Small Molecule Driven Stabilization of Promoter G-quadruplexes and

Subscriber access provided by Kaohsiung Medical University

Article

Small Molecule Driven Stabilization of Promoter Gquadruplexes and Transcriptional Regulation of c-MYC Tania Das, Deepanjan Panda, Puja Saha, and Jyotirmayee Dash Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00338 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Small

Molecule

Driven

Stabilization

of

Promoter

G-

quadruplexes and Transcriptional Regulation of c-MYC Tania Das,‡ Deepanjan Panda,‡ Puja Saha, Jyotirmayee Dash* Corresponding Author’s E-mail: [email protected] Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032

ABSTRACT: G-quadruplexes have been considered as attractive therapeutic targets for the development of anticancer agents. We herein report synthesis of a series of carbazole derivatives by employing modular one-pot Cu(I) catalyzed cycloaddition. These carbazole derivatives are easily synthesizable, soluble in aqueous media and able to strongly interact with quadruplexes. FRET based melting assay and fluorescence titration experiments suggest that a carbazole derivative, Cz-1 preferentially binds c-MYC quadruplex DNA over other investigated quadruplex and duplex DNAs. The biological studies revealed that Cz-1 inhibits cancer cell proliferation by inducing apoptosis. Moreover, Cz-1 inhibits the expression of c-MYC at transcriptional as well as translational level. Exon-specific-assay confirms that the downregulation of MYC expression is mainly driven by the binding of Cz-1 with the promoter G-quadruplex structures. Immunocytochemistry, using quadruplex binding antibody BG4, further suggests that Cz-1 induces and stabilizes G-quadruplexes in cellular system.

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION G-rich DNA sequences are known to

adopt four stranded polymorphic DNA secondary

structures called G-quadruplexes.1-6 It has been reported that quadruplexes present in nuclease hypersensitive regions play important roles for regulation of various oncogenes.3 Genome-wide analysis identified the existence of 376,000 putative G-quadruplex motifs in human genome.7-9 Recent works showed that these structures are also involved in key biological phenomena including telomere maintenance and regulation of oncogenes (c-MYC, c-KIT, kRAS etc.).10-13 It has been demonstrated that small molecules, capable of binding to c-MYC promoter Gquadruplex, can inhibit the c-MYC gene expression.14 G-quadruplexes, including c-MYC have been postulated as anticancer targets for chemical intervention.15-19 However, the biological effect of only a few molecules have been explored in cellular system in vitro.20-23 The reports suggest that most of the ligands suffer from poor selectivity towards different DNA structures. In addition, the mechanism underlying the anticancer nature of the ligands remains largely unknown. Therefore, it is highly desirable to identify new chemical entities that may modulate oncogenic expression by targeting a specific G-quadruplex for the development of anticancer therapeutics. In our present study, we have developed modified carbazole derivatives (Scheme 1) that are smaller in size, easily synthesizable as well as soluble in aqueous medium at physiological conditions. We have found that these derivatives can strongly interact with G-quadruplexes and exhibit remarkable biological activity in cellular system. Our data suggests that one of the derivatives of the new carbazole series, Cz-1 effectively binds c-MYC quadruplex and localizes into cancer cells. The compound effectively downregulates c-MYC transcription and induces


Page 2 of 30

Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concomitant apoptosis in cancer cells. We have further established that the observed c-MYC downregulation is induced by Cz-1 driven formation/stabilization of its promoter quadruplex. RESULTS AND DISCUSSION Design and synthesis. G-rich DNA sequences may adopt a variety of quadruplex structures. Thus, the design and development of small molecules that specifically recognize G-quadruplex structures of certain topology is highly desirable. Usually G-quadruplex binding ligands possess heteroaromatic ring structure that interact with the external G-quartet by π−π stacking interactions.24 The side arm substituents provide further specificity to interact with a particular G-quadruplex DNA via hydrogen bonding and ionic interactions with the groove and loop regions.25 In this context, we attempted to develop a series of synthetically accessible derivatives containing carbazole moiety, having flat aromatic ring system that is highly suitable for quadruplex recognition.26,27 We employed Cu(I) catalyzed 1,3-dipolar azide and alkyne cycloaddition28 of carbazole alkynes 1a-d and azides 2a-b to synthesize four carbazole derivatives Cz-1 to Cz-4. Carbazole alkynes were prepared from commercially available carbazole using Sonogashira coupling as the key step (Scheme S1-S4, Supporting Information, S.I.). Carbazole alkynes 1a-c and azide 2a, containing carboxamide side chain were used to generate monotriazolyl carbazole derivatives Cz-1, Cz-2 and Cz-3 in high yields (Scheme 1). These reactions were carried out under microwave irradiation at 70 °C for 4 h using CuSO4.5H2O in the presence of Na-ascorbate in tertiary butanol and water mixture (3:1). The combined effect of the variation in carbazole core and the triazole link-up might lead to enhanced specificity towards a particular G-quadruplex.29,30 We opted carboxamide-azide 2a containing a tertiary amino group as this side chain provided better selectivity in our previous reports.31,32 For a comparison, we also prepared a small bis-triazolyl carbazole derivative Cz-4 containing two 3 ACS Paragon Plus Environment


aliphatic tertiary amine side chains that lack the extra benzene ring as in carboxamide side chains (Scheme 1). Cz-4 was prepared from alkyne 1d and azide 2b under similar reaction conditions. Compounds Cz-1, Cz-2 and Cz-3 are mono-triazolyl carbazole derivatives containing a single carboxamide side chain.

Scheme 1. Synthesis of carbazole derivatives.


