Triazolyl Dibenzo[a,c]phenazines Stabilize Telomeric G-quadruplex and Inhibit Telomerase

Abstract

We herein report the synthesis and biophysical evaluation of triazolyl dibenzo[a,c]phenazine derivatives as a novel class of G-quadruplex ligands. The aromatic core facilitates π-π interaction and the flexible, protonatable side chains interact with the phosphate backbone of DNA via electrostatic interactions. Förster resonance energy transfer (FRET) melting assay and isothermal titration calorimetry (ITC) studies suggest that these ligands show binding preference for the hTELO G-quadruplex over G-quadruplexes found in the promoter region of various oncogenes and duplex DNA. The in vitro telomeric repeat amplification protocol (Q-TRAP) assay reveals that these ligands reduce telomerase activity in cancer cells.

G-quadruplex (G4) structures are non-canonical higher-order structures formed by guanine-rich (G-rich) DNA sequences.^[1,2] These structures consist of G-quartets, formed by the association of four guanine bases through the hydrogen bonding interactions of Hoogsteen type base-pairing.^[1b] They are prevalent at crucial positions of the genome, including telomeric ends, promoter regions (e.g., cMYC,^[3] BCL-2,^[4] c-KIT,^[5] VEGF,^[6] KRAS^[7]), introns,^[8] untranslated regions (UTRs),^[9,10] etc.^[8,9,11,12] G4s present in the telomeric region in particular, were among the first G4 structures found to be biologically relevant.^[13] In normal cells, single-stranded overhang region containing repetitive hexanucleotide sequence of [TTAGGG]_n in telomeres plays crucial role in maintaining chromosomal stability and prevention of end-to-end fusion.^[14] The length of the telomere is shortened at each cell division, leading to cellular aging, senescence and apoptosis.^[15] Reverse transcriptase telomerase maintains telomere length and this is further protected from degradation by shelterin, a six-subunit protein complex.^[16] In 85-90% malignant cells, telomerase activity is significantly upregulated, resulting in telomere lengthening and immortality of the cell.^[17,18]

Telomerase plays a key role in tumorigenesis by lengthening the G-rich repeats at the telomeric ends and inducing cellular immortalization. The 3’ end of telomeric G-rich sequence can adopt monomeric or multimeric G4 structures. Small molecules can stabilize G4 and indirectly inhibit telomerase activity. The inhibition of telomerase is caused due to the lack of 3’ single-stranded telomeric DNA critical for hybridization with RNA subunit of telomerase enzyme for addition of TTAGGG repeats by the telomerase reverse transcriptase protein (Figure 1). Therefore, the telomeric hexanucleotide repeats represent a potential target in anticancer therapy. As a result, the stabilization of telomeric G-quadruplexes by small molecules has emerged as an attractive strategy for the development of anticancer drugs.^[19–21]

Figure 1. Inhibition of telomerase activity by G-quadruplex formation in telomeric region.

Small molecule 2,6-diamidoanthraquinone was the first telomeric G4 interacting ligand to exhibit telomerase inhibition.^[22] Since then, a number of studies explored the therapeutic potential of telomeric G-quadruplex. Small molecules like telomestatin,^[23] BRACO-19,^[24] 5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4),^[25]] fluoro-isoquinolines,^[26] 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4),^[27] pyridostatin,^[28] berberine,^[29] phenanthroline-dicarboxamide (PhenDC3),^[30] bisquinolinium pyridodicarboxamide (PDC-360A),^[31] Epiberberine (EPI),^[32] naphthalene diimides (NDI),^[33] oxazole telomestatin derivatives (OTD),^[34] phthalocyanine,^[35] BMPQ-1,^[36] and a few more ^[37,38] are known for selective targeting of telomeric G4. We herein report triazole substituted dibenzo[a,c]phenazine derivatives (DPa-c) as hTELO quadruplex ligands with the ability to inhibit telomerase activity.

