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. 2024 Feb 12;15(3):388–395. doi: 10.1021/acsmedchemlett.3c00537

Selective Recognition of c-KIT 1 G-Quadruplex by Structural Tuning of Heteroaromatic Scaffolds and Side Chains

Khushnood Fatma †, Prasanth Thumpati †,‡, Deepanjan Panda †, Ravichandiran Velayutham ‡, Jyotirmayee Dash †,*
PMCID: PMC10945540  PMID: 38505840

Abstract

graphic file with name ml3c00537_0008.jpg

In this study, carbazole (MC) and dibenzofuran (MD) derivatives were synthesized to examine their effect on the biomolecular recognition of G-quadruplex (G4) targets. Biophysical studies revealed that MC-4, a carbazole derivative, exhibits a specific affinity and effectively stabilizes the c-KIT 1 G4. Molecular modeling suggests a stable interaction of MC-4 with the terminal G-tetrad of c-KIT 1 G4. Biological studies demonstrate that MC-4 efficiently enters cells, reduces c-KIT gene expression, and induces cell cycle arrest, DNA damage, and apoptosis in cancer cells. These findings demonstrate MC-4 as a selective c-KIT G4 ligand with therapeutic potential, providing insight into the structural basis of its anticancer mechanisms.

Keywords: G-quadruplex, Small molecule, Anticancer, Gene regulation, Apoptosis, c-KIT inhibition

The c-KIT proto-oncogene encodes a transmembrane receptor tyrosine kinase (RTK), primarily expressed in hematopoietic cells and myeloid cells, which responds to stem cell factor (SCF).1,2c-KIT regulates cell proliferation, survival, migration, pigmentation, and hematopoiesis.3,4 Given its pivotal role in maintaining normal cell function, mutations or overexpression of c-KIT results in oncogenic cellular transformations, including gastrointestinal stromal tumors (GISTs), melanomas, mastocytosis, and acute myeloid leukemia (AML).5,6 Therefore, c-KIT has emerged as a promising molecular target for anticancer therapy.7 Efforts to inhibit c-KIT overexpression have led to the development of small molecule inhibitors like the Food and Drug Administration (FDA)-approved drug imatinib mesylate (Gleevec) and drugs like dasatinib, sunitinib, nilotinib, and sorafenib.8

Using natural or synthetic molecules to regulate aberrant gene expression by targeting noncanonical DNA structures has emerged as an alternative strategy for potential cancer therapeutics.9−11 The human c-KIT oncogene contains two guanine-rich (G-rich) sequences, KIT 1 and KIT 2, upstream of the transcription starting site,12−14 which adopt G-quadruplex (G4) structures and influence cellular functions.15 G4s consist of stacked G-tetrad subunits, with each tetrad containing four coplanar guanines associated by Hoogsteen hydrogen bonds and stabilized by coordination with potassium or sodium cations.16,17 The regulatory role of G-quadruplexes in telomeres (h-TELO) and promoter regions of genes like c-MYC, BCL-2, and c-KIT has been explored.18−21 Studies have indicated that small molecules can stabilize the c-KIT quadruplexes, thereby inhibiting c-KIT transcription.22−24 However, cellular studies focused on c-KIT inhibition through G4 stabilization by carbazole or dibenzofuran derivatives remain scarce.

Small molecule ring systems with optimal side-chain functionalization by rational design can preferentially target biomolecular entities in cellular systems.25,26 The carbazole ring system is a key structural scaffold of many biologically active compounds possessing anticancer, antibacterial, anti-inflammatory, antifungal, anti-HIV, antiprotozoan, or topoisomerase II inhibition ability.27−29 Heteroaromatic dibenzofurans, containing a central furan ring between two benzene rings, have also been devised to exhibit potent anticancer, antibacterial, antifungal, anti-HIV, and anti-inflammatory activities in medicinal chemistry.30 These chemical scaffolds with a plethora of biological activities were used in this study to investigate their interaction with G-quadruplexes.

