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Here we have developed an immunosorbent assay to accurately and sensitively monitor helicase unwinding a broad range of DNA substrate structures. This assay can be utilized to investigate diverse helicases' functions and to identify their inhibitors.

Inter-organelle interactions play a vital role in diverse biological processes. Thus, chemical tools are highly desirable for understanding the spatiotemporal dynamic interplay among organelles in live cells and

The development of triple-negative breast cancer (TNBC) is highly associated with G-quadruplex (G4); thus, targeting G4 is a potential strategy for TNBC therapy. Because concomitant histone deacetylases (HDAC) inhibition could amplify the impact of G4-targeting compounds, we designed and synthesized two novel series of G4/HDAC dual-targeting compounds by connecting the zinc-binding pharmacophore of HDAC inhibitors to the G4-targeting isaindigotone scaffold (

Ligand-Induced duplex-quadruplex transition within the c-MYC promoter region is one of the most studied and advanced ideas for c-MYC regulation. Despite its importance, there is a lack of methods for monitoring such process in cells, hindering a better understanding of the essence of c-MYC G-quadruplex as a drug target. Here we developed a new fluorescent probe ISCH-MYC for specific c-MYC G-quadruplex recognition based on GTFH (G-quadruplex-Triggered Fluorogenic Hybridization) strategy. We validated that ISCH-MYC displayed distinct fluorescence enhancement upon binding to c-MYC G-quadruplex, which allowed the duplex-quadruplex transition detection of c-MYC G-rich DNA in cells. Using ISCH-MYC, we successfully characterized the induction of duplex to G-quadruplex transition in the presence of G-quadruplex stabilizing ligand PDS and further monitored and evaluated the altered interactions of relevant transcription factors Sp1 and CNBP with c-MYC G-rich DNA. Thus, our study provides a visualization strategy to explore the mechanism of G-quadruplex stabilizing ligand action on c-MYC G-rich DNA and relevant proteins, thereby empowering future drug discovery efforts targeting G-quadruplexes.

Recent successes in construction of light-up RNA aptamers allowed fluorescence-based live-cell imaging of RNAs. In addition, light-up aptamers have been converted into signaling aptamers that enable fluorometric detection of small chemicals. To date, only a single target chemical has been detected at a time in cells. In this study, we selected cladogenetic orthogonal light-up aptamers that output three different colors from the RNA library having the same ligand binding core. Two of the three functioned in mammalian cells. These two aptamers, which fluoresce blue and green upon binding of cognate fluorogen, were converted into signaling aptamers. Using these signaling aptamers in combination with a previously described light-up aptamer with red fluorescence, we demonstrated simultaneous detection of multiple chemicals in living cells. The cladogenetic orthogonal light-up aptamers developed in this study and the simple strategy for rational designing of the signaling aptamers will provide innovative advances in the field of RNA-based bioimaging.

Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and in vivo, in-depth studies of the biological roles of this attractive membraneless organelle have been limited. Herein, we report the first small-molecule probe, TASG, for the selective, convenient and real-time monitoring of SGs. This novel molecule can simultaneously bind to SG RNAs, the core SG protein G3BP1, and their complexes, triggering a significant enhancement in fluorescence intensity, making TASG broadly applicable to SG imaging under various stress conditions in fixed and live cells, ex vivo and in vivo. Using TASG, the complicated endogenous SG dynamics were revealed in both live cells and C. elegans. Collectively, our work provides an ideal probe that has thus far been absent in the field of SG investigations. We anticipate that this powerful tool may create exciting opportunities to investigate the underlying roles of SGs in different organisms.

RNA G-quadruplex (rG4) structures in the 5' untranslated region (5'UTR) play crucial roles in fundamental cellular processes. ADAR is an important enzyme that binds to double-strand RNA and accounts for the conversion of Adenosine to Inosine in RNA editing. However, so far there is no report on the formation and regulatory role of rG4 on ADAR expression. Here, we identify and characterize a thermostable rG4 structure within the 5'UTR of the ADAR1 mRNA and demonstrate its formation and inhibitory role on translation in reporter gene and native gene constructs. We reveal rG4-specific helicase DHX36 interacts with this rG4 in vitro and in cells under knockdown and knockout conditions by GTFH (G-quadruplex-triggered fluorogenic hybridization) probes and modulates translation in an rG4-dependent manner. Our results further substantiate the rG4 structure-DHX36 protein interaction in cells and highlight rG4 to be a key player in controlling ADAR1 translation.

