Discovery of a New Four-Leaf Clover-Like Ligand as a Potent c-MYC

Feb 23, 2018 - In this study, through structural modification of aryl-substituted imidazole/carbazole conjugates, a brand-new, four-leaf clover-like l...
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Cite This: J. Med. Chem. 2018, 61, 2447−2459

Discovery of a New Four-Leaf Clover-Like Ligand as a Potent c‑MYC Transcription Inhibitor Specifically Targeting the Promoter G‑Quadruplex Ming-Hao Hu,†,‡,§ Yu-Qing Wang,†,§ Ze-Yi Yu,† Lu-Ni Hu,† Tian-Miao Ou,† Shuo-Bin Chen,*,† Zhi-Shu Huang,† and Jia-Heng Tan*,† †

School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, China

‡

S Supporting Information *

ABSTRACT: Downregulating transcription of the oncogene c-MYC is a feasible strategy for cancer therapy. Stabilization of the G-quadruplex structure present in the c-MYC promoter can suppress c-MYC transcription. Thus, far, several ligands targeting this structure have been developed. However, most have shown no selectivity for the c-MYC G-quadruplex over other Gquadruplexes, leading to uncertain side effects. In this study, through structural modification of aryl-substituted imidazole/ carbazole conjugates, a brand-new, four-leaf clover-like ligand called IZCZ-3 was found to preferentially bind and stabilize the cMYC G-quadruplex. Further intracellular studies indicated that IZCZ-3 provoked cell cycle arrest and apoptosis and thus inhibited cell growth, primarily by blocking c-MYC transcription through specific targeting of the promoter G-quadruplex structure. Notably, IZCZ-3 effectively suppressed tumor growth in a mouse xenograft model. Accordingly, this work provides an encouraging example of a selective small molecule that can target one particular G-quadruplex structure, and the selective ligand might serve as an excellent anticancer agent.

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INTRODUCTION G-quadruplexes are unique four-stranded structures formed by guanine-rich nucleic acid sequences. The basic structural unit is the G-quartet, which is derived from the association of four guanines into a cyclic Hoogsteen hydrogen-bonding arrangement.1,2 G-quadruplexes are widely distributed in many important regulatory regions in the human genome,3−5 notably in telomeres and promoters of oncogenes, such as c-MYC, cKIT, BCL-2, and KRAS.6,7 It is believed that G-quadruplex structures play a significant regulatory role in many biological processes, such as DNA replication, transcription, and genome stability.8−11 Stabilization of G-quadruplexes in oncogene promoters by small molecules leads to downregulation of the expression of their target genes.6 This has developed into a new anticancer drug discovery strategy. However, recent research has revealed that large quantities of potential G-quadruplex-forming sites exist in the human genome, and among them, approximately 10,000 G-quadruplex structures are present in human chromatin, predominantly in regulatory regions,4 which provides an opportunity for a range of drug targets but also represents a potential drug selectivity problem.3,8 In biological processes, G-quadruplex structures may play beneficial roles, © 2018 American Chemical Society

but they can be detrimental to certain processes, such as progression of the replication fork. Thus, G-quadruplexes must be unfolded by helicases during replication or transcription. Highly stable G-quadruplexes (stabilized by small-molecule ligands) could act as kinetic traps that alter efficiency of replication, transcription, or duplex reannealing, which would disturb normal cellular progression and increase genome instability.10 Hence, nonspecific targeting by ligands might alter many genes modulated by G-quadruplexes, leading to unexpected side effects. In addition, it is difficult to validate the relationship between cause and effect using such ligands. Therefore, the current challenge in G-quadruplex-mediated anticancer drug development is to achieve selectivity for a particular G-quadruplex.6,8 c-MYC is well-known as an important oncogene that plays a crucial role in cell growth, proliferation, and apoptosis.12,13 Increased levels of c-MYC expression are observed in 80% of human cancer cells, and such an increase promotes tumorigenesis. The nuclease hypersensitive element III1 (NHE III1), located upstream of the c-MYC promoter, controls 80−90% of Received: November 16, 2017 Published: February 23, 2018 2447

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Figure 1. (A) Lead compound IZCZ-0 and its structural modifications. (B) Synthesis route of the five clover-like compounds. Reagents and conditions: (a) 1-methylpiperazine, K2CO3, DMSO, 90 °C; (b) p-anisidine (or not), 7-diethylaminocoumarin-3-aldehyde, NH4OAc, AcOH, reflux.

MYC G-quadruplex.33 In light of their attractive G-quadruplex selectivity and binding potency, we perceived that incorporation of the carbazole moiety into the triaryl-substituted imidazole scaffold might offer an attractive template for the design of selective c-MYC G-quadruplex ligands. Thus, we synthesized a diaryl-substituted imidazole/carbazole conjugate, IZCZ-0, with two 1-methylpiperazine side chains (Figure 1). The binding of this clover-like compound to G-quadruplexes was then evaluated using fluorescence assays. As shown in Figure S1, IZCZ-0 had considerable selectivity to Gquadruplexes compared with other types of DNAs, but unfortunately, IZCZ-0 had no ability to discriminate between different G-quadruplexes. To enhance the selectivity of IZCZ-0, we embarked on the modification of its structure. On the basis of our experience, two modification directions were pursued. One was to remove the 1-methylpiperazine groups to reduce nonspecific electrostatic interactions.30 In this direction, two compounds (IZCZ-1 and IZCZ-2) were designed and synthesized. The other direction was to introduce phenyl moieties into the center imidazole of IZCZ-0 because such a modification would reduce its binding affinities with antiparallel or hybrid G-quadruplexes, as revealed by our previous study.28 Thus, two four-leaf cloverlike compounds (IZCZ-3 and IZCZ-4) were obtained. Subsequently, these synthesized compounds were screened for selective binding to the c-MYC G-quadruplex through fluorescence spectroscopy. IZCZ-3 was identified as the most promising ligand. The detailed interactions of IZCZ-3 with the c-MYC G-quadruplex were studied using fluorescence titration, CD melting, and molecular modeling studies. The specificity of

c-MYC transcription. This region contains 27 guanine-rich bases that can fold into a G-quadruplex.14 It has been proposed that the G-quadruplex present in NHE III1 is critical for transcriptional silencing.15 c-MYC transcription can be downregulated through stabilization of the G-quadruplex structure using specific ligands. Consequently, the c-MYC proteindependent proliferation is disrupted, ultimately resulting in cancer cell growth inhibition. A number of small molecules have been reported that can stabilize the c-MYC G-quadruplex, including porphyrin derivatives,16,17 berberines,18 quindolines,19−21 carbazoles,22 naphthopyrone,23 and metal complexes.24,25 Nevertheless, few of these ligands have been demonstrated to exhibit specificity toward the c-MYC Gquadruplex over other G-quadruplex structures. However, the diversity of G-quadruplex structures could be used to enhance the selectivity of G-quadruplex ligands.26 Therefore, we were greatly interested in seeking a ligand that can selectively target the c-MYC G-quadruplex. Many small molecules are currently available that stabilize Gquadruplexes, and they can be used as a platform to develop more specific ligands for a particular G-quadruplex.6 Triarylsubstituted imidazole is a G-quadruplex-selective ligand developed by our group.27 Based on this scaffold, we successfully discovered several distinctive probes that bind to particular G-quadruplexes, such as parallel G-quadruplexes28−30 and telomeric multimeric G-quadruplexes.31 In addition, carbazole is a rigid heteroaromatic ring system present in numerous natural products and pharmacologically active compounds.32 Some carbazole derivatives have been demonstrated to act as ligands that show significant binding to the c2448

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Figure 2. Fluorescence spectra of the four compounds with different DNAs: (A) IZCZ-1, (B) IZCZ-2, (C) IZCZ-3, and (D) IZCZ-4. The concentration of the compound was 1 μM, and the DNA concentration was 1 μM.