In this study, copper(I)-catalyzed alkyne–azide cycloaddition was employed to synthesize the MC and MD series of ligands (Scheme 1). Carbazole and dibenzofuran motifs were chosen as building blocks due to their extended aromatic structure, which promotes π–π stacking with the G-tetrads of the folded G4 structures. Iodocarbazole 1a and iododibenzofuran 2a, prepared from commercially available carbazole and dibenzofuran via monoiodination, were employed as precursors for the synthesis of the corresponding alkynes 1b and 2b. These alkynes were synthesized through Sonogashira coupling followed by removal of the trimethylsilyl (TMS) group in high yields. The azide library 3a–g (Scheme S2) included both aliphatic and aromatic azides containing amines (3a, 3b, 3c, 3g), a carboxamide amine side chain (3d), and side chains derived from amino acids, such as prolinamide (3e) and lysinamide (3f). The azides, each bearing distinct substituents, were used to achieve enhanced selectivity as side arms in the ligand scaffold for the recognition of specific G4 structures. Alkyne 1b and azide 3a were initially heated to 70 °C; however, the reaction did not achieve complete conversion, and starting materials were recovered. Subsequently, the reaction was carried out under microwave irradiation,28,31 which drove the reaction toward completion. Thus, the reactions of alkynes 1b and 2b with azides 3a–g were carried out using CuSO4·5H2O and sodium ascorbate in t-BuOH/H2O (3:1) under microwave irradiation at 70 °C for 4 h, providing the corresponding triazole products MC-1–7 and MD-8–14 in high yields.

Scheme 1. Synthesis of Monotriazolyl Carbazole (MC) and Monotriazolyl Dibenzofuran (MD) Derivatives with Respective Side Chain Modifications.

Scheme 1

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In contrast to the previously utilized larger bistriazolyl ligands from our laboratory for targeting G-quadruplexes,32,33 this design of reducing molecular size is intended to address poor permeability of large compounds, facilitating cell membrane permeability while retaining the ability to distinguish specific G4s. A series of biophysical, in silico, and in cellulo investigations were conducted to assess the stabilization potential of these monotriazolyl ligands as potential G4 binders.

The FRET-based melting assay34−37 was employed to evaluate the efficacy of the synthesized small molecules in stabilizing various dual-labeled (5′-FAM and 3′-TAMRA) G-quadruplexes present in the oncogenic promoter (c-MYC, BCL-2, c-KIT 1, c-KIT 2, VEGF) and telomeric (h-TELO) regions. A double-stranded DNA (dsDNA) sequence was used as a control (Figure 1). MC and MD ligands were tested at a 1 μM ligand concentration. Among the MC series, only MC-4 significantly influenced the melting temperature (Tm) of the G-quadruplexes, particularly demonstrating a significant change for c-KIT 1 (ΔTm = 20.1 °C). Furthermore, MC-4 stabilized other G4s to a lesser extent [ΔTm (°C): c-MYC = 3.2, c-KIT 2 = 9.9, BCL-2 = 2.2, VEGF = 3.5, h-TELO = 4.5, and dsDNA = 0.7] (Figures S2 and S3). In comparison, the other MC ligands (MC-1, MC-2, MC-3, MC-5, MC-6, and MC-7) did not exhibit an appreciable change in Tm (ΔTm ≤ 5.5 °C) for the studied G4s. Similarly, the MD ligands (MD-8–14) showed less significant stabilization (≤6.0 °C) for the examined G4s, and MD-11 did not show high stabilization (ΔTm < 3.0 °C) for the G4s.

Figure 1.