Page 4 of 30

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Screening of carbazole derivatives using FRET based melting analysis. The FRET based melting analysis33 was performed to monitor the ability of carbazole alkynes 1a-d and triazole products Cz-1 to Cz-4 to stabilize several oncogenic promoter and telomeric G-quadruplexes. In this study, we have used dual labelled G-quadruplex DNAs (c-MYC, c-KIT, k-RAS, BCL2 and hTELO) and duplex (ds) DNA. The stabilization potential of these ligands was determined by monitoring the difference in the melting temperature (∆Tm) of the DNAs with and without ligands. At a ligand concentration of 1 µM, Cz-1, Cz-2 and Cz-4 exhibited high ∆Tm values for G-quadruplexes in comparison to ds-DNA (Table 1, Figure S1-S3, Supporting Information, S.I.). Bromo derivative Cz-3 non-specifically stabilized all the investigated quadruplexes as well as duplex DNA with high ∆Tm values. Notably, alkynes 1a-d could not stabilize quadruplexes and duplex indicating the importance of side chain for recognition of DNA structures. Thus, Cz-1, Cz-2 and Cz-4 were selected for further biophysical evaluation. The concentration dependent melting experiments displayed that these compounds showed high stabilization potential values for c-KIT and c-MYC G-quadruplexes over other quadruplexes and ds-DNA (Figure 1a). Among these three compounds, mono-triazolyl carbazole Cz-1 exhibited a temperature difference (∆Tm) of 15.8 ± 0.5 °C (i.e a Tm of 94 °C, maximum that could be determined using this method) for the c-MYC at 1 µM ligand concentration. It also showed a ∆Tm value of 29.02 ± 0.6 °C (i.e a Tm of 94 °C) for the c-KIT at 3 µM. In contrast, high amounts of Cz-1 were essentially required to reach the maximum stabilization potential for other G-quadruplex DNAs. Aldehyde substituted mono-triazolyl carbazole Cz-2 exhibited a Tm of 93 °C (maximum that could be determined using this method) for c-MYC at 2 µM and for c-KIT at 5 µM concentration. Bis-triazolyl carbazole Cz-4 also showed highest stabilization for c-MYC and c-KIT quadruplexes at a concentration of 2.2 µM and 3.7 µM, respectively. The normalized stabilization potential values 5 ACS Paragon Plus Environment


Page 6 of 30

indicate that Cz-1 showed a preference for c-MYC G-quadruplex compared to other DNAs (Figure S2-S5, S.I.). The FRET competition assay shows that the most potent stabilizer Cz-1 did not significantly alter the ∆Tm of c-MYC in the presence of 50 equiv. excess of duplex and calf thymus (CT) DNA indicating its excellent selectivity for MYC G-quadruplex compared to dsDNA (Figure S4-S5, S.I.).

Figure 1. (a) Thermal shift profiles of quadruplexes (200 nM) and ds-DNA (200 nM) upon interaction with Cz-1, Cz-2 and Cz-4; in 60 mM potassium cacodylate, pH 7.4. (black)- c-KIT, (green)- k-RAS, (violet)-BCL2, (yellow)- h-TELO, (red)- c-MYC, (blue)- ds-DNA;


(b)

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fluorimetric titration spectra of Cz-1, Cz-2 and Cz-4 (0.5 µM) with 0 –2 equivalent of c-MYC, cKIT and ds-DNA in 100 mM Tris-HCl, 100 mM KCL buffer at pH 7.4. Cz-1 efficiently binds to c-MYC promoter G-quadruplex in vitro. The binding affinities of these carbazole derivatives for biologically important G-quadruplex DNAs were determined using fluorescence spectroscopy.22,34 As shown in Figure 1b, distinct fluorescence response of the carbazole derivatives were observed upon incremental addition of quadruplex DNAs. The emission spectra of Cz-1 displayed two emission peaks at 360 nm and 440 nm, when excited at 280 nm (λex = 280 nm) in 100 mM Tris-HCl containing 100 mM KCl buffer, pH 7.4. Similarly, Cz-2 exhibited two emission peaks at 350 nm and 430 nm (λex = 280 nm). An emission maximum at 387 nm was observed for the bis triazolyl derivative Cz-4 (λex = 280 nm). Upon titrating with c-MYC G-quadruplex (0-2 equivalent), the fluorescence intensity of mono-triazolyl carbazole Cz-1 (0.5 µM) at 360 nm was not altered significantly while a 5-fold enhancement of fluorescence intensity at 440 nm was observed along with a red shift (14–20 nm). However, Cz-1 exhibited nearly 2-fold increase in fluorescence intensity at 440 nm upon addition of 2 equivalents of c-KIT, k-RAS and BCL2 quadruplexes (Figure S6, S.I.). The apparent dissociation constant (Kd) of carbazole derivatives were determined from the typical F/F0 vs [DNA] plots. The Kd value calculated for Cz-1 with c-MYC was 0.21 µM, indicating a 2-4 fold binding preference over c-KIT (Kd = 0.42 µM), k-RAS (Kd = 0.77 µM) and BCL2 (Kd = 0.58 µM) quadruplexes (Table 1). In contrast, it showed very weak fluorescence changes upon addition of h-TELO and ds-DNA. It is interesting to observe that the fluorescence intensity of Cz-1 was not changed even after addition of 10 equivalent of CT-DNA (Figure S7, S.I.). Jobs plot analysis from the fluorescence titration indicates that Cz-1 binds with c-MYC quadruplex with a 1:1 stoichiometry (Figure S8, S.I.). In comparison to Cz-1, Cz-2 (0.5 µM) showed weak binding 7 ACS Paragon Plus Environment


Page 8 of 30

affinities for c-MYC (Kd = 3.19 µM) and BCL2 (Kd = 4.66 µM) quadruplexes, while it showed high affinity for duplex DNA (Kd = 1.55 µM). Negligible difference in fluorescence maxima of Cz-2 were observed after addition of c-KIT, k-RAS and h-TELO quadruplexes (0-2 equivalent). Interestingly, bis-triazolyl carbazole Cz-4 (0.5 µM) displayed a ~3-fold decrease in the emission maximum (λem = 387 nm) upon gradual addition of c-MYC G-quadruplex (0-2 equivalent, Kd = 0.52 µM), indicating a differential binding mode of Cz-4. The fluorescence intensity of Cz-4 was also decreased (1.5 to 2 fold) upon addition of BCL2, k-RAS and h-TELO quadruplexes (Figure S6, S.I.). Moreover, we observed negligible spectral changes of Cz-4 upon gradual addition of cKIT G-quadruplex and duplex DNA. These results demonstrate that Cz-1 and Cz-4 show a binding preference for the c-MYC G-quadruplex compared to ds-DNA and other investigated quadruplexes. Table 1. ∆Tm values (at 1 µM drug concentration) and dissociation constant (KD) values of Cz-1, Cz-2 and Cz-4 for quadruplexes and ds-DNA. ∆Tm [°C]

