Search

Homologous recombination repair (HRR), a crucial approach in DNA damage repair, is an attractive target in cancer therapy and drug design. The Bloom syndrome protein (BLM) is a 3'-5' DNA helicase that performs an important role in HRR regulation. However, limited studies about BLM inhibitors and their biological effects have been reported. Here, we identified a class of isaindigotone derivatives as novel BLM inhibitors by synthesis, screening, and evaluating. Among them, compound 29 was found as an effective BLM inhibitor with a high binding affinity and good inhibitory effect on BLM. Cellular evaluation indicated that 29 effectively disrupted the recruitment of BLM at DNA double-strand break sites, promoted an accumulation of RAD51, and regulated the HRR process. Meanwhile, 29 significantly induced DNA damage responses, as well as apoptosis and proliferation arrest in cancer cells. Our finding provides a potential anticancer strategy based on interfering with BLM via small molecules.

The arginine/glycine-rich region termed the RGG domain is usually found in G-quadruplex (G4)-binding proteins and is important in G4-protein interactions. Studies on the binding mechanism of RGG domains found that small segments (RGG motif) inside the domain contribute greatly to the G4 binding affinity. However, unlike the entire RGG domains that have been broadly explored, the role of the RGG motif remains obscure, with very limited study. Herein, to clarify the role of the RGG motif in G4-protein interactions, we systematically investigated the binding affinity and mode between RGG-motif peptides and G4s. The internal arrangement of RGG repeats and gap amino acids played a more crucial role in the G4-binding mechanism than a critical number of RGG repeats. Arginines and phenylalanines at the exact position of the RGG motif might enable additional hydrogen bonding and π-stacking interaction with nucleobases and strengthen the binding of G4. Impressively, proceeding from a G4-binding RGG peptide, 12, discovered above, we identified the cold-inducible RNA-binding protein (CIRBP) as a new G4 DNA-binding protein both in vitro and in cells. In addition, we found that the key amino acids for G4 binding in peptide 12 and CIRBP were highly similar, and peptide 12 clearly played a key role in the G4 binding of CIRBP. This report is the first in which a G4-binding protein was identified from exploration of the G4-binding RGG motif. Our findings suggest a novel strategy for discovering new G4-binding proteins by exploring key peptide segments.

Telomeric G-quadruplex targeting and telomere maintenance interference are emerging as attractive strategies for anticancer therapies. Here, a novel molecular scaffold is explored for telomeric G-quadruplex targeting. A series of novel schizocommunin derivatives was designed and synthesized as potential telomeric G-quadruplex ligands. The interaction of telomeric G-quadruplex DNA with the derivatives was explored by biophysical assay. The cytotoxicity of the derivatives toward cancer cell lines was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Among the derivatives, compound 16 showed great stabilization ability toward telomeric G-quadruplex DNA and good cytotoxicity toward cancer cell lines. Further cellular experiments indicated that 16 could induce the formation of telomeric G-quadruplex in cells, triggering a DNA damage response at the telomere and causing telomere dysfunction. These effects ultimately provoked p53-mediated cell cycle arrest and apoptosis, and suppressed tumor growth in a mouse xenograft model. Our work provides a novel scaffold for the development of telomeric G-quadruplex ligands.

The promoter of hnRNP K oncogene was found to contain a G/C-rich sequence on the same DNA strand, which can form interconvertible G-quadruplex, i-motif, and hairpin structures. Protein CNBP could bind and stabilize the G-quadruplex, inducing transformation of the hairpin into the G-quadruplex, resulting in down-regulation of hnRNP K transcription. In contrast, Corticosterone could bind and stabilize the hairpin, inducing transformation of the G-quadruplex into the hairpin, resulting in up-regulation of hnRNP K gene transcription.