Figure 1

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FRET melting analysis of different G-quadruplexes (200 nM) and dsDNA (200 nM) with (a) MC-1–7 and (b) MD-8–14. Ligands were added at a concentration of 1 μM. All experiments were performed in 60 mM potassium cacodylate buffer (pH 7.4). The Tm values of dual-fluorophore-tagged G-quadruplexes without ligand were taken as control [Tm (°C): c-MYC = 73 ± 1, BCL-2 = 73.8 ± 1, c-KIT 1 = 63.1 ± 1, c-KIT 2 = 71.5 ± 1, VEGF = 79.5 ± 1, h-TELO = 47.4 ± 1, and dsDNA = 74.5 ± 1].

In addition, a dose-dependent FRET melting assay with increasing concentrations of MC-4 and MD-11 (1, 3, 5, 7, and 10 μM) added to each G4 was performed (Figures 2a and S4). Interestingly, MC-4 exhibited the highest stabilization of c-KIT 1 in a dose-dependent manner. The ΔTm values of c-KIT 1 in the presence of MC-4 at 1, 3, 5, 7, and 10 μM were observed to be 20.1, 22.6, 25.5, 26.5, and 26.5 °C, respectively. In comparison, the stabilization of other G4s by MC-4 was lesser, with ΔTm = 17.3, 12.1, 18.1, 11.2, and 20.3 °C for c-MYC, BCL-2, c-KIT 2, VEGF, and h-TELO, respectively, at the highest concentration of 10 μM. In addition, dsDNA displayed only a small degree of stabilization at 10 μM MC-4. The results suggested that MC-4 exhibited a higher stabilization potential for c-KIT 1 compared to other G4s and dsDNA. In contrast, MD-11 with the same side chain showed less significant alteration in Tm (ΔTm ≤ 5 °C) with the G4s.

Figure 2.

Figure 2

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(a) FRET melting analysis of MC-4 with different G-quadruplexes (200 nM) and dsDNA (200 nM) at different ligand doses (1, 3, 5, 7, and 10 μM). (b) Fluorescence spectra of MC-4 (1 μM) titrated with increasing concentrations of c-KIT 1 G4 until saturation is reached. (c) Fluorometric titration plot of MC-4 with different G-quadruplexes and dsDNA. (d, e) Isothermal heat curve and binding isotherm for the interaction of MC-4 with c-KIT 1 G4. All fluorescence spectroscopy and ITC experiments were performed in 100 mM Tris-KCl buffer (pH 7.4).

Next, a competitive FRET melting assay was performed in the presence of an excess unlabeled calf thymus (CT) DNA competitor to assess the selectivity of MC-4 for c-KIT 1 G4. CT DNA was added at several fold molar equivalents (0, 1, 5, 10, 50, and 100 equiv) excess of the fluorophore-labeled c-KIT 1 DNA in the presence of MC-4 and MD-11 (1 μM) (Figure S5). The ΔTm of c-KIT 1 (20.1 °C) in the presence of MC-4 did not exhibit any significant (≤3.5 °C) alteration with the competitor DNA (up to 100 equiv), suggesting selective stabilization of c-KIT 1 G4 over other DNA substrates. Similarly, MD-11 did not show any significant alteration of Tm (ΔTm ≤ 3.5 °C) of c-KIT 1 G4 in the presence and absence of the CT DNA.

From the initial FRET studies, we considered MC-4 as the lead G4 binder for the c-KIT 1 G-quadruplex, as it exhibited a higher stabilization potential and differential recognition of c-KIT 1 G4. MD-11, featuring the same side chain as MC-4 with the dibenzofuran moiety, did not exhibit substantial stabilization of c-KIT 1 G4 and was further studied as a negative control to illustrate the distinct binding profiles of the different heteroaromatic scaffolds with regard to G4 stabilization.