IZCZ-3 toward the c-MYC G-quadruplex was further evaluated in cells via dual-luciferase reporter assays, exon-specific assays, RT-PCR, and Western blotting. The effects of IZCZ-3 on cancer cell proliferation were investigated using MTT assays, real-time cellular activity (RTCA) assays, colony formation assays, and flow cytometric assays, and its anticancer ability was also evaluated in a mouse xenograft model of cervical squamous cancer.

types of DNAs (such as antiparallel, hybrid G-quadruplex, Gtriplex, and i-motif) compared with the parent IZCZ-0, but this discrimination was not sufficient for further development of IZCZ-1 as a selective ligand for c-MYC G-quadruplex DNA. In addition, IZCZ-2, with no 1-methylpiperazine groups, exhibited a medium emission response only for pu22, revealing that it had high selectivity for the c-MYC G-quadruplex (Figure 2B). However, the fluorescence response induced by IZCZ-2 was much lower than that induced by IZCZ-1, indicating a weak interaction between IZCZ-2 and pu22. Thus, we conclude that to some extent, the strategy of removing the 1-methylpiperazine groups helped to realize selective recognition for the parallel G-quadruplex pu22. However, due to the greatly reduced affinity for the c-MYC G-quadruplex, we could not acquire an ideal ligand through this structural modification. IZCZ-3 showed excellent selectivity as well as a strong emission response to pu22 (Figure 2C). These behaviors differentiated IZCZ-3 from the other compounds and supported the idea that introducing phenyl moieties can enhance the selectivity for the c-MYC G-quadruplex, which proved to be a better strategy for the discovery of more selective ligands based on the triaryl-substituted imidazole scaffold. However, the other compound, IZCZ-4, seemed not to bind to G-quadruplexes effectively (Figure 2D), mostly because it lacked 1-methylpiperazine groups. These results were consistent with SPR assays showing that IZCZ-3 bound to the c-MYC G-quadruplex with both high selectivity and strong affinity (Table S2). Therefore, among the candidates, IZCZ-3 was chosen as the most promising ligand for targeting the cMYC G-quadruplex and thus was used for further detailed investigation. IZCZ-3 Selectively Binds to the c-MYC G-Quadruplex. To further understand the interactions of IZCZ-3 with different G-quadruplexes, fluorescence titration assays were performed. As shown in Figure 3A, IZCZ-3 alone in buffer displayed a

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RESULTS AND DISCUSSION Synthesis of Aryl-Substituted Imidazole/Carbazole Conjugates. The facile synthetic route for the designed clover-like compounds is shown in Figure 1. Intermediates 2a-1 and 2a-2 were obtained by a substitution reaction of 4,4′difluorobenzil and 1-methylpiperazine. The target compounds (IZCZ-0 to IZCZ-4) were prepared via one-pot reactions by condensing the intermediates (2a-1 or 2a-2) with N-ethylcarbazole-3-carbaldehyde, with or without p-anisidine. Compound structures and purities were confirmed by 1H and 13C NMR spectrometry, high-resolution mass spectra (HRMS) spectrometry, and high-performance liquid chromatography (HPLC) analysis (see details in the Experimental Section and Supporting Information). Identification of a Four-Leaf Clover-Like Ligand Selective for the c-MYC G-Quadruplex. To identify the most promising ligand with selectivity for the c-MYC Gquadruplex, the newly synthesized compounds were screened using fluorescence spectroscopy. Seven representative oligonucleotides, namely, single-stranded DNA pu22c, double-stranded DNA hairpin, G-triplex,34 i-motif,35 hybrid telomeric Gquadruplex htg22,36 antiparallel G-quadruplex HRAS,37 and parallel c-MYC G-quadruplex pu22,19 were employed in the assays (Table S1 and Figure S2). As shown in Figure 2A, IZCZ1, which possesses one 1-methylpiperazine group, showed somewhat enhanced discrimination between pu22 and other 2449

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Figure 3. (A) Fluorescence spectra of 1 μM IZCZ-3 with stepwise addition of the c-MYC G-quadruplex pu22 (arrow: 0−1 μM). (B) Fluorescence spectra of 1 μM IZCZ-3 with stepwise addition of the telomeric G-quadruplex htg22 (arrow: 0−10 μM). (C) Fluorescence spectra of 1 μM IZCZ-3 with 1 μM of different promoter G-quadruplexes. (D) Fluorescence titration curves of 1 μM IZCZ-3 with stepwise addition of different promoter Gquadruplexes.

Figure 4. CD melting curves for different types of G-quadruplexes (10 mM Tris-HCl buffer, pH = 7.2) in the absence and presence of 3 equiv of IZCZ-3: (A) parallel c-MYC G-quadruplex pu22 (2 mM KCl), (B) hybrid telomeric G-quadruplex htg22 (50 mM KCl), (C) antiparallel Gquadruplex HRAS (50 mM KCl), (D) parallel G-quadruplex c-kit1 (2 mM KCl), (E) parallel G-quadruplex bcl-2 (2 mM KCl), and (F) parallel Gquadruplex KRAS (2 mM KCl). Different concentrations of KCl were used to ensure that the Tm values of these G-quadruplexes without the ligand were similar.

weak fluorescence emission at 420 nm. With gradual addition of the parallel c-MYC G-quadruplex pu22, the emission peak shifted to 465 nm, accompanied by a sharp fluorescence

enhancement. In contrast, the hybrid telomeric G-quadruplex htg22 also led to a redshift, but the fluorescence signal displayed a very slight enhancement, even when the 2450

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Figure 5. Top view (upper panel) and side view (lower panel) of the binding models of IZCZ-3 with (A) the c-MYC G-quadruplex pu22 (PDB ID: 2L7V), (B) the c-KIT G-quadruplex c-kit1 (PDB ID: 4WO3), and (C) the telomeric G-quadruplex htg22 (PDB ID: 2MB3).

