Selective recognition of c-myc promoter G-quadruplex and down-regulation of oncogene c-myc transcription in human cancer cells by 3,8 a -disubstituted indolizinone

C-myc promoter G-quadruplex is a very important target for developing anti-cancer drugs. However, highly selective recognition of DNA c-myc G-quadruplex with parallel con ﬁ guration is also a very challenging problem. Here, we showed a new type of N-containing alkaloid, 3,8 a -disubstituted indolizinone, which adopted a distorted con ﬁ guration. 6-Methyl-3-(naphthalen-2-yl)-8 a -(4-methylpyridin-2-yl)-indolizinone could selectively recognize DNA c-myc G-quadruplex leading to an obvious ﬂ uorescence enhancement by 11-fold, and meanwhile the G-quadruplex can be stabilized up to 90 (cid:1) C. Further, it could down-regulate the transcription of oncogene c-myc in both human non-small cell line (A549) and human laryngeal epithelial cell line (Hep-2).


Introduction
The G-quadruplex structure formed by tandem guanine (G) nucleotides, which are incorporated in nucleic acid sequences, has illuminated a new role for DNA in biology. 1 G-quadruplexes as one of the higher-order DNA and RNA structures have been identied in G-rich human telomeres and gene promoter regions. G-quadruplex as a therapeutic target has attracted a growing interest, due to its profound roles in a wide spectrum of diseases, such as cancer, diabetes and cardiovascular disorders. 2 Several organic probes have been reported to recognize DNA G-quadruplex via diverse interaction modes. 3 Stabilization of G-quadruplex structures in the promoter region of certain oncogenes is an emerging eld in anticancer drug design. 4 Human c-myc gene is one of these oncogenes, and Gquadruplexes have been proven to be the transcriptional controller of this gene.
Human c-myc gene or its product is a central regulator of cellular proliferation and cell growth, and over-expression of cmyc gene is related to various cancers. 5 C-myc is the rst and most extensively studied system of promoter G-quadruplex formation. The major G-quadruplex formed in the c-myc promoter is a parallel stranded structure with two G3NG3 single-nt strand-reversal loops, and parallel-stranded structures are found to be more common in the promoter G-quadruplexes. 1 Some known G-quadruplex-interactive compounds have become prospective anticancer agents that display relatively low cytotoxicity. In particular, Quaroxin, a rst-in-class G-quadruplex-interactive drug reached phase 2 clinical trials, which was considered to be a c-myc G-quadruplex ligand at rst. 6 Quaroxin is a uoroquinolone derivative with antineoplastic activity, and another known c-myc G-quadruplex ligand quindoline is a derivative of the natural product cryptolepine, 4 and both of them are N-containing alkaloids. Here we reported a new type of N-containing alkaloid, indolizinone (Scheme 1), which can selectively recognize DNA c-myc G-quadruplex (DNA MycG4) and down-regulate oncogene c-myc.
knowledge, any application of which wasn't found to be reported. Recently, we rstly reported a copper-catalyzed domino reaction to construct functionalized 3,8a-disubstituted indolizinones, and this protocol was simple and highly efficient. 8 In the solid state, 3,8a-disubstituted indolizinones adopted distorted conguration, and thus they provided no obviously strong uorescence spectra in solution due to the lack of a larger conjugated p system. Most of DNA MycG4 ligands that have been previously reported are N-heterocycles with planar p systems, which can't distinguish DNA MycG4 from other congurations of G-quadruplexes. In this report, we found 6-methyl-3-(naphthalen-2-yl)-8a-(4-methylpyridin-2-yl)indolizinone (indolizinone) here could selectively recognize DNA MycG4, leading to its greatly enhanced uorescence intensity. We rstly compared the uorescence intensity of indolizinone with ve oligonucleotides of known G4 structures (Table S1 †): intermolecular parallel conguration H7, intramolecular hybrid-type conguration M24, intramolecular antiparallel conguration A24, intramolecular parallel conguration Kit and Myc, relative to the signal of indolizinone alone. As shown in Fig. 1, indolizinone itself at 4 mM gives a very weak uorescence signal. While addition of 8 mM DNA MycG4, and then standing for 24 hours, the uorescence signal of indolizinone lead to an obvious enhancement by 11-fold. Addition of other types of G-quadruplexes, such as intermolecular parallel type H7 led to 0.5-fold enhancement, and for intramolecular types, parallel DNA c-Kit 4.5-fold, hybrid-type M24 1.6-fold and anti-parallel type A24 2.5-fold, respectively. These results indicated that indolizinone preferred to recognize DNA MycG4.
Furthermore, uorescence titrations were carried out to evaluate the binding constants of indolizinone with these ve nucleic acid sequences (Fig. S1-S3 †). A 1 : 1 binding model for indolizinone with DNA G-quadruplex was conrmed by the job's plot analysis. The typical titration curves and tting results according to the 1 : 1 binding model are presented. The binding constants for indolizinone with Myc and Kit are (9.91 AE 0.65) Â 10 5 and (5.77 AE 1.60) Â 10 5 , respectively. The binding strength to Myc was obviously more strong than to Kit. In comparison with other intramolecular DNAG4s, the binding strength to A24 or M24 was much weaker, with binding constants (2.43 AE 0.37) Â 10 4 and (1.88 AE 0.65) Â 10 4 , respectively. Of note, the binding strength to intermolecular H7 was very weak, and the binding constant is less than 10. As shown in Table 1, the trend of the binding constants is consistent with the F/F 0 values.