Fluorescence spectroscopy was performed to determine the binding affinity of lead carbazole MC-4 for each G4. MC-4 was titrated with increasing concentrations of c-MYC, c-KIT 1, c-KIT-2, VEGF, BCL-2, h-TELO, and dsDNA (Figures S6–S8). Fluorimetric titration of MC-4 (1.0 μM) with increasing concentrations of c-KIT 1 G4 showed a 5.2-fold increase (Kd = 1.4 μM) in fluorescence intensity (Figure 2b), which is 3.2 and 2.8 times higher than c-KIT 2 (1.6-fold change, Kd = 2.8 μM) and VEGF (2.1-fold change, Kd = 2.9 μM), respectively (Figure 2c). Negligible changes in fluorescence intensity of MC-4 were observed for BCL-2 (Kd = 4.8 μM), h-TELO (Kd = 3.2 μM), c-MYC (Kd = 2.7 μM), and dsDNA (Kd = 4.0 μM). A low dissociation constant (Kd) of 1.4 μM indicated a high affinity for the binding interaction of MC-4 with c-KIT 1 compared to other G4s and dsDNA. Fluorimetric titration of MD-11 (1.0 μM) with the studied G4s showed higher Kd values than the 1.4 μM exhibited by MC-4 and the lead target G4 (Figure S8 and Table S1).

Structural investigations of the folded c-KIT 1 G4 with lead ligand MC-4 were carried out using circular dichroism (CD) spectroscopy to monitor structural alterations. The CD spectra of c-KIT 1 exhibited characteristic features of parallel G4 with maxima at 262 nm and minima at 240 nm. Moreover, CD titration of c-KIT 1 G4 performed with excess equivalents of MC-4 (0.5, 1, 2, 3, 4, and 5 equiv) showed that the significant peaks of c-KIT 1 were not disrupted even in the presence of excess MC-4, suggesting that MC-4 does not perturb the folded c-KIT 1 G4 structure (Figure S9). Furthermore, c-KIT 1 G4 exhibited an increase of ∼16 °C in Tm in the presence of MC-4 (Figure S10).

Isothermal titration calorimetry (ITC) was performed to probe the thermodynamic binding of lead compound MC-4 (Figures 2d and S11) with different G4s (c-MYC, BCL-2, c-KIT 1, c-KIT 2, VEGF, h-TELO, and dsDNA). It was observed that MC-4 exhibited a Kd of 1.6 μM with c-KIT 1, which is in agreement with the previously observed Kd value obtained from fluorometric titration. Furthermore, the MC-4/c-KIT 1 interaction was enthalpy-driven and exothermic in nature with a stoichiometry of 1.0. In contrast, the thermograms obtained for the reaction of MC-4 with other G4s exhibited high Kd values (c-MYC = 6.2 μM, BCL-2 = 10.8 μM, c-KIT 2 = 20.9 μM, VEGF = 15.9 μM, and h-TELO = 17.4 μM). The thermogram for dsDNA could not be fitted to the model properly, and the Kd value could not be determined. These findings further corroborate the biophysical assessments of MC-4, implying that it can selectively recognize and interact with the specific G4 topology of c-KIT 1 with high affinity.

A structure–activity relationship (SAR) study of lead MC-4 with different G-quadruplexes was performed using AutoDock 4.2 to ascertain the mode of interaction of MC-4 with G-quadruplexes c-KIT 1, c-KIT 2, c-MYC, BCL-2, VEGF, and h-TELO. It was observed that MC-4 interacted with c-KIT 1 G4 with a minimum energy (ΔG) of −6.8 kcal/mol, indicating a strong and energetically favorable interaction (Figure 3a–d). It was found to interact with the 5′-G-tetrad composed of G2, G6, G10, and G13 of c-KIT 1 G4.38 The flexible amine side chain enabled a stronger and more specific interaction with the phosphate backbone of c-KIT 1 G4 (Table S2).

Figure 3.

Figure 3

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(a, b) Molecular docking studies of MC-4 (blue) and c-KIT 1 G4 interaction (PDB ID- 4WO2) as observed from different views: (a) top view; (b) side view. (c) Space-filling model of c-KIT 1 G-quadruplex. (d) Site of DNA–ligand interaction showing the 5′-tetrad guanines where the MC-4 molecules bind via π–π stacking. Bonds are in dots. MC-4 surrounding base pairs are in two-letter code represented in blue.