G-quadruplexes with and without IZCZ-3 obtained through circular dichroism (CD) studies were compared. As shown in Figure 4 and Table S3, IZCZ-3 was highly effective at stabilizing the c-MYC G-quadruplex, showing a ΔTm of 20 °C. In contrast, the presence of IZCZ-3 had a very little effect on the Tm of the other G-quadruplexes, such as the hybrid Gquadruplex htg22, antiparallel G-quadruplex HRAS, and other parallel promoter G-quadruplexes (c-kit1, bcl-2, and KRAS). These results offered additional evidence that IZCZ-3 preferred to bind and stabilize the c-MYC G-quadruplex, indicating that IZCZ-3 could be a promising selective ligand. Binding Mode of IZCZ-3 with the c-MYC G-Quadruplex. To obtain additional details regarding the interactions of IZCZ-3 with G-quadruplexes, a molecular docking study was performed with the AutoDock 4.2 program to understand the binding modes of IZCZ-3 to G-quadruplexes.45 The NMR Gquadruplex structures for pu22,19 c-kit1,41 and htg2236 were used as templates. As shown in Figure 5A, IZCZ-3 stacked perfectly on the terminal G-quartet plane of pu22 via π−π interaction, and the positively charged central imidazole moiety of IZCZ-3 was located in the ion channel of the c-MYC Gquadruplex, leading to a relatively low binding energy of −8.7 kcal/mol. In the complex formed by IZCZ-3 and c-kit1, IZCZ3 did not match the c-kit1 G-quadruplex as well as it did pu22, resulting in a reduced binding affinity (−7.6 kcal/mol, Figure 5B). In the complex formed by IZCZ-3 and htg22, IZCZ-3 bound loosely to the terminal G-quartet of htg22 with a higher binding energy of −6.7 kcal/mol, primarily because the lateral loop across the G-quartet plane hindered IZCZ-3 and htg22 from forming a tight complex (Figure 5C). These findings were in agreement with the trends observed in the fluorescence titration assays and CD melting assays, further supporting the reliability of the models. Taken together, these interaction studies suggested that the high selectivity of IZCZ-3 for the cMYC G-quadruplex arose from its stronger binding affinity. IZCZ-3 Inhibits c-MYC Transcription by Binding and Stabilizing the Promoter G-Quadruplex in Cells. Multiple details regarding and confirming IZCZ-3 targeting of the c-

concentration of htg22 was high (Figure 3B). Additionally, fitting the titration data to the Benesi−Hildebrand equation38 revealed a 1:1 binding stoichiometry (also proved by Job plot analysis, Figure S3), and the dissociation constant (KD) between IZCZ-3 and pu22 was calculated to be 0.1 μM. In contrast, the KD for htg22 could not be determined because of the negligible fluorescence enhancement upon the addition of up to 10 μM DNA, which had a trend similar to the SPR results (Figure S4). Collectively, these data revealed that IZCZ-3 bound to the c-MYC G-quadruplex with a much stronger affinity than to the other G-quadruplexes. The experiments above demonstrated that IZCZ-3 preferentially bound to the parallel c-MYC G-quadruplex rather than the hybrid telomeric G-quadruplex. As we had previously found that such a four-leaf clover-like ligand might be inclined to stack onto the parallel G-quadruplex topology, we then evaluated the interactions of IZCZ-3 with several other parallel promoter Gquadruplexes, including VEGF,39 bcl-2,40 c-kit1,41 KRAS,42 and RET,43 via fluorescence assays. In addition, another promoter G-quadruplex (mixed type, PDGFA)44 was also tested here. As shown in Figure 3C, all these G-quadruplexes induced considerable fluorescence enhancements of IZCZ-3, but these enhancements were much less significant than that induced by the c-MYC G-quadruplex pu22 (Figure S5). Furthermore, we conducted fluorescence titration experiments with these promoter G-quadruplexes (Figure 3D), and the corresponding dissociation constants (KD) were determined to be 0.9 μM for VEGF, 0.8 μM for bcl-2, 0.8 μM for c-kit1, and 1.0 μM for KRAS. These results suggested that IZCZ-3 showed a clear preference for binding to the c-MYC G-quadruplex over other promoter G-quadruplexes. IZCZ-3 Selectively Stabilizes the c-MYC G-Quadruplex. An ideal G-quadruplex ligand should possess two essential features: high G-quadruplex targeting specificity and high Gquadruplex stabilizing ability. Our experiments above demonstrated that to some extent IZCZ-3 selectively targeted the cMYC G-quadruplex over other G-quadruplexes. To test the stabilizing ability of IZCZ-3, the melting temperatures (Tm) of 2451

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MYC G-quadruplex were evaluated in vitro. Then, we asked whether IZCZ-3 selectively targets the c-MYC promoter Gquadruplex and subsequently inhibits gene transcription in cells. To answer this question, we first conducted dualluciferase reporter assays. We constructed a psiCHECK2 plasmid carrying the Renilla luciferase gene with the c-MYC promoter and firefly luciferase downstream within the same plasmid.20 After transfection, SiHa cells were treated with IZCZ-3 for 48 h. As shown in Figure 6, the ratio of Renilla/

plasmid (Figure 6). These results suggested that IZCZ-3 could bind and stabilize the promoter G-quadruplex and thus downregulate c-MYC transcription. To further evaluate whether the observed c-MYC transcription inhibition by IZCZ-3 was dependent on ligandmediated G-quadruplex stabilization in the c-MYC promoter region, a pair of Burkitt’s lymphoma (BL) cell lines were used to perform exon-specific assays.46 The BL reciprocal translocation maintains G-quadruplex-mediated control of c-MYC on both chromosomes in RAJI but only on the nontranslocated chromosome in CA46. The nontranslocated (NT) cells coexist with translocated (T) cells in the CA46 cell line. If the c-MYC transcriptional modulation is mediated by the G-quadruplex, a preferential decrease would be evident in exon 1 but not in exon 2. However, since the entire c-MYC gene is translocated in the RAJI cell line, the changes observed in exons 1 and 2 in RAJI cells would be similar to each other. Using primers specific to the two exons, the mRNA products of exon 1 and exon 2 in the BL cell lines could be examined independently. The results are shown in Figure 7. In the CA46 cells, exon 1 exhibited a low transcription level and was further downregulated by IZCZ-3, while exon 2 exhibited a high transcription level, which was minimally affected by IZCZ-3 (Figure 7A,B). In the RAJI cells, where both exons remain under G4 control irrespective of translocation status, a significant, dose-dependent transcriptional downregulation of both exon 1 and exon 2 was observed, indicating that there was no exon-specific effect (Figure 7C,D). These results again supported the possibility that IZCZ-3 might directly target the c-MYC G-quadruplex in cells. IZCZ-3 Significantly Inhibits Cancer Cell Proliferation by Inducing Cell Cycle Arrest at G0/G1 and Apoptosis. The above results showed that IZCZ-3 had a high binding affinity and a remarkable stabilizing ability specific to the c-MYC G-quadruplex both in vitro and in cells. Accordingly, IZCZ-3 might have the potential to act as a favorable anticancer agent

Figure 6. Relative expression level of the Renilla luciferase (ratio of Renilla luciferase activity to firefly luciferase activity) in plasmids containing the wild-type or mutant c-MYC promoter after the addition of IZCZ-3. The experiments were repeated three times. The data are expressed as the mean ± SEM: (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, significantly different from the control. (ns) Not significantly different from the control.

