Transcriptional regulation of telomeric repeat-containing RNA by acridine derivatives.

Telomere is a specialized DNA-protein complex that plays an important role in maintaining chromosomal integrity. Shelterin is a protein complex formed by six different proteins, with telomeric repeat factors 1 (TRF1) and 2 (TRF2) binding to double-strand telomeric DNA. Telomeric DNA consists of complementary G-rich and C-rich repeats, which could form G-quadruplex and intercalated motif (i-motif), respectively, during cell cycle. Its G-rich transcription product, telomeric repeat-containing RNA (TERRA), is essential for telomere stability and heterochromatin formation. After extensive screening, we found that acridine derivative 2c and acridine dimer DI26 could selectively interact with TRF1 and telomeric i-motif, respectively. Compound 2c blocked the binding of TRF1 with telomeric duplex DNA, resulting in up-regulation of TERRA. Accumulated TERRA could bind with TRF1 at its allosteric site and further destabilize its binding with telomeric DNA. In contrast, DI26 could destabilize telomeric i-motif, resulting in down-regulation of TERRA. Both compounds exhibited anti-tumour activity for A549 cells, but induced different DNA damage pathways. Compound 2c significantly suppressed tumour growth in A549 xenograft mouse model. The function of telomeric i-motif structure was first studied with a selective binding ligand, which could play an important role in regulating TERRA transcription. Our results showed that appropriate level of TERRA transcript could be important for stability of telomere, and acridine derivatives could be further developed as anti-cancer agents targeting telomere. This research increased understanding for biological roles of telomeric i-motif, TRF1 and TERRA, as potential anti-cancer drug targets.

Synthesis and Biological Evaluation of Oleanolic Acid Derivatives as Selective Vascular Endothelial Growth Factor Promoter i-Motif Ligands.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor and plays a key role in tumor progression. The C-rich DNA sequence of VEGF promoter can form i-motif structure, which is a potential target for the development of novel anticancer agents. However, there is a limited number of chemotypes as the selective ligands of VEGF promoter i-motif, which leaves much room for development. Herein, we report the discovery of the natural oleanolic acid scaffold as a novel chemotype for the development of selective ligands of VEGF i-motif. A series of oleanolic acid derivatives as VEGF promoter i-motif ligands were synthesized. Subsequent evaluations showed that 3c could selectively bind to and stabilize VEGF promoter i-motif without significant binding to G-quadruplex, duplex DNA, and other oncogene i-motifs. Cell-based assays indicated that 3c could effectively downregulate VEGF gene transcription and expression in MCF-7 cells, inhibit tumor cells proliferation and migration, and induce cancer cells apoptosis. This work provides evidence of VEGF promoter i-motif as an anticancer target and will facilitate future efforts for the discovery of oleanolic acid-based selective ligands of VEGF promoter i-motif.

Syntheses and evaluation of acridone-naphthalimide derivatives for regulating oncogene PDGFR-ß expression.

Upregulation of platelet-derived growth factor receptor ß (PDGFR-ß) has been found to be associated with development of various types of cancers, which has become an attractive target for anti-tumor treatment. Previously, we have synthesized and studied an acridone derivative B19, which can selectively bind to and stabilize oncogene c-myc promoter i-motif, resulting in down-regulation of c-myc transcription and translation, however its effect on tumor cells apoptosis requires improvement. In the present study, we synthesized a variety of B19 derivatives containing a known anti-cancer fluorescent chromophore naphthalimide for the purpose of enhancing anti-cancer activity. After screening, we found that acridone-naphthalimide derivative WZZ02 could selectively stabilize PDGFR-ß promoter G-quadruplex and destabilize its corresponding i-motif structure, without significant interaction to other oncogenes promoter G-quadruplex and i-motif. WZZ02 down-regulated PDGFR-ß gene transcription and translation in a dose-dependent manner, possibly due to above interactions. WZZ02 could significantly inhibit cancer cell proliferation, and induce cell apoptosis and cycle arrest. WZZ02 exhibited tumor growth inhibition activity in MCF-7 xenograft tumor model, which could be due to its binding interactions with PDGFR-ß promoter G-quadruplex and i-motif. Our results suggested that WZZ02 as a dual G-quadruplex/i-motif binder could be effective on both oncogene replication and transcription, which could become a promising lead compound for further development with improved potency and selectivity. The wide properties for the derivatives of 1,8-naphthalimide could facilitate further in-depth mechanistic studies of WZZ02 through various fluorescent physical and chemical methods, which could help to further understand the function of PDGFR-ß gene promoter G-quadruplex and i-motif.

