Ethidium derivatives bind to G-quartets, inhibit telomerase and act as fluorescent probes for quadruplexes

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we describe ethidium derivatives that stabilize G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure, as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures. In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC₅₀ values of 18–100 nM in a standard TRAP assay.     

Targeting telomerase via its key RNA/DNA heteroduplex

Telomerase is a promising "universal" anticancer target. It has been demonstrated that inhibition of telomerase leads to mortalization and death of previously immortal cell lines. We are interested in targeting telomerase by binding to the RNA/DNA duplex that forms during its catalytic cycle. The RNA strand of this duplex is a component of telomerase and acts as a template to direct the synthesis of the single-stranded DNA telomere. We have hypothesized that molecules that bind to this duplex will inhibit the enzyme by either preventing strand dissociation or by sufficiently distorting the substrate, thereby causing a misalignment of key catalytic residues. To test this hypothesis we have examined the activity of telomerase in the presence of a range of intercalating molecules, known for their broad duplex binding properties. Of the nine compounds we examined, four show promising lead activity in the low micromolar range. A kinetic analysis of the telomeric products suggests that these compounds do not act by stabilizing G-quartets, thereby supporting the telomeric RNA/DNA heteroduplex as the site of action. We anticipate using these lead compounds as the basis for combinatorial variation to increase the affinity and specificity for the target telomerase.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

Amide bond direction modulates G-quadruplex recognition and telomerase inhibition by 2,6 and 2,7 bis-substituted anthracenedione derivatives

G-quadruplex structures of DNA represent a potentially useful target for anticancer drugs. Stabilisation of this arrangement at the ends of chromosomes may inhibit the action of telomerase, an enzyme involved in immortalization of cancer cells. Appropriately substituted amido anthracenediones are effective G-quadruplex stabilizers, but no information is available as yet on the possible modulation of G-quadruplex recognition and telomerase inhibition produced by the direction of the amide bond. To understand the basis of amido anthracenedione selectivity, we have synthesized a number of derivatives bearing the -CO-NH- or -NH-CO- group linked to the planar anthraquinone (AQ) moiety at 2,6 and 2,7 positions. The various isomers were tested in terms of telomerase inhibition, determined by the TRAP assay, G-quadruplex stabilisation measured by the increase in melting temperature of the appropriately folded oligonucleotide using FRET, and conformational and G4 binding properties examined by molecular modelling techniques. In all cases, enzymatic inhibition and G-quadruplex stabilization were directly related, which strongly supports the proposed molecular mechanism of telomerase interference. Interestingly, the AQ-NH-CO- arrangement performs invariantly better than the AQ-CO-NH- arrangement, showing a clear preference among isomeric derivatives. Theoretical calculations suggest that the former amide arrangement is co-planar with the aromatic system, whereas the latter is tilted by about 30 degrees when considering the most stable conformation. A more extended planar surface would allow more efficient stacking interactions with the quadruplex structure, hence more effective telomerase inhibition.     

Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2

We investigated the control of telomere length by the human telomeric proteins TRF1 and TRF2. To this end, we established telomerase-positive cell lines in which the targeting of these telomeric proteins to specific telomeres could be induced. We demonstrate that their targeting leads to telomere shortening. This indicates that these proteins act in cis to repress telomere elongation. Inhibition of telomerase activity by a modified oligonucleotide did not further increase the pace of telomere erosion caused by TRF1 targeting, suggesting that telomerase itself is the target of TRF1 regulation. In contrast, TRF2 targeting and telomerase inhibition have additive effects. The possibility that TRF2 can activate a telomeric degradation pathway was directly tested in human primary cells that do not express telomerase. In these cells, overexpression of full-length TRF2 leads to an increased rate of telomere shortening.     

Targeting the telomere and shelterin complex for cancer therapy: current views and future perspectives

Aberrant telomere homeostasis is essential for cell immortality, enabling cells to evade telomere dependent senescence. Disruption of telomere structure and function in cancer cells is highly toxic as shown by detailed pre-clinical evaluation of telomerase inhibitors. Under telomerase inhibition, cells must divide sufficiently frequently to allow one or more telomeres to shorten to an unprotected length. Functioning telomeres are disguised from the DNA damage machinery by DNA remodelling and other activities of the telomere binding complex shelterin. Direct interference with shelterin has been shown to result in cell killing and small molecules directly targeting telomere DNA also have anti-tumour effects partially dependent on shelterin disruption. However, shelterin components have not generally been regarded as therapeutic targets in their own right. In this review, we explore the possibilities for therapeutic targeting of the shelterin complex.     