G-Quadruplex nucleic acids or G-quadruplexes (G4s) are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. They are widely distributed in functional regions of the human genome, such as telomeres, ribosomal DNA (rDNA), transcription start sites, promoter regions and untranslated regions of mRNA, suggesting that G-quadruplex structures may play a pivotal role in the control of a variety of cellular processes. G-Quadruplexes are viewed as valid therapeutic targets in human cancer diseases. Small molecules, from naturally occurring to synthetic, are exploited to specifically target G-quadruplexes and have proven to be a new class of anticancer agents. Notably, alkaloids are an important source of G-quadruplex ligands and have significant bioactivities in anticancer therapy. In this review, the authors provide a brief, up-to-date summary of heterocyclic alkaloids and their derivatives targeting G-quadruplexes., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

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 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 improve the selectivity of indoloquinoline or benzofuroquinoline derivatives, we previously reported several quinazoline derivatives [17]. These compounds could mimic a tetracyclic aromatic system through intramolecular hydrogen bond. Studies showed that these quinazoline derivatives were effective and selective telomeric G-quadruplex ligands. With this encouragement, here we synthesized a series of N-(2-(quinazolin-2-yl)phenyl)benzamide (QPB) compounds as modified quinazoline derivatives. In this modification, a phenyl group was introduced to the aromatic core. The evaluation results showed that part of QPB derivatives had stronger binding ability and better selectivity for telomeric G-quadruplex DNA than LZ-11, the most potential compound of reported quinazoline derivatives. Furthermore, telomerase inhibition of QPB derivatives and their cellular effects were studied., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

12-N-Methylated and non-methylated 5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized as new series of c-myc G-quadruplex binding ligands. Their interactions with c-myc G-quadruplex were evaluated using fluorescence resonance energy transfer (FRET) melting assay, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), polymerase chain reaction (PCR) stop assay, and molecular modeling. Compared with the non-methylated derivatives, 12-N-methylated derivatives had stronger binding affinity and stabilizing ability to c-myc G-quadruplex structure, and could more effectively stack on the G-quartet surface. All these derivatives had high selectivity for c-myc G-quadruplex DNA over duplex DNA. The reverse transcription (RT) PCR assay showed that compound 21c could down-regulate transcription of c-myc gene in Ramos cell line containing NHE III(1) element, but had no effect in CA46 cell line with NHE III(1) element removed., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A series of 2,4-disubstituted quinazoline derivatives found to be a new type of highly selective ligand to bind with telomeric G-quadruplex DNA, and their biological properties were reported for the first time.Their interactions with telomeric G-quadruplex DNA were evaluated by using fluorescence resonance energy transfer (FRET) melting assay, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and molecular modeling. Our results showed that these derivatives could well recognize G-quadruplex and have high selectivity toward G-quadruplex over duplex DNA. The structure-activity relationships (SARs) study revealed that the disubstitution of quinazoline and the length of the amide side chain were important for its interaction with the G-quadruplex. Furthermore, telomerase inhibition of the quinazoline derivatives and their cellular effects were studied., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

G-quadruplex structures are a new class of attractive targets for DNA-interactive anticancer agents. The primary building block of this structure is the G-quartet, which is composed of four coplanar guanines and serves as the major binding site for small molecules. NMR studies and molecular dynamics simulations have suggested that the planarity of G-quartet surface has been highly dynamic in solution. To better investigate how the planarity of unfused aromatic ligand impacts on its quadruplex binding properties, a variety of planarity controllable isaindigotone derivatives were designed and synthesized. The interaction of G-quadruplex DNA with these designed ligands was systematically explored using a series of biophysical studies. The FRET-melting, SPR, and CD spectroscopy results showed that reducing the planarity of their unfused aromatic core resulted in their decreased binding affinity and stabilization ability for G-quadruplex. NMR studies also suggested that these compounds could stack on the G-quartet surface. Such results are in parallel with subsequent molecular modeling studies. A detailed binding energy analysis indicated that van der Waals energy (ΔE(vdw)) and entropy (TΔS) are responsible for their decreased quadruplex binding and stabilization effect. All these results provided insight information about how quadruplex recognition could be controlled by adjusting the planarity of ligands, which shed light on further development of unfused aromatic molecules as optimal G-quadruplex binding ligands.