Kd [µM]

DNA

Cz-1

Cz-2

Cz-4

Cz-1

c-MYC

15.8 ± 0.5

8.9 ± 0.6

9.46 ± 0.4

0.21 ± 0.1

c-KIT

28.83 ± 1.4

23.86 ± 1.0

28.83 ± 1.4

0.42 ± 0.2

KRAS

23.87 ± 0.6

12.97 ± 0.2

22.87 ± 0.7

0.77 ±

0.1

5.37

±

0.7

0.69

±

0.3

BCL2

9.52 ± 0.4

2.88 ± 0.1

12.52 ± 0.6

0.58

0.1

4.66

±

0.9

1.04

±

0.3

h-TELO

21.12 ± 1.1

0.82 ± 0.4

20.12 ± 1.0

##

0.62

±

0.2

ds-DNA

5.55 ± 0.2

0.94 ± 0.1

5.75 ± 0.1

##

±

Cz-2 3.19

±

Cz-4 0.1

0.52

##

##

## 1.55

±

± 0.3

0.3

##

##: not determined since there are negligible changes in fluorescence intensity upon addition up to 8 ACS Paragon Plus Environment

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6 equivalent quadruplex DNA. Tm of DNA sequences: c-MYC – 77 ± 1 °C, c-KIT – 54 ± 1 °C, KRAS – 47 ± 1 °C, BCL2 – 70 ± 1 °C, h-TELO – 54 ± 1 °C, dsDNA – 61 ± 1 °C.

Circular Dichroism (CD) spectroscopic studies revealed that Cz-1 did not cause any conformational transition of c-MYC quadruplex (Figure S9, S.I.). In contrast, Cz-4 gradually decreased the CD signal with a small spectral shift (from 265 nm to 259 nm), suggesting that Cz4 may stabilize a different topology of c-MYC quadruplex. These observations confer that Cz-1 is the best c-MYC DNA binding ligand among these carbazole derivatives. Then we performed biological characterization and evaluation of ligands Cz-1, Cz-2 and Cz-4 in cellular systems. Gene expression analysis, qRT-PCR, FACS and confocal microscopy experiments were carried out. Cervical carcinoma cell line Hela was opted as a model biological system, as it exhibits high levels c-MYC expression.

Cz-1 localizes into the cells and causes apoptosis. Cellular permeabilization and localization of ligands are important for their biological activity. The localization of the carbazole derivatives was examined using Confocal laser scanning microscopy in Hela cells. Cz-1, Cz-2 and Cz-4 were treated in Hela cells at 1 µM concentration for 24 h. Then cells were fixed and mounted with NucRed Live-647 for co-staining the nucleus. The images revealed that these compounds could efficiently enter and localize into the cells (Figure 2). Cz-1 and Cz-2 mainly localized into the nucleus and gave blue fluorescence response, though these compounds were also found to be present in the cytoplasm to some extent. Cz-4 localizes into the nucleus as well as in the cytoplasm.



Figure 2. Confocal images of Hela cells showing nuclear localization of compound Cz-1, Cz-2 and Cz-4. Scale bar 10 µm. Nucred Live-647 is costained for visualiziation of the nucleus.

XTT assay was then performed to examine the growth inhibitory activity of these compounds in Hela cells. Compound Cz-1 inhibited cell proliferation most efficiently with an IC50 value of 3.4 ± 0.1 µM at 24 h of treatment. In comparison, Cz-2 and Cz-4 exhibited higher IC50 values in Hela cells (11.4 µM and 5.0 µM, respectively) (Figure 3a, Table 2). Similar effect was also observed in HCT116 colon cancer cells. Interestingly, Cz-1 did not significantly inhibit the growth of normal kidney epithelial cells (NKE) even at 30 µM. The bright field microscopic images supported the above observation (Figure 3b). The Cz-1 treated cells showed cell death 10 ACS Paragon Plus Environment

Page 10 of 30

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


whereas Cz-2 treated cells retained their morphology at 3 µM dose. Cz-4 treated cells lost the shape at same concentration. Table 2. . Antiproliferative activities of Cz-1, Cz-2 and Cz-4

IC50 in µM (24 h) Ligands

Hela

HCT116

NKE

Cz-1

3.4

3.2

> 30

Cz-2

11.4

9.5

> 30

Cz-4

5.01

5.3

> 30

The apoptosis assay was further carried out using Annexin V-FITC and propidium iodide (PI) staining in Hela cells (Figure 3c). Cells were exposed to 3 µM concentration of each of the ligands for 24 h and then analyzed using flow cytometry. Cz-1 treated cells showed a significant increase in apoptotic (27.9%) and necrotic (8%) populations compared to control cells. Cz-2 did not show major changes in apoptotic cell population. Cz-4 caused 19.3% cell death of total cell population via apoptosis and necrosis. Next, the distribution of cell cycle was examined by PI mediated FACS analysis. G1 cell population was increased in Cz-1 treated cells, (Figure 3d), whereas a marginal increase in G2/M phase population was observed in Cz-4 treated cells at the same dose. The distribution of the cell population remained unchanged in Cz-2 treated cells. Together, these observations suggest that Cz-1 localizes mainly into the nucleus and induces apoptosis exhibiting a low IC50 value in Hela cells with concomitant arrest in G1 cell cycle.



Figure 3. Cellular effect of the compounds in Hela cells; (a) XTT assay shows only Cz-1 has the significant antiproliferative activity in Hela cell lines after 24 h treatment; (b) bright field microscopic images of ligand treated Hela cells show differential morphological changes at 3 µM concentration of each compound; (c) compound Cz-1 induces significant apoptosis after 24 h treatment (3 µM), as seen by the FACs analysis of FITC-Annexin V/PI stained cells. The average result is plotted in bar diagram; (d) cell cycle analysis with a small increase of G1 phase is observed in Cz-1 treated cells. The avarage result is plotted in bar diagram.