Synthesis, biological evaluation, and molecular docking studies of 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety as potential antitumor agents

A series of new 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety were synthesized. Antiproliferative assay results indicated that compounds 6o and 6u exhibited the most potent activity against gastric cancer cell SGC-7901, which was more potent than the positive control. Especially, compound 6o exhibited significant telomerase inhibitory activity (IC(50)=2.3±0.07μM), which was comparable to the positive control ethidium bromide. Docking simulation was performed to position compound 6o into the active site of telomerase (3DU6) to determine the probable binding model.     

Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity,but less inhibitory activity on telomerase

The template region of human telomerase RNA is a crucial area for regulating telomerase activity and would be a good target for ribozymes. In fact, potent telomerase inhibitory activity of the ribozyme targeting the GUC sequence of the 5(') end of this region (36-ribosome) has been well demonstrated. To search for a more potent ribozyme, we designed a divalent ribozyme to cleave both the phosphodiester bonds following the GUC and the 23 nucleotides downstream of GUA. An in vitro cleavage study showed that this divalent ribozyme cleaved telomerase RNA more efficiently than the 36-ribozyme or the 59-ribozyme to target the GUA. When this ribozyme was introduced into the carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme. The 59-ribozyme showed minimum activity on telomerase. This implies that, although the divalent ribozyme possesses a potent cleavage activity on hTR in vitro, the 36-ribozyme is most potent to suppress telomerase activity.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.     

Telomeres,Telomerase and Cancer: An Endless Search to Target the Ends

Maintenance of functional telomeres, the highly complex nucleo-protein structures, at the end of linear eukaryotic chromosomes appears to be essential for growth and survival of the cells. The compelling correlation between telomerase re-activation and cellular immortalization led to the idea that inhibition of telomerase may provide a way for effective hindrance of cancer cell growth by interfering with telomere maintenance. In addition to targeting the components of telomerase enzyme directly to prevent telomere synthesis, several approaches including disruption of telomeres, interference with telomerase component assembly, translocation of the catalytic component of telomerase etc., have also been under extensive investigation due to the advances in understanding the biology of telomeres and telomerase in recent years. This review will focus on the so far identified approaches to prevent cancer cell growth by targeting telomerase and telomeres with a brief introduction about structure and function of telomeres and telomerase.     

Brought to life: targeted activation of enzyme function with small molecules

Cell-permeable small molecules that are capable of activating particular enzymes would be invaluable tools for studying protein function in complex cell-signaling cascades. But, is it feasible to identify compounds that allow chemical–biology researchers to activate specific enzymes in a cellular context? In this review, we describe some recent advances in achieving targeted enzyme activation with small molecules. In addition to surveying progress in the identification and targeting of enzymes that contain natural allosteric-activation sites, we focus on recently developed protein-engineering strategies that allow researchers to render an enzyme of interest “activatable” by a pre-chosen compound. Three distinct strategies for targeting an engineered enzyme are discussed: direct chemical “rescue” of an intentionally inactivated enzyme, activation of an enzyme by targeting a de novo small-molecule-binding site, and the generation of activatable enzymes via fusion of target enzymes to previously characterized small-molecule-binding domains.     

Inhibition of human-type 1 3beta-hydroxysteroid deshydrogenase/Delta(5)-Delta(4)-isomerase expression using siRNA

Specific inhibition of type 1 3beta-HSD is of particular interest since it will allow us to control the formation of androgens and estrogens in peripheral target tissues without affecting type 2 3beta-HSD, which is responsible for the biosynthesis of glucocorticoids and mineralocorticoids in the adrenals. The high homology between types 1 and 2 3beta-HSD is a major difficulty in the development of specific inhibitors through classical chemical synthesis. In this report, we describe the use of small interference RNA (siRNA) to specifically inhibit human type 1 3beta-HSD. We have constructed three DNA vector-based RNAi vectors that allow us to produce three RNA duplexes of 21 nucleotides targeting three different coding regions of human type 1 3beta-HSD mRNA. The resulting constructs were co-transfected into HEK-293 cells with a vector expressing type 1 3beta-HSD. The results indicate that while the two duplexes that target sequences in the 5'-region do not have a strong inhibitory effect, the duplex that targets the 3'-region efficiently inhibits 3beta-HSD activity. Up to 98% inhibition has been observed. To our knowledge, this is the first report showing successful inhibition of steroidogenic enzymes using siRNA technology.     