Firefly luciferase activity decreased significantly compared with that of the control. To determine whether this inhibitory activity was due to the interaction of IZCZ-3 with the c-MYC G-quadruplex, we constructed another plasmid carrying a mutated c-MYC promoter (Table S1) that could not form a Gquadruplex structure (Figure S6) and then analyzed the activities of the two luciferases, which demonstrated that IZCZ-3 had little effect on the expression of the mutated

Figure 7. Exon-specific assays were performed in the CA46 cell line (A,B) and the RAJI cell line (C,D). The NHE III1 element of the c-MYC gene is removed together with the P1 and P2 promoters in CA46 cells, while RAJI cells retain this element after translocation. The experiments were repeated three times. The data are expressed as the mean ± SEM: (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, significantly different from the control. 2452

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other reported c-MYC G-quadruplex ligands.20,21 Considering the evidence, we proposed that IZCZ-3, as a selective c-MYC G-quadruplex ligand, might block c-MYC transcription and then downregulate c-MYC expression, thereby arresting cell cycle in the G0/G1 phase and causing cancer cell apoptosis. IZCZ-3 Specifically Downregulates c-MYC Transcription in Cancer Cells. The c-MYC protein is considered to be involved in regulating cell cycle and apoptosis. To confirm that the IZCZ-3-induced G0/G1 arrest and apoptosis were caused by its blocking of c-MYC transcription, RT-PCR was performed. SiHa cells were incubated with IZCZ-3 at various concentrations for 6 h. Then, total RNA was extracted and reverse transcribed to cDNA. The cDNA was then used as a template for PCR amplification. As shown in Figure 9A, IZCZ3 showed remarkable inhibitory activity on transcription of the c-MYC gene. In contrast, IZCZ-3 had no effect on the transcription of several other oncogenes whose promoters contain G-quadruplex structures (including VEGF, BCL-2, cKIT, KRAS, RET, PDGFA, and HRAS genes), demonstrating that IZCZ-3 had high selectivity for the c-MYC G-quadruplex in cells (Figures S9 and S10). If a ligand induced any intracellular effects through stabilization of the promoter G-quadruplex and modulation of c-MYC expression, it is expected that the cell lines with high cMYC mRNA expression would be considerably more sensitive than those with low mRNA expression. Therefore, to further verify the IZCZ-3 mechanism of action in cells, we selected several cell lines with different c-MYC mRNA levels, which were identified by RT-PCR experiments (Figure S11), and then evaluated the effects of IZCZ-3 on these cell lines. MTT assays were first used to evaluate the antiproliferation effects of IZCZ3 on these cells. The results are shown in Table S4. We observed that IZCZ-3 exerted a stronger inhibition effect on cells with higher c-MYC transcription. For example, three cell lines (SiHa, U2OS, and BJ cells) had a significant difference in c-MYC transcription, among which SiHa cells had the highest mRNA level and BJ cells had the lowest mRNA level. We found that IZCZ-3 exhibited decreasing inhibitory effects on these three cell lines (IC50 values obtained from MTT assays were 3.3, 6.8, and 15.9 μM for SiHa, U2OS, and BJ cells, respectively). These results were in agreement with the above hypothesis, suggesting that IZCZ-3 preferred to target the cMYC promoter in cells. Furthermore, IZCZ-3 treatment greatly reduced c-MYC transcription in all the cancer cells, which had relatively high c-MYC transcription activity, but presented a slight effect on transcription in primary cultured mouse mesangial cells and BJ cells (Figure S12), further supporting the notion that IZCZ-3 binds to the c-MYC G-quadruplex in cells. In comparison, we observed that the nonspecific ligand IZCZ-0 had a similar inhibition effect on all the tested cell lines (Table 1). All these results demonstrated the binding specificity between IZCZ-3 and c-MYC promoter in vivo. Effects of IZCZ-3 on the Expression of c-MYC Protein and Associated Cell Cycle/Apoptosis Regulators in Cancer Cells. The above data suggested that IZCZ-3 specifically targeted the G-quadruplex structure in the c-MYC oncogene promoter and hence downregulated its transcription. Next, we tested whether the subsequent c-MYC protein expression in cancer cells was modulated by IZCZ-3. Thus, SiHa cells treated with IZCZ-3 for 24 h were collected for Western blotting analysis. As shown in Figure 9B, the c-MYC expression level also decreased significantly upon treatment

by blocking c-MYC transcription. Therefore, we performed a biological evaluation of IZCZ-3 in cancer cells. A short-term (24 h) cell viability assay was first performed to assess the effects of IZCZ-3 on cell proliferation in SiHa, HeLa, Huh7, and A375 cancer cells (with overexpression of c-MYC protein) and in normal BJ fibroblasts and primary cultured mouse mesangial cells (with relatively low expression of c-MYC protein). As shown in Table 1, IZCZ-3 had a significant Table 1. IC50 Values (μM) of All Compounds against Tumor Cells and Primary Cultured Mouse Mesangial Cells As Determined by MTT Assays IZCZ-0 IZCZ-1 IZCZ-2 IZCZ-3 IZCZ-4

SiHa

HeLa

Huh7

A375

BJ

mesangial cells

2.3 30.1 >80 3.3 >80

2.5 15.3 >80 2.1 >80

2.7 12.4 >80 4.1 >80

2.3 7.4 >80 4.2 >80

2.8 13.9 >80 15.9 >80

3.1 17.3 >80 15.6 >80

cytotoxic effect on the cancer cells and induced only weak growth inhibition in the BJ fibroblasts (IC50 = 15.9 μM) and mouse mesangial cells (IC50 = 15.6 μM), suggesting that IZCZ3 was more effective against cancer cells than against c-MYCindependent normal cells. In parallel, we also evaluated the cytotoxicity of the other compounds. Among them, IZCZ-0 displayed significant cytotoxic effects against both cancer cells and normal cells, possibly because it could effectively bind to different G-quadruplexes in cells. IZCZ-1, which has only one 1-methylpiperazine group, had relatively weaker cytotoxicity, while the remaining two compounds possessing no 1methylpiperazine groups (IZCZ-2 and IZCZ-4) showed little cytotoxicity even at concentrations of 80 μM. These data suggested that the effects of IZCZ-3 on cancer cells might be largely c-MYC-dependent. To further evaluate the growth inhibition of cancer cells induced by IZCZ-3, RTCA and colony formation assays were carried out. SiHa cells and BJ fibroblasts were treated with various concentrations of IZCZ-3 in an RTCA for 130 h, and the cell growth curves are shown in Figure 8A. IZCZ-3 caused significant growth arrest of SiHa cells. However, the growth of BJ fibroblasts was not affected by IZCZ-3. Colony formation assays showed a more direct result regarding cell proliferation inhibition by IZCZ-3. As shown in Figure 8B, an 8 day treatment with 0.5 μM IZCZ-3 almost completely inhibited the proliferation of c-MYC-dependent SiHa cells but showed no inhibitory effect on BJ fibroblasts. In addition, to understand the antiproliferative activity of IZCZ-3 in cancer cells, we explored whether IZCZ-3 caused cell cycle arrest or apoptosis using flow cytometry assays. We first analyzed the percentage of cells in different cell cycle phases. As shown in Figure 8C and Figure S7, after a 12 h treatment, IZCZ-3 induced an apparent accumulation of cells in the G0/G1 phase (increasing from 61% to 70%) in a dosedependent manner. Additionally, we observed in Annexin VFITC and PI staining assays that IZCZ-3 induced cellular apoptosis. As shown in Figure 8D and Figure S8, IZCZ-3 induced apoptotic cell death in a dose-dependent manner. In SiHa cells treated with 5.0 μM IZCZ-3 for 24 h, the populations of apoptotic cells in the early stage and late stage were 22% and 52%, respectively. In contrast, the nontreated cells had a negligible population of apoptotic cells. These effects of IZCZ-3 on tumor cells were consistent with the effects of 2453

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Figure 8. Effect of IZCZ-3 on the proliferation of SiHa cells and BJ fibroblasts, measured by (A) RTCA assays and (B) colony formation assays. (C) Cell cycle analysis of SiHa cells after a 12 h treatment with IZCZ-3. The cells were collected and stained with propidium iodide (PI). (D) Apoptosis evaluation of SiHa cells after a 24 h treatment with IZCZ-3. The cells were collected and stained with Annexin V−FITC and PI.

