Jia-Heng Tan

G‐Quadruplexes: Targets in Anticancer Drug Design

G‐quadruplexes are special secondary structures adopted in some guanine‐rich DNA sequences. As guanine‐rich sequences are present in important regions of the eukaryotic genome, such as telomeres and the regulatory regions of many genes, such structures may play important roles in the regulation of biological events in the body. G‐quadruplexes have become valid targets for new anticancer drugs in the past few decades. Many leading compounds that target these structures have been reported, and a few of them have entered preclinical or clinical trials. Nonetheless, the selectivity of this kind of antitumor compound has yet to be improved in order to suppress the side effects caused by nonselective binding. As drug design targets, the topology and structural characteristics of quadruplexes, their possible biological roles, and the modes and sites of small‐ligand binding to these structures should be understood clearly. Herein we provide a summary of published research that has set out to address the above problem to provide useful information on the design of small ligands that target G‐quadruplexes. This review also covers research methodologies that have been developed to study the binding of ligands to G‐quadruplexes.

5-N-Methylated Quindoline Derivatives as Telomeric G-Quadruplex Stabilizing Ligands: Effects of 5-N Positive Charge on Quadruplex Binding Affinity and Cell Proliferation

A series of 5-N-methyl quindoline (cryptolepine) derivatives (2a- x) as telomeric quadruplex ligands was synthesized and evaluated. The designed ligands possess a positive charge at the 5- N position of the aromatic quindoline scaffold. The quadruplex binding of these compounds was evaluated by circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, nuclear magnetic resonance (NMR), and molecular modeling studies. Introduction of a positive charge not only significantly improved the binding ability but also induced the selectivity toward antiparallel quadruplex, whereas the nonmethylated derivatives tended to stabilize hybrid-type quadruplexes. NMR and molecular modeling studies revealed that the ligands stacked on the external G-quartets and the positively charged 5- N atom could contribute to the stabilizing ability. Long-term exposure of human cancer cells to 2r showed a remarkable cessation in population growth and cellular senescence phenotype and accompanied by a shortening of the telomere length.

Turning off Transcription of the bcl-2 Gene by Stabilizing the bcl-2 Promoter Quadruplex with Quindoline Derivatives

Human bcl-2 gene is an apoptosis-related oncogene containing a GC-rich sequence which is located upstream from P1 promoter and has the potential to form G-quadruplex structures. However, the regulatory role of the quadruplex and the effect of its ligands on bcl-2 have not been clarified. Here, we demonstrated that the G-quadruplex structure was disrupted when partial mutation of G --> A was made, resulting in a 2-fold increase in basal transcriptional activity of bcl-2 promoter. Quindoline derivatives, the highly active G-quadruplex ligands developed by our group, could significantly suppress bcl-2 transcriptional activation but had less effect on mutated bcl-2 transcription. These results provided direct evidence that G-quadruplex structure formed in bcl-2 promoter region could function as a transcriptional repressor element, and G-quadruplex specific ligands could regulate the transcription of bcl-2 through stabilization of quadruplex structure. The results further indicated that quindoline derivatives could induce apoptosis of HL-60 tumor cells.

9-N-Substituted berberine derivatives: Stabilization of G-quadruplex DNA and down-regulation of oncogene c-myc

A series of 9-N-substituted berberine derivatives (2a-j) were synthesized and evaluated as a new class of G-quadruplex binding ligands. G-quadruplex of DNA had been proven to be the transcription controller of human c-myc gene. The interaction of 9-N-substituted berberine derivatives with G-quadruplex DNA in c-myc was examined via EMSA, CD spectroscopy, FRET-melting method, PCR-stop assay, competitive dialysis, cell proliferation assay, and RT-PCR assay. The experiment results indicated that these derivatives could selectively induce and stabilize the formation of intramolecular parallel G-quadruplex in c-myc, which led to down-regulation of transcription of the c-myc in the HL60 lymphomas cell line. The related structure-activity relationships were also discussed.