Then we subsequently performed this assay on a larger set of 25 nucleic acid sequences. Structures of these oligonucleotides have been characterized, and these oligonucleotides have been used in the G4-Fluorescence Intercalator Displacement (FID) assay. 9 All sequences are listed in Table 2, and results are presented in Fig. 2. Fieen of the 25 sequences are likely to form G4s. The uorescence enhancements of indolizinone interacted with the parallel conguration G4 in the promoter region (c-myc, c-kit1, c-kit2, bcl-2, AKT1, VAV1, 35B1, 27Kras) were 11fold, 4.0-fold, 3.7-fold, 2.5-fold, 4.0-fold, 5.2-fold, 4.3-fold, and 3.4-fold, respectively. And the human telomeric DNA Gquadruplex 22Ag and 45Ag, which adopt anti-parallel conguration in the presence of K + , led to the uorescence enhancements of indolizinone with the value F/F 0 1.5-fold and 2.8-fold, respectively. And meanwhile, in the presence of another ve Gquadruplex forming sequences (21CTA, TBA, Ceb25, CT4, CGG12), and other non-G-quadruplex DNA templates including single-stranded (G-tripl, dT30), double-stranded (ds26, 19AT, dx12, PS1c), stem-loop (GCdx), triplex, and trinucleotide (CTG12, CAG12), all these oligonucleotide sequences led to a F/F 0 < 2. Indolizinone showed higher response towards intramolecular DNA G-quadruplex with parallel conguration, especially most to DNA MycG4, comparing to other types of Gquadruplex and non-G-quadruplex templates. While it presented very weak response to intermolecular parallel Gquadruplex H7, just leading to 0.5-fold enhancement, probably attributing to its lack of loop-binding sites.
To evaluate the effect of indolizinone on the specic recognition of DNA MycG4, we applied circular dichroism to monitor the melting temperature (T m ) of DNA MycG4 in the presence of 1 equiv. molar ratio of indolizinone. The change of T m in the folded and unfolded quadruplex structure upon interacting with ligand provides evidence of thermal stabilization of DNA structure. 10 DNA MycG4 was incubated with indolizinone for 24 h, the T m values for DNA MycG4 were calculated as shown in Fig. 3. Indolizinone shows the preference to stabilize DNA MycG4 by raising the temperature up to 90 C monitored at 265 nm. In the contrast, indolizinone shows a weak ability obviously to stabilize other types of G-quadruplexes (see Fig. S4 †).
Based on the emission enhancement and the absorption spectral results, uorescence intensity decay measurements  were conducted to demonstrate the strong binding of indolizinone with DNA MycG4. The uorescence decay of indolizinone was fast (<1 ns) in solution due to the highly feasible torsional relation channel in the excited state. However, the lifetimes increased to the nanosecond level in the presence of DNA MycG4. As shown in Fig. 4, the uorescence decay was much lower for indolizinone in the presence of 1 equiv. or 2 equiv. molar ratio of DNA MycG4, with the s value of 3.54 ns and 3.71 ns, respectively. This result indicated that the interaction between indolizinone and DNA MycG4 hinders the rotation of indolizinone and increases its lifetime. c-myc (Myc)  G4-promoter  TGAG 3 TG 3 GAG 3 TG 3 GAA  c-kit1 (Kit)  G4-promoter  G 3 AG 3 CGCTG 3 AGGAG 3  c-kit2  G4-promoter  G 3 CG 3 CGCGAG 3 AGG 3  BCL-2  G4-promoter  G 3 CGCG 3 AG 2 AATTG 3 CG 3  AKT1  G4-promoter  G 3 CG 3 CGGCTCCG 3 CGCG 3  VAV1 G4-promoter G 3 CAG 3 AG 3 AACTG 3 35B1 (Kras) G4-promoter AG 3  Single strand  Oncogene c-myc encodes an important transcriptional regulator c-myc protein, which is involved in cell proliferation, senescence, and apoptosis. 11 Because c-myc promoter region contains G-quadruplex forming G-rich sequence, its binding with indolizinone could increase its stability resulting in downregulation of oncogene c-myc transcription, which would be identied using real-time RT-PCR. 12 To test the above hypothesis and understand the effect of indolizinone on oncogene cmyc, two cancer cell lines with different translocation break points within the oncogene c-myc were tested. Human nonsmall cell line (A549) and human laryngeal epithelial cell line (Hep-2) were obtained from the National Infrastructure of Cell Line Resource (Beijing, China). As shown in Fig. 5, indolizinone could down-regulate the transcription of oncogene c-myc in both A549 and Hep-2 cells, aer treated with indolizinone at 0.5, 1.0, 2.0, 4.0, and 8.0 mM for 48 h. The total RNA was extracted and reverse transcripted to cDNA. This cDNA was then used as a template for specic RCR amplication of the c-myc sequence and controlled by b-actin.

Conclusions
Indolizinones are N-heterocycles that have only recently appeared in the chemical literature, which can be prepared from the simple and achievable starting materials efficiently. Here, we rstly reported the indolizinone has the potential ability to recognize DNA MycG4 by uorescence spectra and varied temperature CD spectra. When indolizinone complexed with DNA MycG4 in the ratio of 1 : 2, and thus it can give an obvious uorescence enhancement by 11-fold, and meanwhile the DNA MycG4 can be stabilized up to 90 C. Further, we found indolizinone could down-regulate the transcription of oncogene c-myc in both of human non-small cell line (A549) and human laryngeal epithelial cell line (Hep-2) by real-time RT-PCR experiment. We believed that indolizinone will have a potential application in the exploration of anticancer drugs.

Conflicts of interest
There are no conicts to declare.