telomeric quadruplex DNA, an enhancement in the fluorescence intensity (Figure S1) was observed indicating a strong interaction between the bound alkaloid with the quadruplex structure. This suggests that the alkaloid may be located in a hydrophobic environment within the ligand–DNA adduct structure.^47,48

Scatchard plots (r/C_f vs r) were obtained from the spectrophotometric and spectrofluorimetric data to determine the equilibrium constant for the alkaloid–quadruplex complexation. The plots (Figure S2) revealed a negative slope at low values of r indicating a noncooperative binding of the alkaloid molecule to both forms of G₄. The intrinsic binding affinity values (K) and number of excluded sites (n) for the complexation from absorbance and fluorescence results are collated in Table 1. Interestingly, results from both techniques indicated that Tel12 had a higher binding affinity than Tel23 to L.

Table 1. Binding Parameters for L Complexation from Absorbance and Fluorescence Data^a.

	Absorbance		Fluorescence
	Ka × 10–5 (M–1)	n	Ka × 10–5 (M–1)	n
Tel12-L	3.20	1.13	3.27	1.06
Tel23-L	0.80	1.14	0.85	1.12
Tel24-BER	1.1
Tel24-4	9.1

^aBinding parameters for berberine (BER) and 4 from ref (45).

This observation is confirmed by the melting profiles (see the SI) of the Tel12 and Tel23 quadruplex structures in the absence and presence of L: a greater stabilization was obtained from the interaction of the alkaloid with Tel12 (ΔT_m increment of 15 K) in comparison to Tel23 (ΔT_m increment of 2 K) at 1:1 molar ratio (Figure S3).

Figure 3 depicts the calorimetric profiles for the titration of the alkaloid into the Tel12 and Tel23 solution at 298.15 K (for full details see the SI and Figure S4).

Figure 3.

ITC titrations for L with (A) Tel12 (10 μM) quadruplex DNA and (B) Tel23 (10 μM) quadruplex DNA at T = 298.15 K. Integrated heat data after correction of heat of dilution against the molar ratio. The data points were fitted to a one site model, and the solid lines represent the best fit data.

The data points show the experimental injection heats as a function of the molar ratio of the alkaloid to G₄, and the solid lines represent the calculated best fits to the experimental data based on the “one set of sites” model that yielded the best fit curve for the obtained data points. The data obtained with the isothermal titration calorimetry studies allow us to determine the thermodynamic parameters reported in Table 2.

Table 2. ITC Thermodynamic Parameters Related to the Binding of L to Tel12 and Tel23 Sequences (298.15 K).

	K (M–1)	n	ΔH°(kcal/mol)	ΔG°(kcal/mol)	TΔS°(kcal/mol)
Tel12	3.16 ± 0.02 x105	1.02 ± 0.01	–7.08 ± 0.09	–7.38 ± 0.08	–0.30 ± 0.01
Tel23	8.15 ± 0.02 x104	1.01 ± 0.01	–8.53 ± 0.09	–6.55 ± 0.09	–1.97 ± 0.09

These findings suggest an enthalpy driven binding of L to both sequences of telomeric quadruplex with a 1:1 complexation molar ratio. In order to obtain structural information useful to rationalize the binding features highlighted by solution studies, crystallization experiments were performed for L using the Tel12 and Tel23 sequences. At the solid state Tel12 and Tel23 DNA form bimolecular and monomolecular G-quadruplex in propeller folding, respectively.⁴⁹ Satisfactory crystals were obtained only for the Tel12-L adduct.

The structure resulted to be isomorphous with the one of the diphenyl-alkyl derivative 4 (Scheme 1) in complex with Tel12.⁴⁵ As shown in Figure 4 the quadruplex unit adopts the expected parallel stranded bimolecular architecture (chain A and chain B). The 5′-end thymine and adenine residues from the two Tel12 chains give an additional tetrad (5′-end TATA) which stacks on the 5′-end G-quartet.

Figure 4.

Columns of alternating L ligands and Tel12 bimolecular G-quadruplexes in the 1:1 Tel12–L adduct (tube color code: gray for chain A, light gray for chain B).

The ligand is sandwiched between two G₄ units, in contact with the 3′-end G-quartet from one unit and the 5′-end TATA tetrad from the other one (Figures 4 and 5).

Figure 5.

Binding site for L at the interface of two symmetry related G₄ units: A, lateral view; B, top view (tube color code: gray for chain A, light gray for chain B).

The pyridine pendant points toward the potassium channel,⁴⁹ and the alkaloid core is in contact with two lateral residues from each tetrad (Figure 5A,B and Figure S5). As in the Tel12–4 complex, both the berberine core and the aromatic pendant are involved in π–π stacking interactions.

However, differently from the phenyl rings from 4, the pyridine nitrogen from L could be involved in additional H-bonds. Unfortunately, no direct H-bond with the target was established and the only water molecule (HOH11, Figure 5B) found nearby this atom is located 3.8 Å apart, and it does not comply with the geometrical requirements to act as a ligand/DNA bridge. Notably, the Fourier difference density map points out the presence of the HOH9 bridging a benzodioxole oxygen atom and the ribose oxygen of the residue A2 (chain A, Figure 5B).