Isaindigotone Derivatives: A New Class of Highly Selective Ligands for Telomeric G-Quadruplex DNA

Four isaindigotone derivatives (5a,b and 6a,b) designed as telomeric G-quadruplex ligands have been synthesized and characterized. The unfused aromatic rings in these compounds allow a flexible and adaptive conformation in G-quadruplex recognition. The interaction of human telomeric G-quadruplex DNA with these designed ligands was explored by means of FRET melting, fluorescence titration, CD spectroscopy, continuous variation, and molecular modeling studies. Our results showed that the adaptive scaffold might not only allow the ligands to well occupy the G-quartet but also perfectly bind to the grooves of the G-quadruplex. The synergetic effect of the multiple binding modes might be responsible for the improved binding ability and high selectivity of these ligands toward G-quadruplex over duplex DNA. Long-term exposure of HL60 and CA46 cancer cells to compound 5a showed a remarkable decrease in population growth, cellular senescence phenotype, and shortening of the telomere length, which is consistent with the behavior of an effective telomeric G-quadruplex ligand and telomerase inhibitor.

Design of selective G-quadruplex ligands as potential anticancer agents.

G-quadruplex structures are promising targets for design of cancer drugs with high selectivity and low toxicity. This review provides an update of the progress made over the last few years in the design of selective G-quadruplex ligands, and a comprehensive summary of the major design strategies and structural characteristics.

Inhibition of Cell Proliferation by Quindoline Derivative (SYUIQ-05) through its Preferential Interaction with c-myc Promoter G-Quadruplex

G-Quadruplex is a special DNA secondary structure and present in many important regulatory regions in human genome, such as the telomeric end and the promoters of some oncogenes. Specially, different forms of G-quadruplexes exist in telomeric DNA and c-myc promoter and play important roles in the pathway of cell proliferation and senescence. The effects of G-quadruplex ligands for either telomeric or c-myc G-quadruplex in vitro have been widely studied, but the specificity of these effects in vivo is still unknown. In the present research, various experiments were carried out to study the effect of G-quadruplex ligand SYUIQ-05 on tumor cell lines and the mechanism of this effect. Our results showed that it preferred to bind with G-quadruplex in c-myc and had rather insignificant effect on G-quadruplex in telomere. Therefore, it is possible that this compound had its antitumor activity for cancer cells mainly through its interaction with c-myc quadruplex.

Design, synthesis and evaluation of novel tacrine-multialkoxybenzene hybrids as dual inhibitors for cholinesterases and amyloid beta aggregation

A new series of tacrine-multialkoxybenzene hybrids (9a-9n) were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. All the synthesized compounds had high acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity with IC₅₀ values at the nanomolar range, which were much better than tacrine alone. A Lineweaver-Burk plot and molecular modeling study showed that these hybrids targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, compounds 9a-9f with methylenedioxybenzene moiety showed higher self-induced Aβ aggregation inhibitory activity than a reference compound, curcumin. These compounds could be selected as multi-potent agents for further investigation to treat AD.

Synthesis and evaluation of 9-O-substituted berberine derivatives containing aza-aromatic terminal group as highly selective telomeric G-quadruplex stabilizing ligands

A series of new 9-O-substituted berberine derivatives (4a-j) as telomeric quadruplex ligands was synthesized and evaluated. The results from biophysical and biochemical assay indicated that introducing of positive charged aza-aromatic terminal group into the side chain of 9-position of berberine significantly improved the binding ability with G-quadruplex, and exhibited the inhibitory effect on the hybridization and on telomerase activity. These derivatives showed excellent selectivity for telomeric G-quadruplex DNA over duplex.

Design, synthesis, and biological evaluation of curcumin analogues as multifunctional agents for the treatment of Alzheimer’s disease

A series of novel curcumin analogues were designed, synthesized, and evaluated as potential multifunctional agents for the treatment of AD. The in vitro studies showed that these compounds had better inhibitory properties against Aβ aggregation than curcumin. Superior anti-oxidant properties (better than the reference compound Trolox) of these compounds were observed by the oxygen radical absorbance capacity (ORAC) method and a cell-based assay using DCFH-DA as a probe. In addition they were able to chelate metals such as iron and copper and decrease metal-induced Aβ aggregation. The structure-activity relationships were discussed. The results suggested that our curcumin analogues could be selected as multifunctional agents for further investigation of AD treatment.