Supplementary Materials forpublication.

The homologous recombination repair (HRR) pathway is critical for repairing double-strand breaks (DSB). Inhibition of the HRR pathway is usually considered a promising strategy for anticancer therapy. The Bloom's Syndrome Protein (BLM), a DNA helicase, is essential for promoting the HRR pathway. Previously, we discovered quinazolinone derivative 9h as a potential BLM inhibitor, which suppressed the proliferation of colorectal cancer (CRC) cell HCT116. Herein, a new series of quinazolinone derivatives with N3-substitution was designed and synthesized to improve the anticancer activity and explore the structure-activity relationship (SAR). After evaluating their BLM inhibitory activity, the SAR was discussed, leading to identifying compound 21 as a promising BLM inhibitor. 21 exhibited the potent BLM-dependent cytotoxicity against the CRC cells but weak against normal cells. Further evaluation revealed that 21 could disrupt the HRR level while inhibiting BLM located on the DSB site and trigger DNA damage in the CRC cells. This compound effectively suppressed the proliferation and invasion of CRC cells, along with cell cycle arrest and apoptosis. Consequently, 21 might be a promising candidate for treating CRC, and the BLM might be a new potential therapeutic target for CRC.

Benzothiazolium and benzoxazolium are common groups for the construction of hemicyanine dyes; however, their isosteric analogue benzoselenazolium have rarely been studied. Here, we report the development of the first benzoselenazolium-based hemicyanine dye for the selective detection of G-quadruplexes. This molecule, SEMA-1, was validated as a red-emitting and activatable fluorescent probe whose fluorescence would only be activated in the presence of G-quadruplexes in buffer solution. Consistent with this, SEMA-1 was found to accumulate in nucleoli and could be used to detect the high abundance of nucleolar rDNA and rRNA G-quadruplexes in fixed HeLa cells. On the other hand, due to the retained mitochondrial membrane potential in live HeLa cells, SEMA-1 was captured by mitochondria and had the potential to detect the mitochondrial G-quadruplexes. Collectively, this work demonstrates the value of developing G-quadruplex-specific fluorescent probes from novel benzoselenazolium-based hemicyanine scaffold.

The mitochondrial DNA G-quadruplex (mtDNA G4) is a potential regulatory element for the regulation of mitochondrial functions; however, its relevance and specific roles in diseases remain largely unknown. Here, we engineered a set of chemical probes, including

DNA damage response (DDR) pathways are crucial for the survival of cancer cells and are attractive targets for cancer therapy. Bloom syndrome protein (BLM) is a DNA helicase that performs important roles in DDR pathways. Our previous study discovered an effective new BLM inhibitor with a quinazolinone scaffold by a screening assay. Herein, to better understand the structure-activity relationship (SAR) and biological roles of the BLM inhibitor, a series of new derivatives were designed, synthesized, and evaluated based on this scaffold. Among them, compound 9h exhibited nanomolar inhibitory activity and binding affinity for BLM. 9h could effectively disrupt BLM recruitment to DNA in cells. Furthermore, 9h inhibited the proliferation of the colorectal cell line HCT116 by significantly triggering DNA damage in the telomere region and inducing apoptosis, especially in combination with a poly (ADP-ribose) polymerase (PARP) inhibitor. This result suggested a synthetic lethal effect between the BLM and PARP inhibitors in DDR pathways.

Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and

Background and purpose Nonalcoholic steatohepatitis (NASH) is the more severe form of metabolic associated fatty liver disease (MAFLD), and no pharmacologic treatment approved as yet. Identification of novel therapeutic targets and their agents are critical to overcome the current inadequacy of drug treatment for NASH. Experimental approach The correlation between heat shock factor 1 (HSF1) levels and the development of NASH and the target genes of HSF1 in hepatocyte were revealed by chromatin-immunoprecipitation sequencing. The effects and mechanisms of SYSU-3d in alleviating NASH were examined in relevant cell models and mouse models (the Ob/Ob mice, high-fat and high-cholesterol diet, the methionine-choline deficient diet fed mice). The drug-like properties of SYSU-3d in vivo were evaluated. Key results HSF1 is progressively reduced with mitochondrial dysfunction in NASH pathogenesis and activation of this transcription factor by its newly-identified activator SYSU-3d efficiently ameliorated all manifestations of NASH in mice. When activated, the phosphorylated HSF1 (Ser326) translocated to nucleus and bound to the promoter of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) to induce mitochondrial biogenesis, thus increasing mitochondrial adaptive oxidation and inhibiting oxidative stress. The deletion of HSF1 and PGC-1α or recovery of HSF1 in HSF1-deficiency cells revealed the HSF1/PGC-1α metabolic axis mainly responsible for the anti-NASH effects of SYSU-3d independent of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). Conclusion and implications Activation of HSF1 is a practicable therapeutic approach for NASH treatment via the HSF1/PGC-1α/mitochondrial axis, and SYSU-3d would take into consideration as a potential candidate for the treatment of NASH.