In addition to the isomorphous Tel12–4 complex, also the crystal structure reported for BER in adduct with Tel23 could be used for interesting comparisons.⁵⁰ Actually, in Tel12–L, Tel12–4, and Tel23–BER, the alkaloid core interacts with the G-quartet in a very similar fashion, pointing out its cationic nitrogen far away from the negative charges of the carbonyl oxygens in the central channel and giving rise to π–π stacking interactions with two guanine residues (Figure 6). Given this structural preference, the carbon atom in the 13th position (Scheme 1) is placed just above the central quadruplex channel, and the alkyl chain needs to form a hump to place the aromatic pendant above the tetrad. In our opinion, this finding is particularly worth mentioning, as it could be relevant for understanding the quadruplex/ligand binding mode of these compounds. Indeed, regardless of the folding, the quadruplex possesses external G-tetrads potentially available for binding. Consequently, the strictly similar interaction mode pointed out by the three crystal structures could potentially mimic the ligand/quartet interaction in diluted solution and could additionally provide relevant information for further drug design to properly tune the linker as well as the nature of the aromatic group in the 13 position of the berberine core.

Figure 6.

Top view of BER and of its derivatives L and 4 stacked on the guanine platforms in their crystal structures. Tel12–L: present work; Tel12–4;⁴⁵ Tel23–BER.⁵⁰

Thus, an important conclusion we can derive from the experimental data is the strong affinity of the berberine core for the guanine quartet, which is in agreement with the binding constants measured in solution (Tables 1 and 2) and with the G₄ vs double helix selectivity data reported in the literature for BER.¹⁴⁻¹⁷ This selectivity clearly stems from the spatial requirement for a large enough aromatic surface to host the alkaloid skeleton, which complied only with the quadruplex structure. Similar considerations can be done to explain the Tel12 vs Tel23 selectivity, observed from both the spectroscopic and the calorimetric results. Structural data suggest that a wide contact area, comprising three (four) residues of the quartet, is needed for L (4) to interact with the target. This is in agreement with the calorimetric results, which overall evidence the exoenthalpic 1:1 DNA/L complex formation and a slight Tel12 vs Tel23 selectivity. This latter could be explained considering that in a diluted solution Tel23 assumes hybrid conformations in which the flanking residues and/or loops make the quartet less available for interaction with the ligands.^51,52 Therefore, in order to clear the three out of four guanines requested to bind L, it undergoes a conformational rearrangement. This affects the affinity constant and the melting temperature ΔT_m which are worse with respect to Tel12. On the other hand, calorimetric studies (Table 2) indicate a slightly more favorable ΔH value for Tel23 with respect to Tel12, suggesting that after the conformational change, some loop residues could be able to interact with L, so balancing and even overcoming the energy loss due to rearrangement. Indeed, spectrofluorimetric studies pointed out the possibility for L to be lodged in a hydrophobic environment, and this assumption could explain the selectivity trend, which is mainly connected with entropic contributions. Entropic terms were found to be less favorable to Tel23, probably because of the receptor stiffening due to the involvement of loops or flanking in the ligand binding. At the same time, it is not possible to exclude that the more negative value of TΔS associated with Tel23 is determined by a greater structuring of the solvent in the regions featuring greater conformational flexibility.

Overall, our studies give a coherent picture for the binding behavior of L with the G₄ structural unit formed by the human telomeric DNA sequence. Both solid state and solution experiments agree in indicating a 1:1 molar ratio, and the spectroscopic and calorimetric experiments evidence a remarkable preference for the bimolecular structure given by Tel12 over the monomolecular one from Tel23. Despite for both targets the favorable enthalpic term is dominant, Tel23 is penalized by a larger unfavorable entropic term, most likely due to structural stiffening upon binding. In any case, the binding values indicate a strong intermolecular association not dissimilar from the values reported for BER and the analogous 13-substituted berberine derivative 4,^14−21,45 suggesting that a relevant contribution to the interaction is to be attributed to the berberine core. This hypothesis is also confirmed by structural data that systematically set the berberine core of BER, 4, and L on two lateral guanine residues with the carbon atom in C13 pointing toward the inner potassium channel. This conservative structural feature makes us confident that a similar interaction mode could be found also in solution, regardless of the type of target (mono- or bimolecular quadruplex).

Solution spectral data support this hypothesis and suggest that the bound alkaloid could be located in a hydrophobic environment. In conclusion, the results here reported accurately describe the binding of an interesting alkylpyridine derivative of natural alkaloid berberine toward human telomeric G-quadruplex structures and supply fruitful information for the design of derivatives featuring side groups at the 13th position with improved binding properties and hopefully better telomerase inhibition profiles.

Glossary

Abbreviations

G₄

G-quadruplex

BER

berberine

XRD

X-ray diffraction

CD

circular dicroism

Tel12

d[TAG₃T₂AG₃T] sequence

Tel23

d[TAG₃(T₂AG₃)₃] sequence

L

NAX075 compound

ITC

isothermal titration calorimetry

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00516.

• Materials and methods: details of used materials, preparation of stock solutions, spectroscopic titration experiments and binding data evaluation (Figures S1 and S2), circular dichroism, thermal melting studies (Figure S3), isothermal titration calorimetry (Figure S4), and X-ray diffraction analysis (Table S1 and Figure S5) (PDF)

Author Present Address

^⊥ Department of Chemistry, Rampurhat College, Rampurhat, Birbhum, West Bengal 731224, India.

Author Contributions

The manuscript was written through contributions of all authors. P.G and C.B initially formulated the concept of the manuscript.

Ente Cassa di Risparmio di Firenze, Italy (2014.0309). FFABR2017-Finanziamento delle attività base di ricerca.

The authors declare no competing financial interest.