Cz-1 transcriptionally downregulates c-MYC. We further explored the functional effect of Cz1 on the transcription of c-MYC gene. Westen Blot results illustrated that Cz-1 could reduce the c-MYC protein levels relative to the control cells (~ 21% at 1 µM and ~ 51% at 3 µM) (Figure 4a). The expression of control housekeeping gene, GAPDH was not affected by Cz-1. 12 ACS Paragon Plus Environment

Page 12 of 30

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Additionally, qRT-PCR analysis revealed that c-MYC mRNA transcription was gradually decreased upon Cz-1 exposure (Figure 4b). In order to confirm the mechanism of G-quadruplex dependent suppression of c-MYC, CA46 exon specific assay was further performed.35, 36

Figure 4. Cz-1 transcriptionally downregulates c-MYC; (a) and (b) concentration dependent downregulation of c-MYC protein and mRNA levels in Hela cells upon exposure to compounds for 24 h; (c) Exon specific assay in CA46 cells upon Cz-1 treatment for 24 h shows that Cz-1 causes G-quadruplex mediated down-regulation of c-MYC. CA46 is a Burkitt’s lymphoma cell line, where a translocation occurs between the c-MYC gene in chromosome 8 and immunoglobulin gene (IgH heavy chain) present in chromosome 14. As a result, the c-MYC expression is differentially regulated from nontranslocated and translocated allele. Therefore, exon 2 of c-MYC gene is translocated into the IgH gene and exon 1 remains under the control of c-MYC promoter. As a result, any quadruplex binding ligand



would reduce exon 1 expression but exon 2 expression would remain unaffected. The expressions of exon 1 and 2 were quantified individually to evaluate the quadruplex stabilization ability of carbazole derivatives. We found that Cz-1 could significantly reduce exon 1 expression (~ 60% at 1 µM and ~ 80% at 3 µM) in a dose dependent manner compared to exon 2 expression. These results indicate that the transcriptional downregulation of c-MYC is due to Cz1 mediated stabilization of G-quadruplex structure (Figure 4c). No significant reduction in the expression of both the exons was observed in Cz-2 and Cz-4 treated CA46 cells (Figure S10, S.I.). Ligand mediated G-quadruplex formation was further corroborated by immunocytochemistry using G-quadruplex binding BG4 antibody.37 BG4 stained cells showed punctuated nuclear dots (red in color) indicating that G-quadruplexes are present in the nucleus (Figure 5). Interestingly, Cz-1 treated cells showed more number of BG4 foci compared to control cells (Figure 5 and Figure S11, S.I.). This observation illustrates that Cz-1 co-localizes with BG4 in the quadruplex forming region. In Cz-2 and Cz-4 treated cells, less numbers of quadruplex structures with low intensity were found compared to Cz-1 treated cells (Figure S12, S.I.). These findings confirm that Cz-1 stabilizes and induces the formation of quadruplexes in cancer cells.


Page 14 of 30

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Immuno-cytochemistry with BG4 antibody shows colocalization of quadruplex DNA and Cz-1. Scale 10 µm. Structure-activity relationship. The heteroaromatic carbazole ring system could stack upon Gquartets of quadruplexes using π-π interactions.31 The presence of a triazole motif and an aromatic carboxamide group in Cz-1, Cz-2, Cz-3 would further facilitate stacking interactions with the nuclear bases of quadruplex structures. The cationic amine side chains could interact with the phosphate backbone of DNA structures due to electrostatic interactions. Carbazole Cz-1 showed the highest binding affinity towards c-MYC compared to Cz-2 containing an electron withdrawing aldehyde group. Cz-3 having a bulky bromo substituent showed non-specific binding to all the DNA structures. Bis-triazolyl carbazole Cz-4 lacking the N-alkylated side chain as well as aromatic carboxamide groups showed comparatively lower binding affinity for quadruplexes than Cz-1. However, Cz-4, due to the presence of two triazolyl amine side chains showed higher affinity for the c-MYC compared to Cz-2. In our previous report, we have synthesized a few bis-triazolyl carbazole derivatives, amongst which BTC-f

has been

established as a promising ligand that stabilizes c-MYC quadruplex and reduces c-MYC expression in HepG2 cells.31 Further experiments like exon specific assay showed that bis15 ACS Paragon Plus Environment


triazolyl carbazole derivative BTC-f was not able to discriminate between exon 1 and exon 2 transcription and thus indicated that the c-MYC repression by these compounds may be due to synergistic effect of G-quadruplex stabilization and other transcription machinery. In the present study, the mono triazolyl carbazole Cz-1 is smaller in size, contains a single triazolyl ring with a single carboxamide side chain and it exerts better biological effect in cancer cells compared to BTC-f. Cz-1 also shows lower IC50 value than BTC-f and localizes easily into the cancer cells inducing significant apoptosis. Our results reveal that the potent G-quadruplex binding ligand Cz-1 efficiently downregulates the c-MYC expression by inhibiting cell growth in cancer cells.

CONCLUSION We have designed and synthesized a series of carbazole derivatives and evaluated their ability to interact with quadruplexes by different biophysical studies. These studies suggest that carbazole derivative Cz-1 shows significant quadruplex-vs.-duplex DNA selectivity and preferentially binds to the c-MYC quadruplex DNA. Western blot, qRT-PCR and CA46 exon specific assays reveal that the downregulation of c-MYC mainly arises due to the binding of Cz-1 with its promoter quadruplex. Moreover, Cz-1 localizes into the nucleus and induces quadruplex formation as visualized by G-quadruplex binding BG4 antibody staining. The prevalence of quadruplex forming sequences in the promoter region of various oncogenes established them as attractive drug targets.1-4 Several studies have already been reported on quadruplex targeting molecules, however, the biological evaluation of many ligands is not yet fully evaluated. Moreover, clinically approved G-quadruplex stabilizing drug is still unavailable. The present study discovers an easily modulable new carbazole scaffold that shows high quadruplex binding affinity and potent anticancer activity. 16 ACS Paragon Plus Environment

