c-MYC is one of the important human proto-oncogenes, and transcriptional factor NM23-H2 can activate c-MYC transcription by recognizing the G-quadruplex in the promoter of the gene. Small molecules that inhibit c-MYC transcription by disrupting the NM23-H2/G-quadruplex interaction might be a promising strategy for developing selective anticancer agents. In recent studies, we developed a series of isaindigotone derivatives, which can bind to G-quadruplex and NM23-H2, thus down-regulating c-MYC ( J. Med. Chem. 2017, 60, 1292−1308). Herein, a series of novel isaindigotone derivatives were designed, synthesized, and screened for NM23-H2 selective binding ligands. Among them, compound 37 showed a high specific binding affinity to NM23-H2, effectively disrupting the interaction of NM23-H2 with G-quadruplex, and it strongly down-regulated c-MYC transcription. Furthermore, 37 induced cell cycle arrest and apoptosis, and it exhibited good tumor growth inhibition in a mouse xenograft model. This work provides a new strategy to modulate c-MYC transcription for the development of selective anticancer drugs.

•A new colorimetric and fluorescent dual probe selective for G-quadruplex was developed.•Carbazole and thiazole orange frameworks were first assembled to construct the probe.•The probe could be further employed in sensing G-quadruplex either in vitro or in cellulo.The development of selective and sensitive probes for sensing G-quadruplexes either in vitro or in cellulo has been the focus of investigation for a long time. Of those investigated, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) is a promising fluorescent probe utilized in many studies investigating G-quadruplex structures. However, its shortcomings, including similar fluorescence responses for G-quadruplex and duplex DNA and green but not red fluorescent emission, might restrict the further application of BMVC. In this study, in order to improve the selectivity and optical properties of BMVC, we engineered a new probe (TO-CZ) by incorporating thiazole orange (TO) into the structure of BMVC. We next found that TO-CZ can act as a colorimetric and red-emitting fluorescent dual probe selective for G-quadruplexes without affecting the G-quadruplex topology. Further experiments showed that a 2:1 binding model involving the external binding of TO-CZ to both ends of the G-quadruplex is the most possible binding mode. Furthermore, we applied TO-CZ in sensing G-quadruplexes both in vitro and in cellulo, and found that TO-CZ can selectively and sensitively visualize G-quadruplexes. Notably, TO-CZ might be used to map DNA and RNA G-quadruplexes in cellulo, showing its great potential in investigating intracellular G-quadruplex structures.

The RNA G-quadruplex is an important secondary structure formed by guanine-rich RNA sequences. However, its folding studies have mainly been studied in vitro. Accurate identification of RNA G-quadruplex formation within a sequence of interest remains difficult in cells. Herein, and based on the guanine-rich sequence in the 5′-UTR of NRAS mRNA, we designed and synthesized the first G-quadruplex-triggered fluorogenic hybridization (GTFH) probe, ISCH-nras1, for the unique visualization of the G-quadruplexes that form in this region. ISCH-nras1 is made up of two parts: The first is a fluorescent light-up moiety specific to G-quadruplex structures, and the second is a DNA molecule that can hybridize with a sequence that is adjacent to the guanine-rich sequence in the NRAS mRNA 5′-UTR. Further evaluation studies indicated that ISCH-nras1 could directly and precisely detect the targeted NRAS RNA G-quadruplex structures, both in vitro and in cells. Thus, this GTFH probe was a useful tool for directly investigating the folding of G-quadruplex structures within an RNA of interest and represents a new direction for the design of smart RNA G-quadruplex probes.