The gut bacterium Akkermansia muciniphila has been increasingly recognized for its therapeutic potential in treating metabolic disorders, including obesity, diabetes, and metabolicdysfunction-associated fatty liver disease (MAFLD). However, its underlying mechanism involved in its well-known metabolic actions needs further evaluation. The present study explored the therapeutic effect and mechanism of A. muciniphila in intervening MAFLD by using a high-fat and high-cholesterol (HFC) diet induced obese mice model. Mice treated with A. muciniphila efficiently reversed MAFLD in the liver, such as hepatic steatosis, inflammatory, and liver injury. These therapeutic effects persisted after long-term drug withdrawal and were slightly weakened in the antibiotics-treated obese mice. A. muciniphila treatment efficiently increased mitochondrial oxidation and bile acid metabolism in the gut-liver axis, ameliorated oxidative stress-induced cell apoptosis in gut, leading to the reshaping of the gut microbiota composition. These metabolic improvements occurred with increased L-aspartate levels in the liver that transported from the gut. The administration of L-aspartate in vitro or in mice displayed the similar beneficial metabolic effects mentioned above and efficiently ameliorated MAFLD. Together, these data indicate that the anti-MAFLD activity of A. muciniphila correlated with lipid oxidation and improved gut–liver interactions through regulating the metabolism of L-aspartate. A. muciniphila could be a potential agent for clinical intervention in MAFLD.

The specific sensing of an exact G-quadruplex structure by small molecules has never been reported. A fluorescent sensor based on the photoinduced electron transfer (PeT) mechanism provides possibilities for such specific, one-to-one recognition, indicated by fluorescence. We have rationally developed a PeT fluorescent sensor IZFL-2 by linking triarylimidazole and fluorescein moieties. IZFL-2 is a distinctive, smart sensor whose fluorescence is tunable by its molecular conformations. We then applied IZFL-2 to sensing G-quadruplexes and found that it could exactly distinguish the wild-type c-MYC G-quadruplex from other types of G-quadruplexes, as shown by the activation of its fluorescence. To understand this behavior, we performed various experiments, including fluorescence assays, absorption assays, and multiscale molecular dynamics simulations, to thoroughly investigate the optimal binding mode of IZFL-2 in the c-MYC G-quadruplex. Then, the corresponding HOMO-LUMO of IZFL-2 was analyzed, and the results demonstrated that the PeT process of IZFL-2 is suppressed only in the wild-type c-MYC G-quadruplex via specific loop interactions, which restores its fluorescence. To our knowledge, this smart molecule provides the first example of and new insights into the development of sensors specific for a particular G-quadruplex structure by utilizing intramolecular PeT-controlled fluorescence switching.

This work introduces a multidisciplinary strategy to characterize the structure, folding status, and function of the RNA G-quadruplex in APP 3′UTR., RNA G-quadruplexes (rG4s) are emerging structural motifs that are of pivotal importance in chemistry and biology; however, the current structural information of rG4s is limited, with their folding status and functions in cells remaining elusive. Here, we develop and employ a multi-disciplinary approach to characterize the structure, formation and function of an individual rG4 of interest in vitro and in cells. We apply this strategy to a biologically important rG4 in amyloid precursor protein (APP) transcript and reveal distinct structural features of APP rG4. Notably, we visualize the formation of APP rG4 in cells using an APP-specific G-quadruplex-triggered fluorogenic hybridization (GTFH) probe and report that the regulatory role of APP rG4 in translation is dependent on rG4 thermostability, providing evidence to the existence and significance of the stable rG4 structure in gene regulation.

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.