Phenazine cores can extensively interact with DNA^[39] and exhibit a wide range of antitumor, antimicrobial, antioxidant and antimalarial activities.^[40] Phenazine derivatives can inhibit the growth of triple-negative breast cancer via dual interaction with cMYC G-quadruplexes and Topoisomerase 1.^[41] Another study shows encouraging results of this core on human colon and epithelial cancer cell lines.^[42] We have chosen this nitrogen containing fused aromatic ring due to its structural simplicity and ease of synthesis as well as it can be easily tailored. We functionalized the parent heterocycles by Cu(I) catalyzed azidealkyne cycloaddition (CuAAC)^[43] to satisfy the basic criteria of G4 selective ligands while retaining the ‘drug-like’ properties. Click chemistry has been applied to synthesize ligands for targeting G-quadruplexes.^[44a] Neidle and Moses group first explored the utility of the click chemistry for the synthesis of ‘first generation’ G-quadruplex stabilizing ligands.^[45] We have designed triazole substituted dibenzo[a,c]phenazine ligands DPa-c, not yet explored for binding to G-quadruplexes. We envisioned that placing triazole rings, considered as amide bond surrogates, would increase C:N ratio as well as the probability of H-bond formation by increasing the polarity of the molecule and impart selectivity towards DNA binding, hence would lead to improved pharmacological properties against tumors. The aromatic core could promote external stacking on G-quartets via π-π interaction and the side chains with dimethyl amino (-NMe₂) groups at the terminal end would aid in binding with the phosphate backbone of DNA via electrostatic interactions. In molecular recognition, structural variations in small molecules result in differential binding profiles for targets. Simple modifications in the side chain of small molecules such as replacement or extension of a protonable side chain would enable more specific binding towards targets.^[46] It has been shown that trisubstituted acridines show higher telomerase inhibition as compared to disubstituted acridines.^[47] Thus, binding specificities of molecules towards quadruplexes could be further improved by fine-tuning of molecular scaffolds.

For the synthesis, commercially available phenanthrene-9,10-dione 1 was brominated at 3 and 6 positions by using Br₂ and catalytic benzoyl peroxide (BPO) at high temperature for 16 h to obtain 3,6-dibromophenanthrene-9,10-dione 2. Compound 2 was further treated with 1,2-diamino benzene 3 in the presence of catalytic PTSA to obtain dibromodibenzo[a,c]phenazine 4. Sonogashira reaction of compound 4 with alkyne 5 and subsequent deprotection of compound 6 produced 3,6-diethynyldibenzo[a,c]phenazine 7. The Cu(I) catalyzed cycloaddition of dialkyne 7 with aryl and alkyl azides 8a-c afforded the corresponding triazole ligands DPa-c (Scheme 1).

Scheme 1. Synthesis of bis-triazolyl dibenzo[a,c]phenazine derivatives.

Next, we employed biophysical studies like Fluorescence ased Förster resonance energy transfer FRET) melting assay,^[48] isothermal titration calorimetry (ITC)^[49] and circular dichroism (CD) assays to ascertain the interaction profile of the synthesized ligands with different G4s. FRET melting assay was employed to study the interaction profiles of pre-folded dual fluorophore 5’-FAM and 3’-TAMRA) tagged G-quadruplexes (G4s). We used telomeric quadruplex (hTELO) and quadruplexes present in the promoter regions (cMYC, c-KIT1, c-KIT2, BCl2, and VEGF) in the presence of increasing concentrations of synthesized dibenzo-phenazine derivatives DPa and DPc (Figure 2a, Table 1). Ligand DPb was sparingly soluble in DMSO and water, so we could not examine biophysical properties of this compound. The ∆T_m values of the dual labeled quadruplexes showed a dose-dependent response with both phenazine derivatives (Figure 2a).

Figure 2.

a) FRET melting profiles of dual labeled G-quadruplexes (0.2 μM) with increasing concentrations of DPa and DPc in 60 mM potassium cacoldylate buffer (pH-7.4). b) FRET competition melting analysis of dual labeled hTELO G4 (0.2 μM) with 3 μM DPa and DPc in the presence of increasing concentrations of unlabeled competitor dsDNA (0-100 eq.) in 60 mM potassium cacoldylate buffer (pH-7.4). The T_m values of the G-quadruplexes in the absence of ligands are cMYC (78 ± 1), BCL2 (78 ± 1), c-KIT 1 (64 ± 1), c-KIT 2 (71 ± 1), VEGF (82 ± 1), and ds DNA (77 ± 1) °C; maximum measurable T_m = 94 °C, in 60 mM potassium cacodylate buffer, pH 7.4.

Table 1.

∆T_m, K_d and ∆G values derived from FRET melting and isothermal titration calorimetry (ITC) titration assays of various DNA sequences with DPa and DPc. Here, n.d. stands for not determined.

G4 DNA	DPa		DPc
	∆Tm (°C) (3 μM)	Kd (μM)	∆Tm (°C) (3 μM)	Kd (μM)
cMYC	0.4	n.d.	15.4	n.d.
BCL 2	1.3	51.8	12.2	n.d.
c-KIT 1	5.1	18.5	29.6	n.d.
c-KIT 2	3.5	n.d.	22.1	21.8
VEGF	1.6	21.7	12.0	n.d.
hTELO	33	2.4	33.5	2.5
dsDNA	0.0	-	0.6	-