In contrast, the interaction of MC-4 with other G4s was energetically less favorable [ΔG (kcal/mol): c-KIT 2 = −5.8, BCL-2 = −5.3, c-MYC = −5.6, VEGF = −2.5, and h-TELO = −1.5; Figure S12]. Therefore, MC-4 consisting of a heteroaromatic carbazole scaffold promotes stacking interactions with the 5′-G-tetrad of c-KIT 1 G4. Carbazole derivatives feature a planar structure with strong π-electron delocalization, enabling a stable conformation for effective π–π stacking interactions with planar G-tetrad in c-KIT 1 G-quadruplexes for stable binding. Furthermore, the nonpolar aromatic regions of carbazole engage in hydrophobic interactions with hydrophobic sites on the G-quadruplexes, promoting stability. The extended side-chain structure of MC-4 further improves the binding affinity via interaction with the phosphate backbone of the G4 and the loops. The monofunctionalized smaller side chain enables optimal interaction with the G4, which is often difficult to achieve with higher-molecular weight molecules. These studies suggested that MC-4 stacks upon the 5′-terminal G-tetrad of c-KIT 1 and its side chain interacts with the loop region of the G4, resulting in selective recognition of c-KIT 1 G4 over other G4s.

Furthermore, molecular dynamics simulation was performed to study the stability profile of MC-4 with c-KIT 1 G-quadruplex. After the completion of the MD simulation, the output files were loaded to VMD for trajectory analysis. The root-mean-square deviation (RMSD) graph was plotted based on the trajectories. RMSD plots are usually used to measure the deviation between the initial structures and the structures obtained during MD simulation. The RMSD graph of the c-KIT 1 DNA showed minor fluctuations between 50000 and 75000 steps, but again regained stability, which is an acceptable range. The RMSD graph of MC-4 also showed minor fluctuations (Figure S13). Fluctuations in only 30000 to 45000 steps were shown. The minor deviations of both DNA and ligand are in favor of the stable binding of MC-4 with c-KIT 1 G4 DNA. The total energy is the sum of the various potential energies and the kinetic energy. The total energy graph demonstrated that the energy has minimal variation. The bond energy of the total system signifies the stability of the bonds between G4 DNA and MC-4 during the simulation.

The biophysical studies with the MC and MD series suggested that MC-4 selectively interacted with c-KIT 1. The effect of this interaction was next studied in the cellular context. The cell cytotoxicity of MC-4 ligand was assessed toward c-KIT-positive myelogenous leukemia cell line (K562). A cancer cell line without overexpression of c-KIT (MCF-7) and a noncancerous cell line (HEK293) were also assessed to determine the cytotoxicity of MC-4 and MD-11 toward these cell lines. It was observed that MC-4 displayed high cytotoxicity toward K562 (IC50 = 6.2 μM) and low cytotoxicity toward MCF-7 (IC50 = 19.8 μM) (Figure S14a) cells. In addition, the ligand MC-4 was nontoxic to normal HEK293 cells with an IC50 value of >50 μM (Figure S14c). This is in line with our previous observations which showed interaction of MC-4 with c-KIT 1 G-quadruplex. In contrast, the dibenzofuran scaffold containing counterpart, MD-11 displayed higher IC50 values (K562 = 11.7 μM, MCF-7 ≥ 19 μM, and HEK293 ≥ 50 μM) (Figure S14b,c). The low IC50 value of MC-4 exclusively in the K562 cancer cell line suggested that MC-4 could effectively permeate the cell and nuclear membranes to stabilize the c-KIT G-quadruplex in cancer cells while inducing a high cytotoxic effect only in the c-KIT overexpressing leukemia cell line. Thus, additional analyses were conducted with MC-4 to elaborate on the nature of the c-KIT 1/MC-4 interactions and to study its effect on cellular systems.