A series of quinolino-benzo-[5, 6]-dihydroisoquindolium compounds (3a, 3f, 3g, and 3j) derived from alkaloid berberine were designed and synthesized as novel G-quadruplex ligands. Subsequent biophysical and biochemical evaluation demonstrated that the addition of pyridine ring and amino group into berberine improved the binding ability and selectivity towards G-quadruplex DNA in comparison with the previously reported 9-N-substituted berberine derivatives. Furthermore, qRT-PCR assay showed compound 3j led the down-regulation of c-myc gene transcription in leukemia cell line HL60, while little effect on normal cell line ECV-304, which was consistent with the behavior of an effective G-quadruplex ligand targeting c-myc oncogene., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences, and they are attractive anticancer drug targets. Understanding the three-dimensional interactions between a G-quadruplex and its ligand in solution is the key to discovering a drug lead. Hence, from crystallographic or NMR structures, molecular dynamics studies have been performed on six ligand-quadruplex complexes. BRACO-19, BSU6039, daunomycin, RHPS4, MMQ1, and TMPyP4 are the six ligands that bind to the G-quadruplex structures in the studies. Based on molecular dynamics simulations and a series of computational analyses, the results suggest that ions move away from the external G-quartet to let the ligand bind to the quadruplex in aqueous solution. The ligand binding can increase the stability of the Hoogseen hydrogen bonds within the G-quartet. However, the G-quartet binding site can only fit one ligand molecule. The ligand can form hydrogen bonds at the loop or flank of the quadruplex. However, not all the interactions will stabilize the ligand-quadruplex complex in aqueous solution. These findings can assist in the design of selective and potent G-quadruplex ligands.

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.

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.

A series of novel oxoisoaporphine alkaloid derivatives, 9-aminoalkanamido-1-azabenzanthrone (general formula Ar-NHCO(CH(2))(n)NR(2), Ar=1-azabenzanthrone, n=1, 2 or 3), had been synthesized. Compared with 1-azabenzanthrone, the derivatives had significantly higher DNA binding affinity with calf thymus DNA, and higher potent cytotoxicity against different tumor cell lines. The cytotoxicity and the structure-activity relationship of the prepared compounds were studied. The derivatives with two methylene groups (n=2), and piperidine or ethanolamine functional group in the side chain exhibited highest DNA binding affinity and cytotoxicity.

The DNA binding properties of three cation-substituted anthrapyrazole derivatives of emodin with calf thymus DNA were characterized by spectroscopic methods and the specific binding modes were elucidated. At low drug and high DNA concentrations, compound 1 with a mono-cationic amino side chain exhibited an intercalative binding mode, 2 with a much longer and more flexible di-cationic side chain exhibited an external binding mode, and 3 with a rigid di-cationic side chain exhibited both intercalative and external binding modes. The DNA binding mode of compounds was altered after structural modification. The molecular structure-DNA binding relationships found from this study may be useful for the design of anthrapyrazole derivatives with desired binding characteristics.

The interaction of berberine and its 9-substituted derivatives with human telomeric DNA d[G(3)(T(2)AG(3))(3)](telo21) has been investigated via CD spectroscopy, fluorescence spectroscopy, PCR-stop assay, competitive dialysis, and telomerase repeat amplification protocol (TRAP) assay. The results indicated that these semisynthesized compounds could induce and stabilize the formation of anti-parallel G-quadruplex of telomeric DNA in the presence or absence of metal cations. Compared with berberine, the 9-substituted derivatives exhibit stronger binding affinity with G-quadruplex and higher inhibitory activity for telomerase. Introduction of a side chain with proper length of methylene and terminal amino group to the 9-position of berberine would significantly strengthen the binding affinity with G-quadruplex, resulting in increasing inhibitory effects on the amplification of telo21 DNA and on the telomerase activity.

Stabilization of G-quadruplex structures in the promoter region of certain oncogenes is an emerging field in anticancer drug design. Human c-myc gene is one of these oncogenes, and G-quadruplexes have been proven to be the transcriptional controller of this gene. In the present study, the interaction of quindoline derivatives with G-quadruplexes in c-myc was investigated. The experimental results indicated that these derivatives have the ability to induce/stabilize the G-quadruplexes in c-myc, which lead to down-regulation of the c-myc in the Hep G2 cell line. It was found that derivatives with terminal amino groups in their side chains would selectively bind to the isomers with the double nucleotide loops in the absence of K+. Molecular modeling studies revealed the binding mode between the derivatives and the G-quadruplexes is end-stacking at the 3'-position, and the positively charged side chain on the quindoline derivatives may contribute to the selectivity to certain loop isomers of topological quadruplexes as well as the improved stabilization action.