DPa showed significantly high stabilization with hTELO G4 with a ∆T_m value of 20.5 °C, at 2 μM and 33.0 °C at 3 μM ligand concentration. However, it showed weak ∆T_m values for cMYC ∆T_m = 0.4 °C), BCl 2 ∆T_m = 1.3 °C), c-KIT 1 ∆T_m = 5.1 °C), c-KIT 2 ∆T_m = 3.5 °C), and VEGF ∆T_m = 1.6 °C) quadruplexes at 3 μM ligand concentration (Figure 2a, Figure S1). Compound DPc also exhibited high stabilization for hTELO G4 ∆T_m = 32.5 °C at 2 μM and 33.5 at 3 μM). In addition, it also showed high stabilization for several other G-quadruplexes such as cMYC ∆T_m= 15.4 °C), BCL2 ∆T_m = 12.2 °C), c-KIT 1 (∆T_m = 29.6 °C), c-KIT 2 (∆T_m = 22.1°C) and VEGF ∆T_m = 12.0°C) at 3 μM. Even though both DPa and DPc showed comparable ∆T_m values of 33.0 °C and 33.5 °C (i.e. the maximum measurable T_m= 94 °C) for hTELO at 3 μM ligand concentration, respectively. DPa shows high selectivity for hTELO in comparison to other G4s including cMYC, c-KIT 1, c-KIT 2, BCL 2, and VEGF. In contrast, DPc non-selectively interacts with all the studied G-quadruplexes. It also showed saturation for c-KIT 1 and c-KIT 2, cMYC, and VEGF at 3 μM concentration. Moreover, both ligands show high selectivity for quadruplexes over duplex DNA. No significant change in the melting profile of duplex hairpin DNA was observed for DPa ∆T_m = 0.0 °C) even at higher concentration of 10 μM while DPc showed a weak stabilization ∆T_m= 3.9 °C) at similar concentration.

For additional insights into the ability of DPa and DPc to stabilize hTELO quadruplex over duplex DNA, the competitive FRET melting assay was performed (Figure 2b). The competition assay was carried out using 3 μM of each ligand in the presence of fluorophore tagged hTELO G-quadruplex with excess of unlabelled dsDNA competitor at incremental values of 0, 1, 5, 10, 50 and 100 equivalents. The G-quadruplex melting profiles did not show significant alteration in ∆T_m in the presence of the DNA competitor for DPa and DPc even at 50 and 100 eq. This observation suggests that DPa and DPc stabilizes telomeric G-quadruplex even in the presence of the duplex competitor and shows high selectivity for the hTELO G4.

This observation prompted us to determine the binding affinities of DPa and DPc for different G4s using ITC studies (Table 1). DPa exhibited a nine-fold binding specificity for hTELO G4 (K_d = 2.4 μM) over c-KIT 1 G4 (K_d = 18.5 μM) Figure S2). It showed lower affinity for other G4s like VEGF (K_d= 21.7 μM) and BCL-2 (K_d= 51.8 μM) (Figure S3). The binding affinity of DPa for c-MYC and c-KIT 2 could not be determined from the binding isotherms. DPc exhibited a comparable binding affinity for the hTELO G4 with a K_d value of 2.5 μM. It showed a weak affinity for c-KIT 2 G4 (K_d = 21.8 μM). Despite showing stabilization for several G-quadruplexes in FRET melting assay, DPc did not show proper fitting of ITC isotherms for cMYC, BCl 2, c-KIT 1 and VEGF G4s and thus, the K_d values could not be determined for these G-quadruplexes (Figure S3). The ITC results suggest that DPa and DPc exhibit energetically favorable interactions with the hTELO among the studied G4s, exhibiting comparable K_d values.

Circular dichroism was carried out to investigate the structural integrity of the hTELO G4 upon interaction with DPa and DPc (Figure S4). The CD signature of hTELO exhibited a minima at ~235 nm and maxima at ~269 nm and ~291 nm,^[50] suggesting that the DNA sequence folds into a characteristic hybrid G4 structure in K⁺ buffer. On addition of increasing concentrations of DPa and DPc (0.3 - 5 eq., Figure S4), no significant alteration was observed in the intensity of the CD signals of hTELO. The CD analysis thus suggests that both DPa and DPc do not disrupt the structural integrity of the hTELO G4 even at high concentrations.

The biophysical studies carried out with the dibenzophenazine derivatives suggest that DPa and DPc exhibited binding affinity towards the telomeric quadruplex structure with DPa showing higher selectivity for hTELO G4 over other G-quadruplexes. To investigate the effect of DPa and DPc on telomerase activity, a three-step modified telomeric repeat amplification protocol (QTRAP) assay was performed. The in vitro TRAP-Lig assay was carried out on telomerase positive HeLa cells to measure the activity of the telomerase enzyme in response to DPa and DPc at different concentrations. The telomerase extract isolated from HeLa cells was treated with increasing doses 5 μM and 10 μM) of DPa and DPc. The final amplification step showed that both DPa and DPc inhibited telomerase activity in a dose dependent manner. The relative telomerase activity was reduced by 57.4% and 61.6% in the presence of DPa at concentrations of 5 μM and 10 μM respectively Figure 3a and Figure S5). On the other hand, DPc also reduced telomerase activity to a lesser extent by showing reduction of 20.6% and 32.5% at 5 μM and 10 μM ligand concentrations, respectively. DPa showed a greater reduction in relative telomerase activity compared to DPc presumably due to its higher selectivity with the hTELO G4 in comparison to other quadruplexes.