As MC-4 exhibited an antiproliferative effect on cancer cells, its ability to permeate the cellular membrane was visualized using cell imaging investigations. Both MC-4 (3 μM) and MD-11 (5.5 μM) were observed to be cell-permeable and localized in the K562 cells with an inherent fluorescence in the green filter range (Figures 4). In addition, the ligand-treated K562 cells were observed under a confocal microscope to evaluate the prevalence of G-quadruplexes. MC-4-treated cells showed a greater number of G-quadruplexes visualized using the G-quadruplex-specific antibody (BG4). The number of BG4 foci increased by 5.4-fold and 1.8-fold in the MC-4- and MD-11-treated cells, respectively, compared to the foci observed in untreated (Figure 4a–c) cells. This direct evidence suggested that treatment with MC-4 leads to greater stabilization of the G-quadruplexes, resulting in their detection in significant numbers within the cells.

Figure 4.

Figure 4

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(a) Cell images of K562 with and without treatment with MC-4 and MD-11. The cell was stained with MC-4 and MD-11 (green), and the G4-specific antibody was visualized as foci (red) present within the cell. (b) Schematic representation of MC-4-stabilized G-quadruplex and BG4 antibody interaction. (c) Graphical representation of the numbers of BG4 foci in the nuclei of untreated and MC-4- and MD-11-treated K562 cells.

G-quadruplex-binding small molecules have been reported to attenuate the gene expression of the proto-oncogene c-KIT by G-quadruplex stabilization.23 Thus, qRT-PCR was performed to examine the effect of each ligand on c-KIT transcriptional regulation (Figures 5a and S15). The well-explored G4 binder TMPyP4 was used as a positive control. K562 cells were treated with MC-4 (3 and 6 μM), MD-11 (5.5 and 11 μM), and TMPyP4 (2 and 4 μM) for 24 h, and the expression of each gene (c-KIT, c-MYC, and BCL-2) was normalized with the expression of housekeeping gene 18s. MC-4 reduced c-KIT oncogenic expression by 28% and 73% at 3 and 6 μM concentrations, respectively. In addition, treatments with MD-11 induced a downregulation of only >1% and 3%, whereas TMPyP4 induced a downregulation of 30% and 48%, respectively. Considering that the G4-mediated stabilization induced by MC-4 results in c-KIT gene repression, we examined the transcriptional regulation of other G4-containing genes like c-MYC and BCL-2. Downregulation of 15% and 19% in the c-MYC gene and decreases of 29% and 39% in the BCL-2 gene were observed after treatment with MC-4 at 3 and 6 μM concentration, respectively. The significant transcriptional downregulation of c-KIT coupled with repression of antiapoptotic BCL-2 gene suggested a potential correlation between the stabilization of c-KIT G-quadruplex by MC-4 and the induction of apoptosis. Conversely, c-MYC showed upregulation of 23% and 31% and BCL-2 showed 8% upregulation and 5% downregulation, respectively, after treatment with MD-11. Expectedly, the G4 binder TMPyP4 (2 and 4 μM) showed dose-dependent downregulation of 11% and 43% in c-MYC and downregulation of 37% and 45% in BCL-2 gene expression, which was in line with general G4-mediated inhibition of gene expression. Thus, the carbazole derivative MC-4 could significantly downregulate the transcription of the c-KIT oncogene up to 73% in leukemia cells.

Figure 5.

Figure 5

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Determination of gene regulation at the level of mRNA and protein in K562 cells treated with different doses of MC-4 (3 and 6 μM), MD-11 (5.5 and 11 μM), and TMPyP4 (2 and 4 μM). (a) Transcriptional regulation using qRT-PCR for the expression of c-KIT, c-MYC, and BCL-2. (b) Schematic representation of the promoter luciferase assay. (c) Relative luciferase expression of c-KIT, c-MYC, and BCL-2 G4 containing luciferase-expressing plasmids transfected in K562 cells. (d) Protein expression of housekeeping gene GAPDH and c-KIT in K562 cells. (e) Bar diagram of relative protein expression of c-KIT gene in K562 cells.