Syntheses and Evaluation of New Bisacridine Derivatives for Dual Binding of G-Quadruplex and i-Motif in Regulating Oncogene c-myc Expression.

The c-myc oncogene is an important regulator for cell growth and differentiation, and its aberrant overexpression is closely related to the occurrence and development of various cancers. Thus, the suppression of c-myc transcription and expression has been investigated for cancer treatment. In this study, various new bisacridine derivatives were synthesized and evaluated for their binding with c-myc promoter G-quadruplex and i-motif. We found that a9 could bind to and stabilize both G-quadruplex and i-motif, resulting in the downregulation of c-myc gene transcription. a9 could inhibit cancer cell proliferation and induce SiHa cell apoptosis and cycle arrest. a9 exhibited tumor growth inhibition activity in a SiHa xenograft tumor model, which might be related to its binding with c-myc promoter G-quadruplex and i-motif. Our results suggested that a9 as a dual G-quadruplex/i-motif binder could be effective in both oncogene replication and transcription and become a promising lead compound for further development with improved potency and selectivity.

Upregulation of BCL-2 by acridone derivative through gene promoter i-motif for alleviating liver damage of NAFLD/NASH.

Nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) are global epidemic public health problems with pathogenesis incompletely understood. Hepatocyte excessive apoptosis is a significant symbol for NAFLD/NASH patients, and therefore anti-apoptosis therapy could be used for NAFLD/NASH treatment. Up-regulation of BCL-2 has been found to be closely related with anti-apoptosis. BCL-2 gene promoter region has a C-rich sequence, which can form i-motif structure and play important role in regulating gene transcription. In this study, after extensive screening and evaluation, we found that acridone derivative A22 could up-regulate BCL-2 transcription and translation in vitro and in cells through selective binding to and stabilizing BCL-2 gene promoter i-motif. Our further experiments showed that A22 could reduce hepatocyte apoptosis in NAFLD/NASH model possibly through up-regulating BCL-2 expression. A22 could reduce inflammation, endoplasmic reticulum stress and cirrhosis in high-fat diet-fed mice liver model. Our findings provide a potentially new approach of anti-apoptosis for NAFLD/NASH treatment, and A22 could be further developed as a lead compound for NAFLD/NASH therapy. Our present study first demonstrated that gene promoter i-motif could be targeted for gene up-regulation for extended treatment of other important diseases besides cancer.

Syntheses and evaluation of new Quinoline derivatives for inhibition of hnRNP K in regulating oncogene c-myc transcription.

Aberrant overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key feature in oncogenesis and progression of many human cancers. hnRNP K has been found to be a transcriptional activator to up-regulate c-myc gene transcription, a critical proto-oncogene for regulation of cell growth and differentiation. Therefore, down-regulation of c-myc transcription by inhibiting hnRNP K through disrupting its binding to c-myc gene promoter is a potential approach for cancer therapy. In the present study, we synthesized and screened a series of Quinoline derivatives and evaluated their binding affinity for hnRNP K. Among these derivatives, (E)-1-(4-methoxyphenyl)-3-(4-morpholino-6-nitroquinolin-2-yl)prop-2-en-1-one (compound 25) was determined to be the first-reported hnRNP K binding ligand with its KD values of 4.6 and 2.6 µM measured with SPR and MST, respectively. Subsequent evaluation showed that the binding of compound 25 to hnRNP K could disrupt its unfolding of c-myc promoter i-motif, resulting in down-regulation of c-myc transcription. Compound 25 showed a selective anti-proliferative effect on human cancer cell lines with IC50 values ranged from 1.36 to 3.59 µM. Compound 25 exhibited good tumor growth inhibition in a Hela xenograft tumor model, which might be related to its binding with hnRNP K. These findings illustrated that inhibition of DNA-binding protein hnRNP K by compound 25 could be a new and selective strategy of regulating oncogene transcription instead of targeting promoter DNA secondary structures such as G-quadruplexes or i-motifs.

Syntheses and evaluation of new acridone derivatives for selective binding of oncogene c-myc promoter i-motifs in gene transcriptional regulation.

We synthesized a series of acridone derivatives for specific binding ligands of i-motifs. Subsequent evaluations showed that B19 could selectively bind to and stabilize the c-myc promoter i-motif without significant binding to the G-quadruplex and duplex DNA. This caused down-regulation of c-myc transcription and expression, resulting in tumor cell apoptosis.