Figure 3.

a) Q-TRAP analysis showing relative telomerase activity (RTA) of telomerase extract treated with 5 μM and 10 μM of DPa and DPc. b) Molecular docking studies showing ball-stick model (left side) and space-filling model (right side) of DPa (green) with hTELO G-quadruplex (PDB ID: 2JPZ).

We further evaluated the growth inhibitory activities of DPa and DPc in different mammalian cell lines. Human cancer cell lines like human cervical cancer (HeLa) (Figure S6) breast cancer (MCF-7) (Figure S7), and myelogenous leukemia (K562) (Figure S8) as well as normal kidney epithelial (NKE) (Figure S8) cell lines were used to carry out the study. Despite being selective for hTELO and inhibiting telomerase activity in vitro, both DPa and DPc were found to be non-toxic to the investigated cell lines. The non-cytotoxic nature of these ligands could possibly be attributed to their low cell permeability and cellular uptake. Moreover, though the side chains conjugated to the aromatic core make the molecules soluble in DMSO and water, further side chain modification would enable their entry into cancer cells, thereby enhancing cellular toxicity of this class of compounds. Instances of phenazine derivatives exhibiting very high or undetermined IC₅₀ values have also been reported.^[51] These ligands represent a class of potential therapeutic small molecules, which by themselves may be non-toxic to cancerous cells but could enhance the potency of other drugs when used in combination therapy. Combination therapy may emerge as a promising approach to combine different types of telomerase inhibitors with other chemotherapeutics,^[52] to inhibit telomerase activity by amplifying the effect of individual molecules when present in appropriate combinations.

Molecular docking was carried out in order to ascertain the structure activity relationship and the mode of interaction between the dibenzo[a,c]phenazine derivatives DPa and DPc and the hTELO G-quadruplex (Figure 3b and Figure S9). The docking studies showed that both DPa and DPc interact with hTELO G4 (PDB ID: 2JPZ) through π-π interaction with the terminal G-quartet of the quadruplex with ∆G values of -7.6 kcal mol^-1 and -5.8 kcal mol^-1, respectively. The dibenzophenazine core of ligands stack on external G-quartet and the benzamide side chains of DPa show improved interaction with hTELO G4 by providing increased electrostatic interactions compared to the aliphatic amine side chains of DPc. The docking studies of DPa and DPc were also carried out with the other G-quadruplexes under investigation (cMYC, BCl2, VEGF, c-KIT 1 and c-KIT 2), which revealed that DPa exhibits minimum binding energy for hTELO G4 (-7.6 kcal mol^-1) whereas DPc exhibits minimum binding energy for c-KIT 2 G4 (-6.3 kcal mol^-1) (Figure S10 and Figure S11). This observation further corroborates with other biophysical studies like FRET and ITC, suggesting DPa as a specific ligand for the telomeric G-quadruplex.

Though, both ligands interact with hTELO, the structural variation of the side chains results in significant differences in their binding properties. DPa, with an extended structure due to the presence of benzamide side chains, shows enhanced interaction and selectivity for the hTELO among the G-quadruplexes as compared to the shorter amine side chains of DPc that interacts with several G4 structures and lacks specificity to discriminate between different G-quadruplexes. Thus, DPa inhibits telomerase strongly in comparison to DPc.

In summary, we report the synthesis of bis-triazolyl dibenzo[a,c]phenazine derivatives as a new class of ligands that stabilize human telomeric G-quadruplex. The ligands are prepared from inexpensive and commercially available starting materials by using simple and modular methods. The dibenzophenazine ligand with benzamide sidechains shows appreciable stabilization as well as a higher degree of binding preference for the telomeric G4 over other promoter G4s and dsDNA. Both these ligands inhibit telomerase activity, most probably by stabilizing the higher order structure of telomeric sequence. We also highlight the differential selectivity observed by simple side chain modifications such as the presence of either benzamide or amine side chains. Dibenzo-phenazine derivatives show promising interaction profiles in biophysical and in vitro studies with telomeric G-quadruplex. Additional structural modifications could further improve cell penetration and antiproliferative properties of this class of ligands as well as telomerase inhibition in cancer cells by selective interaction with the telomeric region of the eukaryotic chromosome. This study presents a class of potential therapeutic small molecules and may hold a significance in combination therapy with other less potent drugs to inhibit telomerase, an important hallmark of cancer cells.