A new series of quindoline derivatives (4a-j) were designed and synthesized to develop novel and potent telomerase inhibitors. The interaction of the G-quadruplex of human telomere DNA with these newly designed molecules was examined via circular dichroism spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative (4a) and G-quadruplex or duplex DNA was investigated by competition dialysis. These new compounds as inhibitors of telomerase were also investigated through the utilization of modified telomerase repeat amplification protocol (TRAP) assay. The results revealed that the introduction of electron-donating groups such as substituted amino groups at the C-11 position of quindoline significantly improved the inhibitory effect on telomerase activity ((Tel)IC50 > 138 microM for quindoline, 0.44-12.3 microM for quindoline derivatives 4a-j). The quindoline derivatives not only stabilized the G-quadruplex structure but also induced the G-rich telomeric repeated DNA sequence to fold into quadruplex.

Since many of the pivotal questions confronting chemical biology concern the interaction of more than one analyte, it is essential to investigate multiple analytes simultaneously. While commercial fluorescent dyes capable of detecting and quantitating DNA or RNA are widely used in biological research, few probes can be applied in DNA and RNA together. Distraction from each other is also a puzzling question for individual detection. Here, we have discovered a dual-responsive probe, namely, DR1 , which can selectively recognize DNA by emitting out red fluorescence in the nucleus, while an entirely different stain pattern by labeling cytoplasm and nuclei with distinct cyanine fluorescence is observed owing to binding with RNA. Mechanism study found that DR1 binds to DNA in a monomer state with red fluorescence, and therefore the cyanine fluorescence may derive from its aggregated state while it binds to RNA. Then, DR1 was applied to quantify DNA and RNA contents and evaluate the effects of several inhibitors comprehensively on high-content screening platforms. Our study provided a new tool for simultaneously monitoring cellular DNA and RNA, and gaining new insights into developing dual-channel probes for multiple biomolecules. • The dual-color imaging probe DR1 was developed to monitor DNA and RNA simultaneously in cells. • Dual-responsive mechanism of DR1 was discovered to be related to aggregation and monomer status. • A high-content imaging method of evaluating DNA and RNA modulators effects was established based on our probe. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: A series of bisaryldiketene derivatives were designed and synthesized as a new class of specific G-quadruplex ligands. The ligand–quadruplex interactions were further evaluated by FRET, ITC, and PCR stop assay. In contrast to most of the G-quadruplex ligands reported so far, which comprise an extended aromatic ring, these compounds are neither polycyclic nor macrocyclic, but have a non-aromatic and relative flexible linker between two quinoline moieties enabling the conformation of compounds to be flexible. Our results showed that these bisaryldiketene derivatives could selectively recognize G-quadruplex DNA rather than binding to duplex DNA. Moreover, they showed promising discrimination between different G-quadruplex DNA. The primary binding affinity of ligand M2 for c-myc G-quadruplex DNA was over 200 times larger than that for telomere G-quadruplex DNA. [ABSTRACT FROM AUTHOR]

Abstract: Mg2+ is one of the most important cations in cells, affecting the structures and functions of the proteins and nucleic acids. It should be noted that Mg2+ is indispensable in DNA transcription, where G-quadruplex is believed to be actively involved. Therefore, it is important to investigate the influence of Mg2+ on G-quadruplex. Here we studied the effect of Mg2+ on G-quadruplex DNA with CD, FRET, EMSA, and PCR-stop assay. We found that various G-quadruplexes could be differentiated through simultaneous addition of both K+ and Mg2+, which could be used for selective identification of G-quadruplexes in promoter oncogene but not in telomere. Mg2+ at physiological relevant concentration not only greatly enhanced the thermostability of oncogene G-quadruplexes but also efficiently protected them from unfolding by their complementary strands, which revealed the great impact of Mg2+ on the equilibrium between promoter G-quadruplex and duplex DNA. The PCR-stop assay further confirmed that Mg2+ could affect gene transcription by stabilizing promoter G-quadruplex. The above studies were carried out for various G-quadruplexes of varying sequences in promoter oncogenes and telomeric region. Our results suggest that Mg2+ may be a key regulator for G-quadruplexes of oncogene promoter, which can subsequently affect the expression of related genes. [ABSTRACT FROM AUTHOR]