Figure 9. (A) RT-PCR was used to determine the levels of c-MYC transcription in SiHa cells treated with various concentrations of IZCZ-3 for 6 h. (B) Western blotting was used to determine the c-MYC expression in SiHa cells treated with various concentrations of IZCZ-3 for 24 h. (C) Western blotting was used to determine the expression of c-MYC-related cell cycle and apoptosis regulators in SiHa cells treated with various concentrations of IZCZ-3 for 24 h.

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Figure 10. Compound IZCZ-3 inhibits tumor growth in a SiHa xenograft model in vivo. After treatment with IZCZ-3 at 20, 10, or 5 mg/kg or with doxorubicin at 1 mg/kg for 24 days, the mice were sacrificed, and the tumors were weighed. (A) Images of excised tumors from each group when the treatment ended. (B) Weights of the excised tumors from each group when the treatment ended. (C) Tumor volumes of the mice in each group during the treatment period. (D) Body weights of the mice in each group during the treatment period. (E) Viscera weights of the mice in each group when the treatment ended. The data are presented as the mean ± SEM: (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001, significantly different from the negative control based on Student’s t test; n = 10. (F) Expression of c-MYC protein (brown area) in the tumor tissues from the doxorubicin and IZCZ-3 groups, as determined by immunohistochemistry (IHC). The percentages of c-MYC-expressing cells in the three groups were analyzed.

with IZCZ-3, which was consistent with the trend observed in transcription modulation. How did the reduced c-MYC expression cause cell cycle arrest and apoptosis? We analyzed the expression of certain cell cycle and apoptosis regulators associated with c-MYC protein in cells treated with IZCZ-3 for 24 h by Western blotting. As shown in Figure 9C, we observed a clear, dose-dependent downregulation of Cyclin D1 and CDK6, along with upregulation of cyclin-dependent kinase inhibitors, such as p15 and p27, indicating that IZCZ-3 stalled cell cycle progression. Furthermore, an apoptosis marker protein, cleaved PARP, was upregulated in IZCZ-3-treated cells relative to its level in the untreated group. Together, these results indicated that through downregulating c-MYC expression, IZCZ-3 disturbed the functions of cell cycle and apoptosis regulators, thereby inducing G0/G1 arrest and apoptosis. IZCZ-3 Inhibits Tumor Growth in a Human Cervical Squamous Cancer Xenograft. IZCZ-3 presented effective anticancer activity in vitro, which prompted us to investigate its in vivo antitumor activity against human cervical squamous cancer xenografts in nude mice. The tumor-bearing mice were divided into five groups (10 mice in each group) and treated with saline (negative control), doxorubicin (positive control, at a dose of 1 mg/kg), and IZCZ-3 (three experimental groups, at doses of 20, 10, and 5 mg/kg) every other day for 24 days. The

results are presented in Figure 10. Compared with the negative control group, treatment with IZCZ-3 at 20, 10, and 5 mg/kg resulted in a significant reduction in tumor weight with tumor growth inhibition (TGI) of 69%, 64%, and 57%, respectively (Figures 10A,B). IZCZ-3 also displayed time-dependent inhibition of tumor growth (Figure 10C). During the experiment, all of the mice appeared healthy with no visible signs of pain, distress, or discomfort. There was no significant difference in body weight or viscera weight between the negative control group and the IZCZ-3-treated groups, indicating that IZCZ-3 was tolerated well at these doses (Figure 10D,E). Although treatment with doxorubicin led to better tumor growth inhibition, its toxicity was apparent, as evidenced by the losses in body weight and viscera weight. All of the data showed that IZCZ-3 exhibited good antitumor ability by inhibiting cervical squamous cancer growth in nude mice with SiHa xenografts. To determine whether the expression of c-MYC was consistently affected within tumors during treatment with IZCZ-3, we assessed the c-MYC expression (brown area in Figure 10F) in tumor tissues using immunohistochemistry (IHC). The IZCZ-3-treated group exhibited significantly decreased c-MYC expression compared with that in the negative control group. We noted that doxorubicin reduced c-MYC expression to some extent but not as significantly as 2455

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cooling, the mixture was treated with 3 M NaOH to reach pH 8, and the product was extracted with EtOAc (20 mL × 5). The combined organic phase was dried over Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified using flash column chromatography to obtain the final product. 3-(4,5-Bis(4-(4-methylpiperazin-1-yl)phenyl)-1H-imidazol-2-yl)-9ethyl-9H-carbazole (IZCZ-0). Pale yellow solid (62% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 8.83 (s, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.53−7.31 (m, 5H), 7.23 (t, J = 7.4 Hz, 1H), 7.05−6.78 (m, 4H), 4.55−4.37 (m, 2H), 3.26−2.98 (m, 8H), 2.49−2.32 (m, 8H), 2.20 (s, 6H), 1.34 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 149.99, 146.77, 140.37, 140.01, 128.65, 125.95, 123.53, 123.12, 123.00, 121.40, 120.75, 119.16, 117.74, 115.74, 108.64, 55.06, 48.75, 46.11, 37.65, 13.86. HRMS (ESI) m/z: calcd for C39H43N7: 650.3653 [M + H]+. Found 650.3654 [M + H]+. 9-Ethyl-3-(5-(4-fluorophenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-1H-imidazol-2-yl)-9H-carbazole (IZCZ-1). Pale yellow solid (65% yield). 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.11 (t, J = 8.2 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.71−7.55 (m, 2H), 7.54−7.32 (m, 6H), 7.02 (t, J = 8.2 Hz, 2H), 6.92 (d, J = 7.0 Hz, 2H), 4.38 (q, J = 6.9 Hz, 2H), 3.38−3.16 (m, 4H), 2.67−2.53 (m, 4H), 2.37 (s, 3H), 1.46 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.94, 149.80, 146.31, 139.98, 139.41, 129.11, 128.70, 125.99, 123.56, 122.23, 121.62, 120.21, 119.07, 117.09, 115.15, 114.92, 109.28, 109.14, 54.24, 47.34, 45.27, 37.05, 13.67. HRMS (ESI) m/z: calcd for C34H32FN5: 530.2715 [M + H]+. Found 530.2707 [M + H]+. 3-(4,5-Bis(4-fluorophenyl)-1H-imidazol-2-yl)-9-ethyl-9H-carbazole (IZCZ-2). White solid (68% yield). 1H NMR (400 MHz, DMSOd6) δ 8.60 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.59−7.35 (m, 7H), 7.23 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 8.4 Hz, 4H), 4.35 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.31, 146.82, 140.05, 139.57, 129.68, 126.03, 123.60, 122.27, 121.45, 120.24, 119.12, 117.25, 115.35, 109.35, 109.22, 37.10, 13.70. HRMS (ESI) m/z: calcd for C29H21F2N3: 450.1776 [M + H]+. Found 450.1776 [M + H]+. 9-Ethyl-3-(1-(4-methoxyphenyl)-4,5-bis(4-(4-methylpiperazin-1yl)phenyl)-1H-imidazol-2-yl)-9H-carbazole (IZCZ-3). Purple solid (43% yield). 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.50−7.33 (m, 3H), 7.25− 7.14 (m, 2H), 7.07−6.96 (m, 4H), 6.85 (d, J = 8.8 Hz, 2H), 6.81−6.70 (m, 4H), 4.32 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 3.33−3.13 (m, 8H), 2.68−2.50 (m, 8H), 2.36 (s, 6H), 1.40 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 158.86, 150.09, 149.50, 147.67, 140.28, 139.59, 137.56, 132.00, 130.65, 129.74, 128.16, 126.87, 126.73, 125.73, 123.11, 122.63, 121.91, 121.78, 121.44, 120.53, 119.02, 115.72, 115.21, 114.13, 108.53, 107.91, 55.36, 55.07, 55.04, 48.91, 48.32, 46.05, 46.03, 37.59, 13.82. HRMS (ESI) m/z: calcd for C46H49N7O: 358.7072 [M + 2H]2+. Found 358.7056 [M+2H]2+. 3-(4,5-Bis(4-fluorophenyl)-1-(4-methoxyphenyl)-1H-imidazol-2yl)-9-ethyl-9H-carbazole (IZCZ-4). White solid (43% yield). 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.94 (d, J = 7.7 Hz, 1H), 7.58 (dd, J = 8.6, 5.6 Hz, 2H), 7.52−7.42 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.26−7.17 (m, 2H), 7.12 (dd, J = 8.5, 5.5 Hz, 2H), 7.03−6.90 (m, 6H), 6.77 (d, J = 8.8 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 163.43, 160.91, 159.23, 148.37, 140.34, 139.82, 137.22, 132.98, 130.64, 130.05, 129.62, 129.27, 129.14, 126.91, 126.71, 125.89, 123.04, 122.75, 121.47, 121.03, 120.55, 119.16, 115.62, 115.10, 114.35, 108.60, 108.03, 55.41, 37.64, 13.80. HRMS (ESI) m/z: calcd for C36H27F2N3O: 556.2195 [M + H]+. Found 556.2190 [M + H]+. Materials. All oligonucleotides (Table S1) were dissolved in TrisHCl buffer, and their concentrations were determined based on absorbance at 260 nm using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). To obtain G-quadruplexes, oligonucleotides were annealed in relevant buffers containing 100 mM KCl by heating at 95 °C for 5 min, followed by gradual cooling to room temperature. G-quadruplex formation was determined by circular dichroism (CD). Stock solutions of compounds (10 mM) were dissolved in DMSO and stored at −80 °C. Further dilutions to the