In order to investigate whether the repression of c-KIT is due to MC-4-mediated promoter G4 stabilization, a luciferase promoter assay was carried out using a plasmid construct containing the wild-type c-KIT promoter region upstream of the luciferase gene. K562 cells were transfected with the c-KIT plasmid construct and treated with different doses of MC-4 (3 and 6 μM), MD-11 (5.5 and 11 μM), and TMPyP4 (2 and 4 μM), followed by measurement of luciferase expression (Figure 5b,c). In MC-4-treated cells, there was dose-dependent downregulation of the c-KIT gene, with reductions of 22% at 3 μM dose and a substantial decrease of 69% at 6 μM dose. Conversely, MD-11 showed a decrease of only 3.3% at 5.5 μM and 7.6% at 11 μM. To obtain a comparative analysis, plasmid vectors containing other promoter G4s (c-MYC and BCL-2) were also investigated. Smaller changes were observed in c-MYC and BCL-2 construct transfected cells treated with 3 and 6 μM MC-4 (c-MYC = 6.0% and 12.3%, BCL-2 = 7.8% and 15.8%) and 5.5 and 11 μM MD-11 (c-MYC = 6.2% and 10.1%, BCL-2 = 9.5% and 14%). TMPyP4 (2 and 4 μM)-treated cells showed nonspecific downregulation of genes (c-KIT = 20.1% and 34.1%, c-MYC = 24.4% and 45.3%, and BCL-2 = 20.7% and 39.7%), suggesting that the G4-binding TMPyP4 downregulates the expression of promoter G-quadruplex-containing oncogenes. These observations further corroborate that the stabilization of c-KIT G4 by MC-4 induces downregulation of the c-KIT gene expression in cancer cells.

Western blotting assay was performed to confirm the results of c-KIT protein inhibition by the lead ligand. MC-4-treated K562 leukemia cells showed dose-dependent downregulation of c-KIT with 23.3% and 65.5% expression at 3 and 6 μM (Figure 5d,e). MD-11 induced c-KIT 1 downregulation of only 7.8% at 5.5 μM and upregulation of 15.8% at 11 μM. Expectedly, TMPyP4 showed downregulation of c-KIT (∼10% and 20%), suggesting that it inhibited protein expression of the c-KIT gene but at a much lesser extent. The upregulation of c-MYC and BCL-2 genes observed in MD-11-treated K562 cells may be a result of G-quadruplex-independent mechanisms which either affected the c-MYC gene or its upstream effectors.

Flow cytometry studies were conducted to investigate the relationship between the inhibitory effects of MC-4 and the possible arrest of cancer cells in any phase of the cell cycle. MC-4 (3 and 6 μM)- and MD-11 (5.5 and 11 μM)-treated K562 cells were stained with propidium iodide (PI) to determine the number of cells in each phase of the cell cycle (Figure S16). Cells treated with 3 and 6 μM MC-4 exhibited cell cycle arrest in a dose-dependent manner, with arrest of 74.1% and 79.7% of cells in the G0/G1 phase. This observation supports current literature, where G4-binding ligands were observed to arrest cells at G1 phase of the cell cycle.39 In addition, the MD-11-treated cells did not show any significant changes in the cell populations in any cycle phase. Additional flow cytometry studies to evaluate ligand-induced DNA damage were conducted using AlexaFluor 645-tagged γH2AX antibodies (Figure 6). Flow cytometry detection of γH2AX in cells is a reliable method for the quantification of DNA damage. Analysis of MC-4 (3 and 6 μM)-treated K562 cells showed DNA damage in 38.6% and 67.1% of the cell population, respectively. In contrast, MD-11 (5.5 and 11 μM)-treated cells showed less DNA damage (27.6 and 30.3%, respectively).