•New derivatives of thiazole orange were designed and synthesized.•Hydrocarbon rings were introduced into thiazole orange.•This simple modification improved selective detection of G-quadruplex.•Planarity and affinity were key factors responsible for the selective detection.Thiazole orange is a commonly used cyanine dye for binding to nucleic acids. Recently, it has been used for the detection of G-quadruplexes. However, thiazole orange is non-selective for G-quadruplex and other nucleic acids, thus hampering its further application. Herein, we designed and synthesized new fluorescent probes by incorporating hydrocarbon rings into the chromophore of thiazole orange. This simple modification dramatically improved selective binding to certain G-quadruplexes. The most promising probe, the cyclopentane fused analogue, exhibited significant fluorescence enhancement when treated with G-quadruplexes but retained weak fluorescence in the presence of double-stranded and single-stranded DNA. The cyclopentane fused probe also displayed considerable selectivity for parallel G-quadruplexes. These modifications reduced the quantum yield of thiazole orange. Further study of the mechanism revealed that the introduction of a hydrocarbon ring altered the planarity of the chromophore as well as the binding affinities for G-quadruplexes, and therefore, influenced the ability to detect G-quadruplexes.

•A novel colorimetric and fluorescent dual probe for parallel G-quadruplexes was discovered.•A dual-channel and accurate high-throughput method for identifying parallel G-quadruplexes was developed based on the probe.•The probe could be further employed in the high-throughput classification of parallel and anti-parallel G-quadruplexes.G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G‐quadruplex‐forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.

To efficiently identify small molecules binding to a G-quadruplex structure while avoiding binding to duplex DNA, we performed a multistep structure-based virtual screening by simultaneously taking into account G-quadruplex DNA and duplex DNA. Among the 13 compounds selected, one outstanding ligand shows significant selectivity for G-quadruplex binding as determined using SPR, FRET-based competition and luciferase activity assay.

G-quadruplexes are higher-order nucleic acid structures that have attracted extensive attention because of their biological significance and potential applications in supramolecular chemistry. An ever-increasing interest in G-quadruplexes has promoted the development of selective and sensitive fluorescent probes as research tools for these structures. However, most current studies primarily focus on the improved selectivity of probes for G-quadruplexes. Their detection limits or ways to improve their detection limits are rarely described. In this study, a new set of di-substituted triarylimidazole fluorescent probes were designed and synthesized, with the aim of upgrading the detection limit of a lead triarylimidazole IZCM-1 for G-quadruplexes. Among these compounds, IZCM-7 was the most promising candidate. The limit of detection (LOD) value of IZCM-7 for the G-quadruplex was up to 3 nM in solution and up to 5 ng in a gel matrix. These values were significantly improved in comparison with those of IZCM-1. Further biophysical studies revealed that the fluorescence quantum yield and binding affinity of IZCM-7 for G-quadruplexes were markedly increased, and these two factors might be responsible for the significantly improved detection limit of IZCM-7. In addition, the sensitive and selective fluorescence performance of IZCM-7 for G-quadruplexes remained the same even in the presence of large amounts of non-G-quadruplex competitors, suggesting its promising application prospect.

A tailor-made colorimetric and red-emitting fluorescent dual probe for G-quadruplex nucleic acids was developed by incorporating a coumarin–hemicyanine fluorophore into an isaindigotone framework. The significant and distinct changes in both the color and fluorescence of this probe enable the label-free and visual detection of G-quadruplex structures.

A novel 2,4,5-triaryl-substituted imidazole (IZCM-1) has been found to display distinct and specific fluorescence enhancement upon binding to parallel G-quadruplexes. Such a sensitive and topology-specific probe is able to light up without affecting the topology or thermal stability of the G-quadruplex sample. Thus, these advantages distinguish IZCM-1 from other G-quadruplex probes.