IZCZ-3. The results indicated that IZCZ-3 might inhibit tumor growth by specifically downregulating c-MYC expression.

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CONCLUSION Increased c-MYC expression promotes tumorigenesis. Transcription of the c-MYC gene can be suppressed by stabilizing the G-quadruplex structure present in its promoter. To date, many ligands have been reported to stabilize the c-MYC Gquadruplex and thereby inhibit cancer cell proliferation, making them potential anticancer drugs. However, in most cases, in addition to the c-MYC G-quadruplex, these ligands might also stabilize other G-quadruplexes, leading to unexpected side effects. In addition, it is difficult to validate the relationship between cause and effect in cancer cells using such ligands. Thus, we were inspired to search for a selective ligand for the cMYC G-quadruplex. In this study, based on the triaryl-substituted imidazole scaffold, we designed and synthesized IZCZ-0, which lacked the ability to discriminate among different G-quadruplexes. To obtain a selective ligand, we set about modifying the IZCZ-0 structure. Hence, by applying two modification directions, four compounds (IZCZ-1 to IZCZ-4) were designed. Next, we evaluated these compounds with regard to their binding to Gquadruplexes using fluorescence assays and confirmed that our modification strategy was effective for the discovery of a selective ligand. IZCZ-3 seemed to have excellent selectivity for the c-MYC G-quadruplex over other G-quadruplexes and double-/single-stranded DNAs. Further fluorescence titrations, melting assays, and molecular modeling studies revealed that, among several parallel promoter G-quadruplexes, IZCZ-3 preferred to bind and stabilize the c-MYC G-quadruplex. In line with the in vitro assays, subsequent bioassays, including dual-luciferase reporter assays, RT-PCR, exon-specific assays, and Western blotting, were performed and revealed that IZCZ-3 effectively downregulated c-MYC gene transcription and expression in cancer cells by specifically targeting the cMYC G-quadruplex rather than other promoter G-quadruplexes. We hence evaluated the effects of IZCZ-3 on cancer cells, demonstrating that it could provoke cell cycle arrest and apoptosis and inhibit cancer cell growth, which might be ascribed to downregulation of c-MYC expression. IZCZ-3 also exhibited good antitumor ability by inhibiting cervical squamous cancer growth in nude mice. To the best of our knowledge, this is the first ligand that possesses high selectivity for the c-MYC G-quadruplex. This work provides new insights for the development of selective chemical probes or anticancer drugs that could specifically target the c-MYC G-quadruplex structure and further disrupt c-MYC related pathways.

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EXPERIMENTAL SECTION

Synthesis and Characterization. 1H and 13C NMR spectra were recorded using TMS as the internal standard in CDCl3 or DMSO-d6 with a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. Mass spectra (MS) were recorded on a Shimadzu LCMS2010A instrument with an ESI mass selective detector. HRMS data were acquired on a Shimadzu LCMS-IT-TOF instrument. The purity of synthesized compounds was confirmed to be higher than 95% using analytical HPLC performed with a dual pump Shimadzu LC-20 AB system. The intermediates 2a-1 and 2a-2 were synthesized according to our previous reports.27,30 General Method for Synthesis of IZCZ-0−IZCZ-4. A mixture of 1a (or 2a-1, 2a-2) (1.2 mmol), p-anisidine (4.8 mmol), Nethylcarbazole-3-carbaldehyde (1.5 mmol), NH4OAc (20.0 mmol), and AcOH (5 mL) was stirred at reflux temperature for 5 h. After 2456