Advances in pretargeting biotechnology

A major focus of current drug research is to improve drug targeting to internal target sites such as to solid tumors or specific organs. The objective of drug targeting, especially for cancer chemotherapy and radioimmunotherapy, is to enhance the effectiveness of the drug by concentrating it at the target site and minimizing its effects in nontarget sites. Although tumor targeting has been obtained with large long-circulating radiolabeled antibody molecules, normal organ activity, especially in the blood kidneys, liver, and bone marrow is a significant problem. Over the last 20 years, studies to improve the therapeutic use of antibodies have included the use of antibody fragments, chase molecules, metabolizable linkers, antibody-directed enzyme prodrugs (ADEPT), local delivery, and pretargeting. Here, we will review the most interesting recent advances in pretargeting biotechnology.     

The 5'-end of hTERT mRNA is a good target for hammerhead ribozyme to suppress telomerase activity

Because the expression level of hTERT, a catalytic subunit of human telomerase, is a rate-limiting determinant of telomerase activity, hTERT mRNA would be an excellent target of hammerhead ribozymes for the regulation of telomerase activity. We studied the efficiency of several hammerhead ribozymes targeting hTERT mRNA by transient and stable transfection procedures. To screen the potency of the ribozymes, transient ribozyme transfection and telomerase determination were performed. The ribozyme targeting 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to target 59 nucleotides upstream from the poly(A) tail showed clear activity. A stable transfection study confirmed that the 13-ribozyme suppressed telomerase. These observations suggest that the 13-ribozyme can regulate telomerase activity and may possess potential for cancer therapy.     

Proteolysis targeting chimeras (PROTACs) for epigenetics research

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules and allow selective protein degradation by addressing the natural ubiquitin proteasome system. As this new strategy of chemically induced protein degradation can serve as a biological tool and provides new possibilities for drug discovery, it has been applied to a variety of targets including (nuclear) receptors, kinases, and epigenetic proteins. A lot of PROTACs have already been designed in the field of epigenetics, and their synthesis and characterization highly contributed to structural optimization and improved mechanistic understanding of these molecules. In this review, we will discuss and summarize recent advances in PROTAC discovery with focus on epigenetic targets.     

Bisubstrate analogue structure-activity relationships for p300 histone acetyltransferase inhibitors

p300 and CBP are important histone acetyltransferases (HATs) that regulate gene expression and may be anti-cancer drug targets. Based on a previous lead compound, Lys-CoA, we have used solid phase synthesis to generate a series of 11 new analogues and evaluated these compounds as HAT inhibitors. Increased spacing between the CoA moiety and the lysyl moiety generally decreases inhibitory potency. We have found two substituted derivatives that show about 4-fold increased potency compared to the parent compound Lys-CoA. These structure-activity studies allow for a greater understanding of the optimal requirements for potent inhibition of HAT enzymes and pave the way for a novel class of anti-cancer therapeutics.     

Synthesis,biological evaluation, 3D-QSAR studies of novel aryl-2H-pyrazole derivatives as telomerase inhibitors

A series of novel aryl-2H-pyrazole derivatives bearing 1,4-benzodioxan or 1,3-benzodioxole moiety were designed as potential telomerase inhibitors to enhance the ability of aryl-2H-pyrazole derivatives to inhibit telomerase, a target of anticancer. The telomerase inhibition tests showed that compound 16A displayed the most potent inhibitory activity with IC₅₀ value of 0.9 μM for telomerase. The antiproliferative tests showed that compound 16A exhibited high activity against human gastric cancer cell SGC-7901 and human melanoma cell B16-F10 with IC₅₀ values of 18.07 and 5.34 μM, respectively. Docking simulation showed that compound 16A could bind well with the telomerase active site and act as telomerase inhibitor. 3D-QSAR model was also built to provide more pharmacophore understanding that could be used to design new agents with more potent telomerase inhibitory activity.