The c-KIT G-quadruplex structures are a novel class of attractive targets for the treatment of gastrointestinal stromal tumor (GIST). Herein, a series of new quinazolone derivatives with the expansion of unfused aromatic ring system were designed and synthesized. Subsequent biophysical studies demonstrated that the derivatives with adaptive scaffold could effectively bind to and stabilize c-KIT G-quadruplexes with good selectivity against duplex DNA. More importantly, these ligands further inhibited the transcription and expression of c-KIT gene and exhibited significant cytotoxicity on the GIST cell line HGC-27. Overall, these quinazolone derivatives represent a new class of promising c-KIT G-quadruplex ligands. The experimental results have also reinforced the idea of inhibition of c-KIT expression through targeting c-KIT G-quadruplex DNA.Keywords: adaptive scaffold; biophysical study; c-KIT G-quadruplex; cellular study; quinazolone derivatives;

The rapid and convenient method for identification of all kinds of G-quadruplex is highly desirable. In the present study, a novel colorimetric indicator for a vast variety of G-quadruplex was designed and synthesized on the basis of thiazole orange and isaindigotone skeleton. Its distinct color change enables label-free visual detection of G-quadruplexes, which is due to the disassembly of dye H-aggregates to monomers. This specific detection of G-quadruplex arises from its end-stacking interaction with G-quartet. On the basis of this universal indicator, a facile approach for large-scale identification of G-quadruplex was developed.

A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. The synthetic compounds had IC50 values at micro or nano molar range for cholinesterase inhibition, and some compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much better than the isaindigotone derivatives previously reported by our group. Most of these compounds showed higher self-induced Aβ aggregation inhibitory activity than a reference compound curcumin. The structure–activity relationship studies revealed that the derivatives with higher inhibition activity on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibition for both AChE and self-induced Aβ aggregation was further studied using CD, EM, molecular docking and kinetics.A variety of new isaindigotone derivatives were synthesized. Their structure–activity relationships were studied, and compound 6c was found to be an excellent dual inhibitors for acetylcholinesterase and amyloid beta aggregation.

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.A series of novel curcumin analogues were designed, synthesized, and biologically evaluated as multifunctional agents targeting β-amyloid, oxidative stress, and redox-active metal ions. Compound A4 was found to have the most potent Aβ aggregation inhibitory activity (IC50 = 2.5 μM).

Discovery of potent and selective ligands for telomeric G-quadruplex DNA is a challenging work. Through a combination approach of pharmacophore model construction, model validation, database virtual screening, chemical synthesis and interaction evaluation, we discovered and confirmed triaryl-substituted imidazole TSIZ01 to be a new telomeric G-quadruplex ligand with potent binding and stabilizing activity to G-quadruplex DNA, as well as a 8.7-fold selectivity towards telomeric G-quadruplex DNA over duplex DNA.

A series of graveoline and graveolinine derivatives were synthesized. The biological results showed that most of graveoline derivatives possessed higher cytotoxicity and better inhibitive effect against the adhesion and migration of human umbilical vein endothelial cell (HUVEC) than graveolinine derivatives. Among these compounds, 8d was the most potent agents that also showed significant anti-angiogenesis activities in chick embryo chorioallantoic membrane (CAM) assay.

A tailor-made colorimetric and red-emitting fluorescent dual probe for G-quadruplex nucleic acids was developed by incorporating a coumarin–hemicyanine fluorophore into an isaindigotone framework. The significant and distinct changes in both the color and fluorescence of this probe enable the label-free and visual detection of G-quadruplex structures.

To efficiently identify small molecules binding to a G-quadruplex structure while avoiding binding to duplex DNA, we performed a multistep structure-based virtual screening by simultaneously taking into account G-quadruplex DNA and duplex DNA. Among the 13 compounds selected, one outstanding ligand shows significant selectivity for G-quadruplex binding as determined using SPR, FRET-based competition and luciferase activity assay.

A novel 2,4,5-triaryl-substituted imidazole (IZCM-1) has been found to display distinct and specific fluorescence enhancement upon binding to parallel G-quadruplexes. Such a sensitive and topology-specific probe is able to light up without affecting the topology or thermal stability of the G-quadruplex sample. Thus, these advantages distinguish IZCM-1 from other G-quadruplex probes.