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working concentrations were performed with the relevant buffer immediately prior to use. Fluorescence Spectroscopic Assays. Fluorescence studies were performed on a FluoroMax-4 luminescence spectrophotometer (HORIBA, USA). A quartz cuvette with a 2 mm × 10 mm path length was used for spectral recording at 3 nm excitation and emission slit widths unless otherwise specified. For titration experiments, small aliquots of an oligonucleotide stock solution were added to solution containing a compound at a fixed concentration (5 μM) in Tris-HCl buffer (10 mM, pH = 7.2) with 100 mM KCl. After each addition, the reaction was stirred and allowed to equilibrate for at least 1 min, and the fluorescence measurement was read when excited at 350 nm. SPR Studies. SPR measurements were performed on a ProteOn XPR36 Protein Interaction Array system (Bio-Rad Laboratories, CA) using a streptavidin-coated GLH sensor chip. Biotinylated DNA was attached to the chip. In a typical experiment, biotinylated DNA was folded in filtered and degassed running buffer (50 mM Tris-HCl, 100 mM KCl, pH 7.2). The DNA samples were then captured (approximately 1000 RU) in flow cells, leaving one flow cell as a blank. Compound solutions were prepared with running buffer through serial dilutions of stock solution. Five concentrations were injected simultaneously at a flow rate of 50 μL/min for 300 s of association phase, followed by 300 s of dissociation phase at 25 °C. The GLH sensor chip was regenerated with a short injection of 1 M KCl between consecutive measurements. The final graphs were obtained by subtracting blank sensorgrams from different DNA sensorgrams. The data were analyzed with ProteOn manager software, using the Langmuir model for fitting kinetic data. Circular Dichroism Spectroscopic Assays. Circular dichroism (CD) studies were performed on a Chirascan circular dichroism spectrophotometer (Applied Photophysics, UK). A quartz cuvette with a 4 mm path length was used to record the spectra over a wavelength range of 230−330 nm with a 1 nm bandwidth, 1 nm step size, and a time of 0.5 s per point. The DNA samples were set at a concentration of 3 μM. CD melting assays were performed at a fixed G-quadruplex concentration (3 μM), either with or without a fixed concentration (15 μM) of IZCZ-3 in Tris−HCl buffer (10 mM, pH = 7.2) with 2 mM KCl (50 mM KCl for htg22). The data were recorded at intervals of 5 °C over a range of 25−95 °C with a heating rate of 1 °C/min. The final analysis of the data was conducted using Origin 9.0 (OriginLab Corp.). Molecular Docking Process. The structure of IZCZ-3 was constructed and optimized with Gaussian 03 using the HF/6-31G* basis set. The NMR G-quadruplex structures (pu22, c-kit2, and htg22) were used as templates for the docking studies. The docking simulations were performed using Schrodinger software to determine the binding sites. Dual-Luciferase Reporter Assay. Briefly, SiHa cells were transfected with 200 ng of psiCHECK2 luciferase plasmid (Promega, USA) containing the wild-type or mutant c-MYC promoter using Lipofectamine 2000 (Invitrogen, USA). After 4 h, IZCZ-3 was added to the cells at different concentrations. The cells were incubated at 37 °C with CO2 for another 24 h, and the transfected cells were first washed with ice-cold PBS to reduce background signals from the medium. Luciferase assays were subsequently performed according to the manufacturer’s instructions using a dual-luciferase assay system (Promega, USA). After a 3 s delay, secreted luciferase signals were collected for 10 s using a microplate reader (Molecular Devices, Flex Station 3, USA). Quantification was performed using a multimode reader (Molecular Devices). The secreted Renilla luciferase activity was normalized to the firefly luciferase activity. Exon-Specific Assay. RAJI and CA46 cells were treated with IZCZ-3 for 6 h at various concentrations. Total RNA was extracted and used as a template for reverse transcription using the following protocol: each 20 μL reaction contained 4 μL of 5× M-MLV buffer, 1 μL of 10 mM dNTP, 1 μL of 50 μM oligo dT18 primer, 200 U of MMLV reverse transcriptase, 20 U of RNase inhibitor, DEPC-H2O, and 2 μg of total RNA. Next, the mixtures were incubated at 42 °C for 1 h and then at 85 °C for 5 min. Finally, the reacted solution was stored at −20 °C. Exon 1 and exon 2 were amplified using a real-time PCR

apparatus, and the PCR products were analyzed via electrophoresis on a 1% agarose gel at 120 V for 20 min. The primers used in the RTPCR were exon 1 A (5′-CGTCCCTGGCTCCCCTCCT-3′),exon-1 S (5′-GCTCCCTCTGCCTCTCGCTG-3′), exon 2 A (5′-CCGAAGGGAGAAGGGTGTGA-3′), and exon 2 S (5′-CCAGCGAGGATATCTGGAAGAA-3′). MTT Assay. Cells were seeded in 96-well plates (5.0 × 103 cells/ well) and exposed to various concentrations of IZCZ-3. After a 24 h treatment, 20 μL of 2.5 mg/mL methylthiazolyl tetrazolium (MTT) solution was added to each well, and the cells were further incubated for 4 h. The cells in each well were then treated with dimethyl sulfoxide (DMSO) (100 μL per well), and the optical density (OD) was recorded at 570 nm. All experiments were performed in parallel and in triplicate, and the IC50 values were derived from the curves of the mean OD values of the triplicate tests plotted against the drug concentrations. Real-Time Cellular Activity Assay. SiHa cells or primary cultured mouse mesangial cells were seeded at 2000 cells per well in E-Plate 16-well plates (Roche Applied Science, Indianapolis, IN). After seeding, cells were allowed to settle for 20 min at room temperature before being inserted into the xCELLigence RTCA DP instrument. After 24 h of RTCA profiling, the assay was paused, and the E-Plate was removed from the xCELLigence system. The medium was carefully removed and replaced with new medium with different concentrations of IZCZ-3. The E-Plate was placed back into the xCELLigence system, and cells were monitored for a total of 130 h. The results were plotted using RTCA software 1.2 (Roche Applied Science, Indianapolis, IN). The data expressed in cell index (CI) units were exported to Microsoft Excel software (Microsoft, Redmond, WA) for mathematical analysis and normalization. The data were normalized to a starting CI of 1.0 at the time point immediately prior to compound addition. Colony Formation Assay. SiHa cells and primary cultured mouse mesangial cells were seeded on six-well plates (300 cells per well) and exposed to IZCZ-3 at various concentrations at 37 °C in a 5% CO2 incubator. Different concentrations of IZCZ-3 were replaced every 2 days. Cells were fixed with methanol and dyed with crystal violet after culture for 8 days. Cell Cycle Analysis. SiHa cells treated with IZCZ-3 at various concentrations were harvested and washed in PBS and fixed with 70% ethanol at 4 °C overnight. Then, the cells were centrifuged and resuspended in a staining solution (50 μg/mL propidium iodide (PI), 75 KU/mL RNase A in PBS) for 30 min at room temperature in the dark. The cells were analyzed by flow cytometry using an EPICS XL flow cytometer (Beckman Coulter, USA). For each analysis, 1.6 × 104 events were collected. The cell cycle distribution was analyzed using EXPO32 ADC software. Apoptosis Analysis. SiHa cells treated with IZCZ-3 were harvested and washed in PBS. Then, they were centrifuged and resuspended in Annexin-binding buffer. After that, the cells were incubated with Annexin V-FITC and PI for 15 min at room temperature in the dark and immediately analyzed by flow cytometry using an EPICS XL flow cytometer (Beckman Coulter, USA). For each analysis, 1.6 × 104 events were collected. The data are presented as biparametric dot plots showing PI red fluorescence against Annexin V-FITC green fluorescence. RT-PCR. SiHa cells (2.0 × 105 cells) were incubated in six-well plates with IZCZ-3 at various concentrations for 6 h. After that, total RNA was extracted and used as a template for reverse transcription with the following protocol: each 20 μL reaction contained 4 μL of 5× M-MLV buffer, 1 μL of 10 mM dNTP mixture, 1 μL of 50 μM oligo dT18 primer, 200 U of M-MLV reverse transcriptase, 20 U of RNase inhibitor, DEPC-H2O, and 2 μg of total RNA. The mixtures were incubated at 42 °C for 1 h and then at 85 °C for 5 min. Afterward, PCR was performed on a PCR apparatus. The 20 μL RT-PCR reaction mixtures contained 10 μL of 2× HiFiTaq PCR StarMix (GenStar), 1 μL each of the forward and reverse primers (10 μM), 1 μL of cDNA, and nuclease-free water to volume. The program used for all genes consisted of a denaturing cycle of 5 min at 95 °C and 30 cycles of PCR (95 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s). The PCR 2457