Page 16 of 30

Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION Chemistry. Column chromatography was carried out using silica gel (100-200 mesh). TLC was performed using Kieselgel 60 F254 plates and spots were visualized by UV. 1H NMR spectra were measured on 400 and 500 MHz instruments and 13C spectra were measured on 100 and 125 MHz instruments using deuterated solvents. Chemical shifts are reported in parts per million (ppm) and are referred to the residual solvent peak. The following notations are used: singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m); broad (br). Coupling constants are given in Hertz (Hz) and are denoted as J. Mass spectra were recorded on a Q-TOF (ESI) spectrometer by positive mode electrospray ionization. The final compounds showed ≥ 95% purity. General procedure for the azide and alkyne cycloaddition (GP1). To a solution of alkyne (1a-d) in tertiary butanol and water (3:1) was added and CuSO4•5 H2O and Na-ascorbate. The mixture was stirred for 10 min followed by the addition of the corresponding azide (2a-b). The resulting mixture was subsequently heated with stirring in a microwave vial for 4 h at 70 °C. Then it was cooled to room temperature, solvent was concentrated under reduced pressure. The residue was purified by column chromatography (from 100% CH2Cl2 to 10:1 CH2Cl2/MeOH to 10:1:0.5 CH2Cl2/MeOH/NH4OH) to obtain the triazole products Cz-1 to Cz-4 as yellow liquids. Preparation of Cz-1. Using the GP1, 1a (50 mg, 0.181 mmol), 1:2 mixture of t-BuOH/H2O (4 mL), CuSO4·5H2O (4.52 mg, 0.0181 mmol), Na-ascorbate (3.59 mg, 0.0181 mmol) and 2a (67.22 mg, 0.169 mmol) afforded 72 mg of Cz-1 (76%). 1H NMR (500 MHz, DMSO-d6): δ 9.38(s, 1H), 8.74(s, 1H), 8.67 (s, 1H), 8.21 (d, 1H, J = 8.0), 8.10 (s, 4H), 8.06 (d, 1H, J = 8.6), 7.74 (d, 1H, J = 8.6), 7.63 (d. 1H, J = 8.0), 7.54 (d, 1H, J = 8.6), 7.49 (t, 1H, J = 8.6), 4.45 (t, 2H, J = 6.8), 3.33 (t, 2H, J = 6.8), 2.35( t, 2H, J = 6.8), 2.23 (t, 2H, J = 6.8), 2.20-2.15 (m, 12H), 1.94 (t, 2H, J = 6.8), 1.71 (t, 2H, J = 6.8).

13

C NMR (100 MHz, DMSO-d6): δ 165.1, 148.7, 140.6, 17

ACS Paragon Plus Environment


140.1, 138.4, 134.3, 128.9, 128.6, 126.1, 123.6, 122.4, 122.1, 120.9, 120.4, 119.4, 118.6, 117.3, 112.6, 109.8, 56.7, 56.0, 44.9, 37.7, 37.4, 26.8, 26.4. (ESI) calculated for [C31H38N7O] 524.3138, Found 524.3129. Preparation of Cz-2. Using the GP1, 1b (100 mg, 0.33 mmol), CuSO4·5H2O (8.2 mg, 0.033 mmol), Na-ascorbate (6.5 mg, 0.033 mmol), 3:1 t-BuOH/H2O and 2a (122.4 mg, 0.495 mmol) yielded Cz-2 (136.5 mg, 75%).1H NMR (500 MHz, DMSO-d6): δ 10.09 (s, 1H), 9.45 (s, 1H), 8.89 (s, 1H), 8.81 (s, 1H), 8.77 (s, 1H), 8.14-8.11 (m, 5H), 8.02 (d, 1H, J = 8.4), 7.84 (d, 1H, J = 8.4), 4.53 (t, 2H, J = 6.9), 3.33 (t, 2H, J = 6.9), 2.49 (2H, merged with DMSO peak), 2.31 (s, 8H), 2.19 (s, 6H), 1.99 (t, 2H, J = 6.9), 1.76 (t, 2H, J = 6.9). 13C NMR (500 MHz, DMSO-d6): δ 191.8, 165.1, 148.3, 144.0, 140.8, 138.4, 134.1, 129.0, 128.6, 126.8, 124.4, 124.0, 122.7, 122.4, 122.2, 119.3, 118.9, 117.7, 110.6, 110.1, 56.0, 55.5, 52.0, 44.6, 44.0, 39.7, 37.4, 26.0. (ESI) calculated for [C32H38N7O2] 552.3087, Found 552.3091. Preparation of Cz-3. Using the GP1, a mixture of 1c (50 mg, 0.141 mmol), 1:2 mixture of tBuOH/H2O (4 mL), CuSO4·5H2O (3.52 mg, 0.0141 mmol) and Na-ascorbate (2.79 mg, 0.0141 mmol) and 2a (41.84 mg, 0.169 mmol) yielded Cz-3 (64.6 mg, 76%). 1H NMR (500 MHz, DMSO-d6): δ 9.38 (s, 1H), 8.81 (s, 1H), 8.78 (s, 2H), 8.44 (s, 1H), 8.13-8.08 (m, 5H), 7.77 (d, 1H, J = 8.4), 7.63 (d, 1H, J = 8.4), 4.46 (t, 2H, J = 6.7), 3.35 (t, 2H, J = 6.7), 2.58 (t, 2H, J = 7.6), 2.37-2.32 (m, 8H), 2.22 (s, 6H), 1.96 (t, 2H, J = 6.7), 1.78 (t, 2H, J = 7.6). 13C NMR (100 MHz, DMSO-d6): δ 165.1, 148.5, 140.3, 139.2, 138.4, 134.2, 129.0, 128.4, 124.3, 124.0, 122.9, 121.5, 121.4, 119.3, 118.7, 117.8, 111.6, 111.2, 110.1, 56.0, 55.5, 44.5, 44.0, 40.8 (merged with DMSOd6 peak), 37.3, 26.0, 25.9. HRMS (ESI) calculated for [C31H37BrN7O] 602.2243, Found 602.2284.


