Insights from Binding on Quadruplex Selective Carbazole Ligands

Abstract

Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.

Keywords: G-quadruplexes; NMR spectroscopy; biophysics; carbazole ligands; ligand design.

Figure 1

(a) G‐quadruplex (G4) formation via stacking of Hoogsteen hydrogen bonded G‐tetrads. (b) Structure and polymorphism of major type of G4s.

Figure 2

Chemical structures of carbazole and its derivatives that show potent pharmacological activities such as anti‐inflammatory, anti‐microbial (Murrayanine), ^[8b] antiproliferative (Mukonine), ^[9b] antioxidant (Carbazomadurin A), and antitumor (Ellipticine, Celiptium, Alectinib ^[17] ).

Figure 3

Schematic drawing of binding modes found for carbazole ligands. They have been divided into two main categories: Binding with local and binding with global conformational changes. The first category includes ligands that induce changes around the binding pocket and they were either designed to bind to the major conformation (A) of the target sequence or to target solely in a topology selective manner. Whereas in the conformational selection mechanism, the ligand binds to a pre‐existing minor conformation (b) and influences the conformational equilibrium in favour to the binding competent form. Further, the ligand can induce a change in the overall topology of the binding partner and follow an induced fit mechanism.

Figure 4

Chemical structures of carbazole ligands capable of binding to the major conformation of G4 DNA.

Figure 5

Chemical structure of carbazole 4e and Cz1.

Figure 6

Imino region of 1D ¹H NMR spectrum of the c‐MYC22 G‐quadruplex DNA with increasing [ligand]:[DNA] molar ratio of (a) 4e and (b) Cz1. b) The spectra were recorded at 298 K, 600 MHz. Experimental conditions: 100 μM DNA in 25 mM Tris ⋅ HCl (pH 7.4) buffer containing 100 mM KCl in 5 % d⁶‐DMSO/95 % H₂O. c) sequence of c‐MYC22 with the numbering used for assignment that has been transferred from Ambrus et al.. d) and e) Mapping of the observed changes in 1D ¹H NMR spectra upon addition of 4e and Cz1, respectively, on the solution NMR structure of c‐MYC22 (PDB:1XAV). T, A and G are light blue, dark blue and grey, respectively. GH1 and GH8, TH6, AH8 and AH2 that experience chemical shift perturbation are highlighted with red, light blue and dark blue spheres, respectively. The mapping reveals that both ligands affect similar signals, which are located at the external tetrads and the groove formed by the G‐stretches 8–9‐10 and 13–14‐15. Therefore, an identic binding site for both ligands is expected. However, Cz1 shows stronger binding as compared to 4e.

Figure 7

(a) Chemical structure of ligand BMVC. (b) top view onto the 5’‐end of the 1 : 1 BMVC:c‐MYC22 complex (PDB: 6JJ0). (c) side view of the 1 : 1 complex and (d) side view of the 2 : 1 BMVC : c‐MYC22 complex (PDB: 6O2L), BMVC* indicates the weaker binding site. A‐, T‐ and G‐residues are depicted in green, blue and grey, respectively.

Figure 8

Structures of topology selective ligand BMVC‐1c‐Br and specific G4‐conformation selective carbazole derivatives BTC, BTC f and carbazole‐thiazole orange conjugate Cz‐TO.

Figure 9

Imino region of 1D ¹H NMR spectrum of the c‐MYC22 G‐quadruplex DNA with increasing [Ligand]:[DNA] molar ratio of (a) BTC and (b) BTC f. The spectra were recorded at 298 K, 600 MHz. Experimental conditions: 100 μM DNA in 25 mM Tris ⋅ HCl (pH 7.4) buffer containing 100 mM KCl in 10 % D₂O/90 % H₂O. Both ligands bind to a pre‐existing minor conformation and induce a change in conformational equilibrium of c‐MYC22. The numbering is according to Figure 6c). a) is adapted with permission from Ref. [36]. Published by The Royal Society of Chemistry. b) is adapted with permission from Ref. [37]. Copyright 2015, Springer Nature.

Figure 10

Induced‐fit binding mechanism of G4‐interactive ligand. Ligand BMVC‐8C3O induces conformational transition from (a) hybrid‐I and (b) hybrid‐II telomeric G4 topology to (d) parallel topology.

Figure 11

Chemical structures of other G4‐interactive carbazole ligands: the carbazole derived (a) fluorescent probes and (b) anti‐cancer agents.