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Notes

products were confirmed with agarose gel electrophoresis. The primers used in the RT-PCR are shown in Table S5. Western Blotting Assay. SiHa cells (2.0 × 105 cells) were incubated in six-well plates with IZCZ-3 at various concentrations for 24 h. After that, cells were washed with PBS, lysed with extraction buffer at 4 °C for 30 min, and then centrifuged at 15,000 rpm at 4 °C for 15 min to harvest the supernatant. The protein concentration was calculated with a BCA protein assay kit (Thermo Fisher Scientific). An equal amount of protein (30 μg) was electrophoresed on a 10% SDS− PAGE gel and transferred to a nitrocellulose membrane at 80 V for 2 h. The membranes were blocked for 1 h with a 5% nonfat dry milk solution in TBS containing 1% Tween-20 at room temperature. Membranes with the samples were incubated overnight at 4 °C with primary antibodies. After three washes in TBST, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies at room temperature for 2 h. Xenograft Animal Model and Drug Treatment. BALB/c nude mice (5 weeks old) were purchased from and housed at the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China) and maintained in pathogen-free conditions (12 h light−dark cycle at 24 ± 1 °C with 60−70% humidity and provided food and water ad libitum). SiHa cells were harvested during log-phase growth and resuspended in FBS-free DMEM at 8 × 107 cells/mL. Each mouse was injected subcutaneously in the right flank with 1 × 107 cells. Tumor growth was examined twice a week after implantation until the tumor volume reached approximately 50 mm3. The volume of the tumor mass was measured with an electronic caliper and calculated as 1/2 × length × width2 in mm3. The mice were randomly divided into four groups of 10 animals and treated intraperitoneally with various regimens every other day for the entire observation period (24 days). Mice in the IZCZ-3-treated group were administered a dose of 5, 10, or 20 mg/kg, those in the doxorubicin-treated group were given a dose of 1 mg/kg, and those in the control group were treated with an equivalent volume of saline. The tumor size and the body weight of the mice were measured every day after drug treatment, and growth curves were plotted using the average tumor volume within each experimental group. At the end of the observation period, the animals were euthanized by cervical dislocation, and the tumors were removed and weighed. The inhibition rate (IR) was calculated according to the following formula: IR = (1 − mean tumor weight of the experimental group/mean tumor weight of the control group) × 100%.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21672268 and 81330077), the Natural Science Foundation of Guangdong Province (2015A030306004 and 2017A030308003), the 111 Project (B16047), and Guangdong Provincial Key Laboratory of Construction Foundation (2011A060901014).

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ABBREVIATIONS USED CD, circular dichroism; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; NHE III1, nuclease hypersensitive element III1; RTCA, real-time cellular activity assay; RT-PCR, reverse transcription polymerase chain reaction

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(1) Burge, S.; Parkinson, G. N.; Hazel, P.; Todd, A. K.; Neidle, S. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 2006, 34, 5402−5415. (2) Yang, D.; Okamoto, K. Structural insights into G-quadruplexes: towards new anticancer drugs. Future Med. Chem. 2010, 2, 619−646. (3) Chambers, V. S.; Marsico, G.; Boutell, J. M.; Di Antonio, M.; Smith, G. P.; Balasubramanian, S. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 2015, 33, 877−881. (4) Hansel-Hertsch, R.; Beraldi, D.; Lensing, S. V.; Marsico, G.; Zyner, K.; Parry, A.; Di Antonio, M.; Pike, J.; Kimura, H.; Narita, M.; Tannahill, D.; Balasubramanian, S. G-quadruplex structures mark human regulatory chromatin. Nat. Genet. 2016, 48, 1267−1272. (5) Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 2013, 5, 182−186. (6) Balasubramanian, S.; Hurley, L. H.; Neidle, S. Targeting Gquadruplexes in gene promoters: a novel anticancer strategy? Nat. Rev. Drug Discovery 2011, 10, 261−275. (7) Wu, Y.; Brosh, R. M., Jr. G-quadruplex nucleic acids and human disease. FEBS J. 2010, 277, 3470−3488. (8) Neidle, S. Quadruplex nucleic acids as targets for anticancer therapeutics. Nat. Rev. Chem. 2017, 1, 0041. (9) Valton, A. L.; Prioleau, M. N. G-quadruplexes in DNA replication: a problem or a necessity? Trends Genet. 2016, 32, 697− 706. (10) Mendoza, O.; Bourdoncle, A.; Boule, J. B.; Brosh, R. M., Jr.; Mergny, J. L. G-quadruplexes and helicases. Nucleic Acids Res. 2016, 44, 1989−2006. (11) Rhodes, D.; Lipps, H. J. G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res. 2015, 43, 8627−8637. (12) Dang, C. V. c-Myc target genes involved in cell growth, apoptosis, and metabolism. Mol. Cell. Biol. 1999, 19, 1−11. (13) Pelengaris, S.; Khan, M.; Evan, G. c-MYC: more than just a matter of life and death. Nat. Rev. Cancer 2002, 2, 764−776. (14) Ambrus, A.; Chen, D.; Dai, J.; Jones, R. A.; Yang, D. Solution structure of the biologically relevant G-quadruplex element in the human c-MYC promoter. Implications for G-quadruplex stabilization. Biochemistry 2005, 44, 2048−2058. (15) Siddiqui-Jain, A.; Grand, C. L.; Bearss, D. J.; Hurley, L. H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 11593−11598. (16) Seenisamy, J.; Rezler, E. M.; Powell, T. J.; Tye, D.; Gokhale, V.; Joshi, C. S.; Siddiqui-Jain, A.; Hurley, L. H. The dynamic character of the G-quadruplex element in the c-MYC promoter and modification by TMPyP4. J. Am. Chem. Soc. 2004, 126, 8702−8709. (17) Seenisamy, J.; Bashyam, S.; Gokhale, V.; Vankayalapati, H.; Sun, D.; Siddiqui-Jain, A.; Streiner, N.; Shin-ya, K.; White, E.; Wilson, W.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01697. Additional experimental results; 1H NMR, 13C NMR, HPLC, and HRMS spectra for final compounds (PDF) Molecular formula strings and additional data (CSV) Coordinate information for IZCZ-3/2L7V (PDB), IZCZ-3/4WO3 (PDB), and IZCZ-3/2MB3 (PDB)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(S.-B.C.) Phone: 8620-39943068. E-mail: chenshb8@mail. sysu.edu.cn. *(J.-H.T.) Phone: 8620-39943053. E-mail: [email protected]. edu.cn. ORCID

Tian-Miao Ou: 0000-0002-8176-4576 Zhi-Shu Huang: 0000-0002-6211-5482 Jia-Heng Tan: 0000-0002-1612-7482 Author Contributions §

These authors contributed equally to this work. 2458

DOI: 10.1021/acs.jmedchem.7b01697 J. Med. Chem. 2018, 61, 2447−2459

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.7b01697 J. Med. Chem. 2018, 61, 2447−2459