Novel mansonone F derivative MSN54 (9-bromo-2,3-diethylbenzo[de]chromene-7,8-dione) exhibited significant cytotoxicity against twelve human tumor cell lines in vitro, with particularly strong potency against HL-60/MX2 cell line resistant to Topo II poisons. MSN54 was found to have IC50 of 0.69 and 1.43 µM against HL-60 and HL-60/MX2 cells, respectively. The resistance index is 10 times lower than that of the positive control VP-16 (etoposide). Various biological assays confirmed that MSN54 acted as a Topo IIα specific non-intercalative catalytic inhibitor. Furthermore, MSN54 exhibited good antitumor efficacy and low toxicity at a dose of 5 mg/kg in A549 tumor xenograft models. Thus, compound MSN54 is a promising candidate for the development of novel antitumor agents.

Background and Purpose The human gut bacterium Akkermansia muciniphila has been increasingly recognized for its therapeutic potential in treating metabolic disorders. However, its efficacy in preventing non-alcoholic fatty liver disease (NAFLD) and the mechanism involved in its well-known metabolic actions are unknown. The present study explored the therapeutic effect and novel mechanism of A. muciniphila in intervening NAFLD. Experimental Approach The anti-NAFLD activity of A. muciniphila was evaluated in an obese mouse model induced by high-fat and cholesterol (HFC) diets using three different interventions. The gut microbiota composition, beneficial metabolic effects in the gut-liver axis were explored. The level and beneficial metabolic effects of L-aspartate in vitro and in vivo were further determined. Key Results Mice treated with A. muciniphila efficiently reversed NAFLD in the liver, such as hepatic steatosis, inflammatory, and liver injury. These therapeutic effects persisted after long-term drug withdrawal and were slightly weakened in a germ-free mouse model. A. muciniphila treatment efficiently increased mitochondrial oxidation and bile acid metabolism in the gut-liver axis, ameliorated oxidative stress- induced cell apoptosis in the gut, leading to the reshaping of the gut microbiota composition. These metabolic improvements occurred with increased L-aspartate levels in the liver that transported from the gut. The administration of L-aspartate in vitro or in mice displayed the similar beneficial metabolic effects mentioned above. Conclusion and Implications The anti-NAFLD activity of A. muciniphila correlated with lipid oxidation and improved gut-liver interactions through regulating L-aspartate metabolism. A. muciniphila would be a potent agent for clinical intervention in NAFLD.

Discovery of novel anti-obesity agents is a challenging and promising research area. Based on our previous works, we synthesized 40 novel β-indoloquinazoline analogues by altering the skeleton and introducing preferential side chains, evaluated their lipid-lowering activity and summarized the structure-activity relationships. In combination with an evaluation of the lipid-lowering efficacies, AMP-dependent activated protein kinase (AMPK) activating ability and liver microsomal stability, compound 23 (named as IQZ23) was selected for further studies. IQZ23 exerted a high efficacy in decreasing the triglyceride level (EC50 = 0.033 μM) in 3T3-L1 adipocytes. Mechanistic studies revealed the lipid-lowering activity of IQZ23 was dependent on the AMPK pathway by modulating ATP synthase activity. This activation was accompanied by mitochondrial biogenesis and oxidation capacity increased, and insulin sensitivity enhanced in pertinent cell models by various interventions. Correspondingly, IQZ23 (20 mg/kg, i.p.) treatment significantly reversed high fat and cholesterol diet (HFC)- induced body weight increases and accompanying clinical symptoms of obesity in mice but without indicative toxicity. These results indicate that IQZ23 could be a useful candidate for the treatment of obesity and related metabolic disorders.

Recent years have witnessed an ever-increasing interest in G-quadruplexes for diagnostic and therapeutic applications. This calls for the development of highly selective probes that can differentiate a particular structure among large quantities of G-quadruplexes. However, to this day, there are few probes that show satisfactory selectivity for a particular G-quadruplex structure. In this study, we engineered a new probe called TO-BTZ by introducing an additional benzothiazole moiety on a commercial cyanine dye thiazole orange (TO). Such a modification of TO significantly tuned its selectivity toward the hybrid telomeric G-quadruplex DNA by specifically targeting the unique binding site formed near the 5′-end G-quartet. Such specific interactions disassembled the TO-BTZ H-aggregate to its monomers and further hindered the molecular rotation of the TO-BTZ monomer, thus leading to fluorescence enhancement. Furthermore, we observed that TO-BTZ has the potential to sense telomeric G-quadruplexes in cells. Taken together, this work will shed light on the search for a new generation of probes that can distinguish between different G-quadruplex structures.

Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes.