Page 18 of 30

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation of Cz-4. Using the GP1, a mixture of 1d (100 mg, 0.464 mmol), CuSO4·5H2O (11.6mg, 0.0465 mmol), Na-ascorbate (9.2 mg, 0.0465 mmol), 3:1 t-BuOH/H2O, 2b (148.7 mg, 1.16 mmol) yielded Cz-4 (155.7 mg, 71%). 1H NMR (500 MHz, DMSO-d6): δ 8.66 (s,2H), 8.56 (s, 2H), 7.91 (d, 2H, J = 8.2), 7.56 (d, 2H, J = 8.2), 4.44(t, 4H, J = 6.3), 2.25 (t, 4H, J = 6.3), 2.16 (s, 12H), 2.05 (t, 4H, J = 6.3),

13

C NMR (100 MHz, DMSO-d6): δ 147.5, 139.8, 123.6, 123.4,

122.8, 121.9, 120.4, 120.2, 117.1, 116.9, 111.5, 55.7, 47.7, 45.1, 27.7. HRMS (ESI) calculated for [C26H34N9] 472.2937, Found 472.2897. FRET based melting experiments: Compounds were dissolved in Milli Q water and 1 µM concentration of each carbazole derivatives was prepared. Labeled DNA oligonucleotide sequences were diluted in 60 mM potassium cacodylate buffer, pH 7.4. DNA sequences used in the FRET experiment are given as follows. c-MYC: 5′-FAM- TGAG3TG3TAG3TG3TA2-TAMRA-3′ , c-KIT: 5′ -FAM- G3AG3CGCTG3AG3AG3-TAMRA-3, BCL2: 5′-FAM- AG4CG2GCGCG3AG2AAG5CG3AGCG4CTG-TAMRA-3′, KRAS: 5′-FAM- AG3CG2TGTG3A2GAG3A2GAG5AG2-TAMRA-3′, h-TELO: 5′-FAM- T2G3T2AG3T2AG3T2AG3A-TAMRA-3′, dsDNA: 5′-FAM- C2AGT2CGTAGTA2C3-HEG-G3T2ACTA CGA2 CTG2-TAMRA-3′

All the DNAs (400 nM) were annealed at 95 °C for 5 min followed by cooling. The experiment was executed in 96-well plates using 200 nM dual labelled DNA with 1uM each of the eight compounds. Increasing concentration of Cz-1, Cz-2 and Cz-4 (0-10µM) were added to 200 nM of each of the DNA sequences. Measurements were recorded in an excitation at 483 nm and a detection at 533 nm using a LightCycler® 480-II RT-PCR system (Roche). 19 ACS Paragon Plus Environment


Fluorimetric titration assay: Fluorescence spectra were recorded in Horiba Jobin Yvon Fluoromax 3 instrument at 25 °C in a 10 mm path-length quartz cuvette. The experiments were carried out by gradual addition of the DNA into 500 nM ligand solution in 100 mM Tris-HCl, 100 mM KCl buffer. The DNA sequences used in the titration are given in the Supporting Information.

Cell culture and Cell cytotoxicity Human cervical cancer cell line HeLa, were procured from NCCS, Pune, India. Human colorectal carcinoma cell line HCT116 was gifted by Dr susanta Rochoudhry, IICB and normal kidney epithelial (NKE) cell line was a kind gift from Dr. Prosenjit Sen, IACS. The human cervical cancer cells (Hela), human colon carcinoma cells (HCT116) were cultured in DMEM (high glucose), 10% FBS at pH 7.4. NKE cells were cultured in RPMI 1640 (GIBCO) with 10% FBS. Cells were grown in 5% CO2 level and 37 °C atmosphere. 104 numbers of Hela and HCT116 cells were plated and exposed to various concentrations of Cz-1, Cz-2 and Cz-4 for 24 h. XTT solution was added to each well 4 h at 37 °C. Absorbance of the samples were recorded in a micro plate reader at 450 nm. The background absorbance was subtracted from each data. The percentage of viable cells was calculated by the following equation: (%) Viable cells =

Absorbance of treated cells Absorbance of untreated cells

× (100)

Western Blot Analysis Hela cells were treated with each of the carbazole derivatives for 24 h in respective doses and lysed in TritonX100 lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA in 0.5% Triton X100). Total proteins were estimated by Folin-Lowry method. SDS gel electrophoresis were 20 ACS Paragon Plus Environment

Page 20 of 30

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


performed with 50 µg of each protein sample and transferred to a nitrocellulose membrane (Millipore MA, USA) using semi dry TRANS-BLOTR followed by incubation with primary antibodies against GAPDH and c-MYC (Invitrogen) at 4 °C for overnight. Appropriate enzyme conjugated secondary antibody was used and signals were detected using respective enzyme substrate (MP). The comparative intensities of the protein bands were determined and calculated using ImageJ software and the GAPDH normalized band intensity of c-MYC protein was plotted in microsoft excel.

RNA extraction and RT-PCR Hela cells were seeded and incubated with various concentrations of Cz-1, Cz-2 and Cz-4 for 24 h and mRNA was prepared from treated and untreated cells using the Trizol kit as per the manufacturer’s protocol (Thermo Fisher Scientific, catalogue number 15596018). cDNA Reverse Transcription Kit (Applied Biosystems, catalogue number 4368814) was used to prepare the cDNA library. The real-time PCR was performed on Roche LightCycler 480 by using SYBR Premix (Applied Biosystems), according to the manufacturer’s protocol. The primers used for the qRT-PCR analyses are provided as follows. c-MYC (Fwd primer): 5′-CTGCGACGAG2AG2AG2ACT-3′ c-MYC (Rev primer): 5′-G2CAGCAGCTCGA2T3CT2-3′ GAPDH (Fwd primer): 5′-GACG2C2GCATCT2CT2GT-3′ GAPDH (Rev primer): 5′-CACAC2GAC2T2CAC2AT4-3′ Finally, the relative mRNA level of c-MYC was calculated using the arithmetic calibrator (2−∆∆CT). The difference in c-MYC mRNA level was expressed as fold changes.