Figure 6.

Figure 6

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Flow cytometric analysis of DNA damage after treatment of K562 cells with MC-4 (3 and 6 μM) and MD-11 (5.5 and 11 μM); bar diagram (right panel) representing the percentage of cells exhibiting DNA damage.

Flow cytometry was carried out to investigate the mode of death induced by MC-4 and MD-11 in cancer cells (Figure S17). K562 cells treated with MC-4 (3 and 6 μM) and MD-11 (5.5 and 11 μM) were analyzed upon annexin V and PI dual staining. Dot plot analysis indicated that MC-4 induced apoptosis in K562 cells in a dose-dependent manner, with increasing rates of apoptotic cells and increasing ligand concentrations. At 3 μM concentration, MC-4 induced 35% cell death, whereas at the second dose of 6 μM, an increase of apoptotic cells to 63% was observed with a more significant shift from early to late apoptosis. In contrast, MD-11 induced apoptosis in 1% and 11% of the cell population. Overall, the flow cytometry studies revealed that MC-4 is capable of arresting the K562 cells in the G0/G1 phase of the cell cycle. It also triggered DNA damage and led to apoptosis in cells, likely by interacting with c-KIT 1 G4.

In conclusion, we synthesized 14 small molecules with carbazole (MC) and dibenzofuran (MD) scaffolds and identical side-chain modifications to explore their roles in G-quadruplex recognition and stabilization. Among the MC molecules, MC-4 emerged as the lead ligand for c-KIT 1 G4, while MD molecules did not stabilize G4s. Biophysical studies revealed that though MC-4 exhibited moderate binding affinity (1.4 μM), it showed high specificity for the c-KIT 1 quadruplex.40 These results suggested that both the heteroaromatic scaffold and the side chain play crucial roles in G4 binding, with the aromatic scaffold primarily responsible for binding. MC-4 also stabilized G4 structures in cellulo, as evidenced by a significant increase in cellular G-quadruplexes in K562 cells. Flow cytometry revealed that MC-4 induced cell cycle arrest and DNA damage and induced apoptosis. Furthermore, MC-4 also regulated gene expression by reducing the level of c-KIT gene transcription and translation.

The specific recognition exhibited by MC-4 for c-KIT 1 G4, as opposed to its dibenzofuran analogue, emphasized the pivotal role of the molecular scaffold in these interactions. Furthermore, this study is the first to highlight a carbazole-derived small molecule’s potential to influence the transcription and translation of the c-KIT gene, likely through stabilization of the c-KIT 1 G-quadruplex. Moreover, it paves the way for further functionalization of carbazoles by rational fine-tuning of the side chains to achieve a higher degree of selectivity for diverse DNA structures of distinct topologies and regulate the expression of cancer and other aberrant genes.

Acknowledgments

K.F. thanks UGC for a fellowship. P.T. thanks NIPER-Kolkata for a fellowship. J.D. thanks Wellcome Trust/DBT India Alliance Fellowship [Grant IA/S/18/2/503986], SERB (CRG/2021/004525) and Technical Research Centre, IACS for funding. The authors thank Dr. Sayantan Pradhan (IACS) for the docking and simulation experiments, Chanchal Kumar Das (IACS) for operating the FACS instrument, and Mrs. Shreetoma Kundu (NIPER Kolkata) for operating the Leica DMI8 Stellaris 5 microscope.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00537.

  • Additional data and supplementary figures (PDF)

Author Contributions

§ K.F. and P.T. contributed equally.

The authors declare no competing financial interest.

Special Issue

Published as part of ACS Medicinal Chemistry Lettersvirtual special issue “Many Faces of Medicinal Chemistry”.

Supplementary Material

ml3c00537_si_001.pdf (4MB, pdf)

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