Design of small molecules targeted at human telomeric G-quadruplex DNA is an extremely active research area. Interestingly, the telomeric G-quadruplex is a highly polymorphic structure. Changes in its conformation upon small molecule binding may be a powerful method to achieve a desired biological effect. However, the rational development of small molecules capable of regulating conformational change of telomeric G-quadruplex structures is still challenging. In this study, we developed a reliable ligand-based pharmacophore model based on isaindigotone derivatives with conformational change activity toward telomeric G-quadruplex DNA. Furthermore, virtual screening of database was conducted using this pharmacophore model and benzopyranopyrimidine derivatives in the database were identified as a strong inducer of the telomeric G-quadruplex DNA conformation, transforming it from hybrid-type structure to parallel structure.

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 dicyanoisophorone-based probe with two-photon absorption and NIR emission was developed for the in vivo fluorescence imaging of amyloid-β plaques, which exhibited high selectivity toward Aβ aggregates over other intracellular proteins. The detection limit was calculated to be as low as 109 nM. In vivo imaging studies indicated that the probe could penetrate the blood–brain barrier and label Aβ plaques in the living transgenic mice, and its specific binding to cerebral Aβ plaques was further confirmed by one- and two-photon ex vivo fluorescence imaging. All these results featured its promising application prospects for amyloid-β sensing in basic research and biomedical research.

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.

Two neutral merocyanine-based near-infrared fluorescent probes were for the first time developed through rational engineering of the classical cationic cyanine scaffold IR-780 for in vivo imaging of amyloid-β plaques. In vivo NIRF imaging revealed that the probe could penetrate the blood-brain barrier and efficiently differentiate the living transgenic and wild-type mice.

Multiple G-quadruplex units in the 3΄-terminal overhang of human telomeric DNA can associate and form multimeric structures. The specific targeting of such distinctive higher-order G-quadruplexes might be a promising strategy for developing selective anticancer agents with fewer side effects. However, thus far, only a few molecules were found to selectively bind to telomeric multimeric G-quadruplexes, and their effects on cancer cells were unknown. In this study, a new triaryl-substituted imidazole derivative called IZNP-1 was synthesized and found to specifically bind to and strongly stabilize telomeric multimeric G-quadruplexes through intercalating into the pocket between the two quadruplex units. The pocket size might affect the binding behavior of IZNP-1. Further cellular studies indicated that IZNP-1 could provoke cell cycle arrest, apoptosis and senescence in Siha cancer cells, mainly because of telomeric DNA damage and telomere dysfunction induced by the interactions of IZNP-1 with telomeric G-quadruplexes. Notably, IZNP-1 had no effect on the transcriptional levels of several common oncogenes that have the potential to form monomeric G-quadruplex structures in their promoter regions. Such behavior differed from that of traditional telomeric G-quadruplex ligands. Accordingly, this work provides new insights for the development of selective anticancer drugs targeting telomeric multimeric G-quadruplexes.

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 visualization of RNA G-quadruplexes in cells is critical to studying and understanding their biological functions. However, while various fluorescent probes have been developed for RNA imaging, very few RNA G-quadruplex-specific fluorescent probes have been found. To address this challenge, we reasoned that turning an RNA fluorescent probe into an RNA G-quadruplex-specific fluorescent probe by rational structural modification to improve the specificity would be an efficient new strategy. Therefore, the benzothiazole-indole-based RNA fluorescent probe BEDO-0 was selected to test this approach. Based on its sensing mechanism, we designed several candidate probes with the aim of improving their G-quadruplex binding selectivity while maintaining the specific fluorescence response. Among them, BEDO-3, which possesses an extended aromatic ring and amino side chain targeting the G-quartet and phosphate backbone, is the best candidate with the strongest G-quadruplex binding affinity and specificity. The relationships between the candidate structure and imaging performance were examined in detail. Furthermore, the practicability of using BEDO-3 as an RNA G-quadruplex-specific fluorescent probe was determined. These findings fully validate the feasibility of our proposed efficient and rational strategy, which will illuminate the development of brand-new fluorescent probes for RNA G-quadruplexes and even for other RNAs.

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.

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.

A novel phenanthroimidazole ethylenediamine Pt(II) complex with coumarin derivative (1) was synthesized and showed higher affinity, selectivity and thermal stabilization for mixed-type dimeric G-quadruplexes (G2T1) over monomeric G-quadruplexes (G1) and duplex DNA. Complex 1 could bind to G-quadruplexes via end-stacking and external-binding modes.

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.