FACS analysis of apoptosis and cell cycle Apoptosis assay was carried out using the manufacturer’s protocol. Briefly, 106 Hela cells were exposed to 3 µM concentration of carbazole derivatives for 24 h in fresh growth medium at 37 °C and 5% CO2. Cells were then harvested by mild trypsinization, washed with 1X PBS and the cell pellet was resuspended in 1 X Anexin V binding buffer and then treated with FITC tagged Annexin V and Propidium Iodide (PI). Each sample was analyzed immediately using fluorescence–activated cell sorting (BD-LSR-FACS) analysis (BD Biosciences, Mountain View, CA, USA) after incubation for 5 min at room temperature. Approximately 104 Hela cells were analyzed for each sample. For the cell cycle analysis, cells were incubated with compounds Cz1, Cz-2 and Cz-4 at 3 µM for 24 h. After treatment, cells were trypsinized and fixed with 70% ethanol (ice-cold) at 4 °C for overnight. Cells were then resuspended in PBS containing 100 µg/Ml propidium iodide. The cell cycle analysis was performed using BD-LSR-FACS apparatus. Exon-specific assay CA46 cells were cultured and exposed to Cz-1 at increasing concentration for 24 h, washed in PBS twice and the mRNA was prepared using the trizol kit as per the manufacturer’s protocol. High capacity reverse transcription kit was used to prepare the cDNA library. The cDNA was then amplified using Power SYBR Green PCR Master Mix with the exon 1 and exon 2 primers using Light Cycler 480 II system. The relative mRNA expression was normalized by the GAPDH expression. Exon 1 and exon 2 primer sequences are as follows. Exon 1 Fwd primer: 5′-CACGA3CT3GC3ATAGC-3′ Exon 1 Rev primer: 5′-GCA2G2AGAGC2T3CAGAG-3′ Exon 2 Fwd primer: 5′-C3TCA2CGT2AGCT2CAC2-3′ 22 ACS Paragon Plus Environment

Page 22 of 30

Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Exon 2 Rev primer: 5′-AGCAGCTCGAAT3CT2C2A-3′

Immunocytochemistry with BG4 antibody Cells were seeded in 6 well cell culture plate containing cover slips and incubated with 1 µM of each Cz-1, Cz-2 and Cz-4 inCO2 (5%) incubator at 37 °C for 24 h. Cells were washed with PBS thrice and fixed in ice-cold 1:1 aceto-methanol. It was finally stained with NucRed 647 live. For immunocytochemistry, cells were first fixed with acetomethanol, blocked with 3% BSA in PBS and incubated overnight with BG4 antibody. Alexafluor-647 conjugated secondary antibody (Invitrogen) was used and finally cells were mounted with anti-fed solution. Images were taken in confocal laser scanning microscope. The raw data was analyzed in Flouview FV-1000 V4.1 software.

ASSOCIATED CONTENT Supporting Information Supporting Information. Additional experimental results; 1H NMR, 13C NMR and HPLC spectra, CD spectroscopic data and molecular modeling analysis. Molecular formula strings and additional data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: +91 33 2473 4971, Ext 1405. E-mail: [email protected].



Author Contributions ‡ Equal contribution by the authors. Notes The authors declare no competing financial interests. ACKNOWLEDGMENT We thank Department of Science and Technology (DST) and Deaprtment of Biotechnology (DBT), India for funding. JD thanks DST for a SwarnaJayanti fellowship. TD thanks DBT for a research fellowship. DP and PS thank DST for their fellowship. The authors thank Mr Tanmoy Dalui for FACS analysis, Indian Institute of Chemical Biology, Kolkata, Mr Arijit Pal for confocal microscopy, DBT-IPLS unit, University of Calcutta. REFERENCES (1) Neidle, S. (2017) Quadruplex nucleic acids as targets for anticancer therapeutics. Nat. Rev. Chem. 1, 0041. (2) Hurley, L. H., Wheelhouse, R. T., Sun, D., Kerwin, S. M., Salazar, M., Fedoroff, O. Y., Han, F. X., Han, H. Y., Izbicka, E. and Von Hoff, D. D. (2000) G-quadruplexes as targets for drug design. Pharmacol. Therapeut. 85, 141-158. (3) Han, H. Y. and Hurley, L. H. (2000) G-quadruplex DNA: a potential target for anti-cancer drug design. Trends in Pharmacol. Sci. 21, 136-142. (4) Balasubramanian, S. and Neidle, S. (2009) G-quadruplex nucleic acids as therapeutic targets. Curr. Opin. Chem. Biol. 13, 345-353. (5) Bochman, M. L., Paeschke, K. and Zakian, V. A. (2012) DNA secondary structures: stability and function of G-quadruplex structures. Nat. Rev. Genet. 13, 770-780. 24 ACS Paragon Plus Environment

Page 24 of 30

Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Neidle, S. (2016) Quadruplex nucleic acids as novel therapeutic targets. J. Med. Chem. 59, 5987-6011. (7) Huppert, J. L. and Balasubramanian, S. (2005) Prevalence of quadruplexes in the human genome. Nucleic Acids Res. 33, 2908-2916. (8) Murat, P. and Balasubramanian, S. (2014) Existence and consequences of G-quadruplex structures in DNA. Curr. Opin. Genet. Dev. 25, 22-29. (9) Chambers, V. S., Marsico, G., Boutell, J. M., Di Antonio, M., Smith, G. P. and Balasubramanian, S. (2015) High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 33, 877-881. (10) Simonsson, T., Pecinka, P. and Kubista, M. (1998) DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res. 26, 1167-1172. (11) Qin, Y. and Hurley, L. H. (2008) Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie 90, 11491171. (12) Balasubramanian, S., Hurley, L. H. and Neidle, S. (2011) Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat. Rev. Drug Discovery 10, 261−275. (13) Zahler, A. M., Williamson, J. R., Cech, T. R. and Prescott, D. M. (1991) Inhibition of telomerase by G-quartet DNA structures. Nature 350, 718-720. (14) Siddiqui-Jain, A., Grand, C. L., Bearss, D. J. and Hurley, L. H. (2002) Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. P. Natl. Acad. Sci. 99, 11593-11598. (15) Seenisamy, J., Bashyam, S., Gokhale, V., Vankayalapati, H., Sun, D., Siddiqui-Jain, A., Streiner, N., Shin-ya, K., White, E., Wilson, W. D., et al. (2005) Design and synthesis of an 25 ACS Paragon Plus Environment


expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure. J. Am. Chem. Soc. 127, 2944–2959. (16) Ou, T. M., Lin, J., Lu, Y. J., Hou, J. Q., Tan, J. H., Chen, S. H., Li, Z., Li, Y. P., Li, D., Gu, L. Q., et al. (2011) Inhibition of cell proliferation by quindoline derivative (SYUIQ-05) through its preferential interaction with c-myc promoter G-Quadruplex. J. Med. Chem. 54, 5671–5679. (17) Felsenstein, K. M., Saunders, L. B., Simmons, J. K., Leon, E., Calabrese, D. R., Zhang, S., Michalowski, A., Gareiss, P., Mock, B. A. and Schneekloth Jr, J. S. (2015) Small molecule microarrays enable the identification of a selective, quadruplex-binding inhibitor of MYC expression. ACS Chem. Biol. 11, 139-148. (18) Chauhan, A., Paul, R., Debnath, M., Bessi, I., Mandal, S., Schwalbe, H. and Dash, J. (2016) Synthesis of fluorescent binaphthyl amines that bind c-MYC G-quadruplex DNA and repress cMYC expression. J. Med. Chem. 59, 7275–7281. (19) Zeng, D. Y., Kuang, G. T., Wang, S. K., Peng, W., Lin, S. L., Zhang, Q., Su, X. X., Hu, M. H., Wang, H. G., Tan, J. H., et al. (2017) Discovery of novel 11-triazole substituted benzofuro[3,2-b]quinolone derivatives as c-myc G-Quadruplex Specific Stabilizers via Click Chemistry. J. Med. Chem. 60, 5407–5423. (20) Seenisamy, J., Rezler, E. M., Powell, T. J., Tye, D., Gokhale, V., Joshi, C. S., Siddiqui-Jain, A. and Hurley, L. H. (2004) The dynamic character of the G-quadruplex element in the c-MYC promoter and modification by TMPyP4. J. Am. Chem. Soc. 126, 8702-8709. (21) Liu, J. N., Deng, R., Guo, J. F., Zhou, J. M., Feng, G. K., Huang, Z. S., Gu, L. Q., Zeng, Y. X. and Zhu, X. F. (2007) Inhibition of myc promoter and telomerase activity and induction of delayed apoptosis by SYUIQ-5, a novel G-quadruplex interactive agent in leukemia cells. Leukemia 21, 1300-1302. 26 ACS Paragon Plus Environment

Page 26 of 30

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(22) Alzeer, J., Vummidi, B. R., Roth, P. J. C. and Luedtke, N. W. (2009) Guanidinium-modified phthalocyanines as high-affinity G-quadruplex fluorescent probes and transcriptional regulators. Angew. Chem. Int. Ed. 48, 9362-9365. (23) Brooks, T. A. and Hurley, L. H. (2010) Targeting MYC expression through Gquadruplexes. Genes & Cancer 1, 641-649. (24) Monchaud, D. and Teulade-Fichou, M. P. (2008) A hitchhiker's guide to G-quadruplex ligands. Org. Biomol. Chem. 6, 627-636. (25) Collie, G. W. and Parkinson, G. N. (2011) The application of DNA and RNA Gquadruplexes to therapeutic medicines. Chem. Soc. Rev. 40, 5867-5892. (26) Maji, B., Kumar, K., Kaulage, M., Muniyappa, K. and Bhattacharya, S. (2014) Design and synthesis of new benzimidazole–carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells. J. Med. Chem. 57, 69736988. (27) Chang, C. C., Wu, J. Y., Chien, C. W., Wu, W. S., Liu, H., Kang, C. C., Yu, L. J. and Chang, T. C. (2003) A fluorescent carbazole derivative: high sensitivity for quadruplex DNA. Anal. Chem. 75, 6177–6183. (28) Rostovtsev, V. V., Green, L. G., Fokin, V. V. and Sharpless, K. B. (2002) A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599. (29) Moorhouse, A. D., Santos, A. M., Gunaratnam, M., Moore, M., Neidle, S. and Moses, J. E. (2006) Stabilization of G-quadruplex DNA by highly selective ligands via click chemistry. J. Am. Chem. Soc. 128, 15972-15973.



(30) Lombardo, C. M., Martinez, I. S., Haider, S., Gabelica, V., Pauw, E., Moses, J. E. and Neidle, S. (2010) Structure-based design of selective high-affinity telomeric quadruplex-binding ligands. Chem. Comm. 46, 9116-9118. (31) Panda, D., Debnath, M., Mandal, S., Bessi, I., Schwalbe, H. and Dash, J. (2015) A Nucleusimaging probe that selectively stabilizes a minor conformation of c-MYC G-quadruplex and down-regulates c-MYC transcription in human cancer Cells. Sci. Rep. 5, 13183. (32) Panda, D., Saha, P., Das, T. and Dash, J. (2017) Target guided synthesis using DNA nanotemplates for selectively assembling a G-quadruplex binding c-MYC inhibitor. Nat. Commun. 8, 16103. (33) De Cian, A., Guittat, L., Kaiser, M., Sacca, B., Amrane, S., Bourdoncle, A., Alberti, P., Teulade-Fichou, M. P., Lacroix, L. and Mergny, J. L. (2007) Fluorescence-based melting assays for studying quadruplex ligands. Methods 42, 183−195. (34) Grande, V., Doria, F., Freccero, M. and Würthner, F. (2017) An aggregating amphiphilic squaraine: A light‐up probe that discriminates parallel G‐quadruplexes. Angew. Chem., Int. Ed. 56, 7520–7524. (35) Brown, R. V., Danford, F. L., Gokhale, V., Hurley, L. H. and Brooks, T. A. (2011) Demonstration that drug-targeted down-regulation of MYC in non-hodgkins lymphoma is directly mediated through the promoter G-quadruplex. J. Biol. Chem. 286, 41018-41027. (36) Boddupally, P. V., Hahn, S., Beman, C., De, B., Brooks, T. A., Gokhale, V. and Hurley, L. H. (2012) Anticancer activity and cellular repression of c-MYC by the G-quadruplex-stabilizing 11piperazinylquindoline is not dependent on direct targeting of the G-quadruplex in the c-MYC promoter. J. Med. Chem. 55, 6076-6086.


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(37) Biffi, G., Tannahill, D., McCafferty, J. and Balasubramanian, S. (2013) Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 5, 182-186.



TOC


Page 30 of 30

Small Molecule Driven Stabilization of Promoter G-quadruplexes and

Recommend Documents