Downregulating transcription of the oncogene c-MYC is a feasible strategy for cancer therapy. Stabilization of the G-quadruplex structure present in the c-MYC promoter can suppress c-MYC transcription. Thus, far, several ligands targeting this structure have been developed. However, most have shown no selectivity for the c-MYC G-quadruplex over other G-quadruplexes, leading to uncertain side effects. In this study, through structural modification of aryl-substituted imidazole/carbazole conjugates, a brand-new, four-leaf clover-like ligand called IZCZ-3 was found to preferentially bind and stabilize the c-MYC G-quadruplex. Further intracellular studies indicated that IZCZ-3 provoked cell cycle arrest and apoptosis and thus inhibited cell growth, primarily by blocking c-MYC transcription through specific targeting of the promoter G-quadruplex structure. Notably, IZCZ-3 effectively suppressed tumor growth in a mouse xenograft model. Accordingly, this work provides an encouraging example of a selective small molecule that can target one particular G-quadruplex structure, and the selective ligand might serve as an excellent anticancer agent.

G-quadruplexes are four-stranded nucleic acid secondary structures that are formed in guanine-rich sequences. G-quadruplexes are widely distributed in functional regions of the human genome and transcriptome, such as human telomeres, oncogene promoter regions, replication initiation sites, and untranslated regions. Many G-quadruplex-forming sequences are found to be associated with cancer, and thus, these non-canonical nucleic acid structures are considered to be attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this mini review, we summarize recent advances made by our lab in the study of G-quadruplex-targeted natural alkaloids and their derivatives toward the development of potential anticancer agents.

c-MYC is an important oncogene that is considered as an effective target for anticancer therapy. Regulation of this gene's transcription is one avenue for c-MYC-targeting drug design. Direct binding to a transcription factor and generating the intervention of a transcriptional programme appears to be an effective way to modulate gene transcription. NM23-H2 is a transcription factor for c-MYC and is proven to be related to the secondary structures in the promoter. Here, we first screened our small-molecule library for NM23-H2 binders and then sifted through the inhibitors that could target and interfere with the interaction process between NM23-H2 and the guanine-rich promoter sequence of c-MYC. As a result, a quinazolone derivative, SYSU-ID-01: , showed a significant interference effect towards NM23-H2 binding to the guanine-rich promoter DNA sequence. Further analyses of the compound-protein interaction and the protein-DNA interaction provided insight into the mode of action for SYSU-ID-01: . Cellular evaluation results showed that SYSU-ID-01: could abrogate NM23-H2 binding to the c-MYC promoter, resulting in downregulation of c-MYC transcription and dramatically suppressed HeLa cell growth. These findings provide a new way of c-MYC transcriptional control through interfering with NM23-H2 binding to guanine-rich promoter sequences by small molecules.

During hernioplasty focal thickened tissue containing smooth muscle is found at the neck of the hernia sac in most patients with indirect inguinal hernia. These thickenings may be related to the processus vaginalis and reveal the etiology of indirect inguinal hernia. The study included 50 male adults with indirect inguinal hernia and 50 male adults with direct inguinal hernia, all of them were initial cases. Hernioplasty and excision of the hernia sac were performed, meanwhile anatomical features of the hernia sac and the spermatic cord were recorded, then followed by histological investigation of the hernia sacs. Focal thickenings were observed at the neck of the hernia sac in 88 % of adults with indirect inguinal hernia. Dense adhesion between the hernia sac and the spermatic cord was found where the thickening located. Histological examination identified smooth muscle cells in 57 % of the thickened tissues. No similar findings were observed in patients with direct inguinal hernia. The focal thickening which contains smooth muscle tissue may be remnant of the processus vaginalis after its obliteration. In other word, the presence of the thickening means that fusion of the processus vaginalis has previously taken place. Thus, most indirect inguinal hernias in adults may represent acquired diseases.

The sensitive and selective detection of nucleic acids is important for basic research and many applied fields. Herein, a series of new BODIPY-benzofuroquinoline conjugates were designed, synthesized and evaluated as DNA intercalating dyes. All compounds were characterized by using 1H, 13C NMR, IR, UV–Vis and fluorescence spectroscopy, and DNA binding properties of these conjugates to calf thymus DNA were studied by using fluorescence titration, UV titration, isothermal titration calorimetry and CD analysis. Significant enhancement of the fluorescent quantum yield was observed for all the conjugates in the presence of calf thymus DNA, and one compound showed excellent sensitivity and selectivity offering its potential application as a DNA specific fluorescent probe. Our results showed that these conjugates could intercalate into calf thymus DNA with high binding affinities. The properties of these dyes as fluorescent probes for living cells imaging were also investigated.

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.

G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.