Shikonin derivatives: synthesis and inhibition of human telomerase

We synthesized DL-shikonin, shikonin, alkanin, and their cyclo-derivatives and acyl-derivatives. These compounds have low cytotoxicity, as well as inhibitory activity against the telomerase enzyme, except cyclo-derivatives.     

Recognition of hairpin-containing single-stranded DNA by oligonucleotides containing internal acridine derivatives.

Oligodeoxynucleotides with an internal intercalating agent have been targeted to single-stranded sequences containing hairpin structures. The oligonucleotide binds to nonadjacent single-stranded sequences on both sides of the hairpin structure in such a way as to form a three-way junction. The acridine derivative is inserted at a position that allows it to interact with the three-way junction. The melting temperature (Tm) of complexes formed between the hairpin-containing target and oligonucleotides containing one internal acridine derivative was higher than that obtained with the same target and an unmodified oligonucleotide (DeltaTm = +13 degrees C). The internal acridine provided the oligonucleotide with a higher affinity than covalent attachment to the 5' end. Oligonucleotides could also be designed to recognize a hairpin-containing single-stranded nucleic acid by formation of Watson-Crick hydrogen bonds with a single-stranded part and Hoogsteen hydrogen bonds with the stem of the hairpin. An internal acridine derivative was introduced at the junction between the two domains, the double helix domain with Watson-Crick base pairs and the triple helix domain involving Hoogsteen base triplets in the major groove of the hairpin stem. Oligonucleotides with an internal acridine or an acridine at their 5' end have similar binding affinities for the stem-loop-containing target. The bis-modified oligonucleotide containing two acridines, one at the 5' end and one at an internal site, did not exhibit a higher affinity than the oligonucleotides with only one intercalating agent. The design of oligonucleotides with an internal intercalating agent might be of interest to control gene expression through recognition of secondary structures in single-stranded targets.     

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.     

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.     

Human telomerase inhibition and cytotoxicity of regioisomeric disubstituted amidoanthraquinones and aminoanthraquinones

Telomerase is an attractive target for the rational design of new anticancer drugs due to its central role in the control of cellular proliferation. A number of 1,4-disubstituted amidoanthraquinones and 1,5-disubstituted aminoanthraquinones that are related to mitoxantrone and ametantrone have previously been prepared. The present study details the effects on human telomerase of these new classes of 1,4- and 1,5-difunctionalized tricyclic anthraquinone compounds. We have used cytotoxicity assay, reporter SEAP assay to monitor the hTERT expression, and TRAP-G4 assay to measure the relative activity of these compounds, and have examined how the attached substituents affect their ability to influence telomerase. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Structural and activity relationships indicated that the position of disubstituent side chains is important for its inhibitory effect. Moreover, a primary amine or tertiary amine on the substitution group appears to be required for the telomerase inhibitory effect. There is no significant correlation between telomerase activity and cytotoxicity. These symmetrical disubstituted anthraquinones may represent useful leads for the development of human telomerase inhibitors as potential anticancer agents, and the exact mode of intercalative binding is dictated by the positional placement of substituent side chains for effective telomerase inhibition.     

Accumulation of acridine amino derivatives by electron-dense granules in thrombocytes

The uptake and release of dense bodies' content were studied using fluorescent amines. Real-time measurements of fluorescence spectra by a device developed on the basis of a multichannel optical analyzer and computer technique of spectrum deconvolution made it possible to investigate the kinetics of monoamine uptake and release in intact cells.     

Potent inhibition of human telomerase by U-73122

Summary Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC₅₀ value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 μM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC₅₀ values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.     

The action of acridine derivatives on penicillinase production by staphylococcus aureus]

A study was made of a possibility of inhibition of biosynthesis of penicillinase in Staph. aureus by acridine derivatives. Acetone preparations of penicillinase were obtained from the cultures of staphylococcus strains 16/160 and 8325 (p11(147) pen 1220) grown in the presence of various subbacterial concentrations of acridine derivatives. The activity of the enzyme was studied in experiment and control by the microiodometric method. Acriflavine and proflavine inhibited the penicillinase biosynthesis from the 4th hour of growth, and rivanol, acrichine, acridines No. 27 and 37--from the 12th hour of the culture growth.     

Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG

The ends of human chromosomes are protected from the degradation associated with cell division by 15-20 kb long segments of hexameric repeats of 5'-TTAGGG-3' termed telomeres. In normal cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; however, most cancer cells bypass this lifespan restriction through the expression of telomerase. hTERT, the catalytic subunit essential for the proper function of telomerase, has been shown to be expressed in approximately 90% of all cancers. In this study we investigated the hTERT inhibiting effects of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea catechins, in MCF-7 breast cancers cells and HL60 promyelocytic leukemia cells. Exposure to EGCG reduced cellular proliferation and induced apoptosis in both MCF-7 and HL60 cells in vitro, although hTERT mRNA expression was decreased only in MCF-7 cells when treated with EGCG. Furthermore, down-regulation of hTERT gene expression in MCF-7 cells appeared to be largely due to epigenetic alterations. Treatment of MCF-7 cells with EGCG resulted in a time-dependent decrease in hTERT promoter methylation and ablated histone H3 Lys9 acetylation. In conjunction with demethylation, further analysis showed an increase in hTERT repressor E2F-1 binding at the promoter. From these findings, we propose that EGCG is effective in causing cell death in both MCF-7 and HL60 cancer cell lines and may work through different pathways involving both anti-oxidant effects and epigenetic modulation.     

Synthesis, human telomerase inhibition and anti-proliferative studies of a series of 2,7-bis-substituted amido-anthraquinone derivatives

Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone were designed and synthesized. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and cytotoxicity. In the series, compounds (6, 10, 13, 16, 18, 19, 20-22, and 24) showed potent telomerase inhibitory activity, while compounds 19, 21, and 22 activated hTERT expression in normal human fibroblasts. The results indicated that 2,7-diamidoanthraquinones represent an important class of compounds for telomerase-related drug developments. Compounds 8, 16, 18, 26, and 32 were also selected by the NCI for Screening Program and demonstrated high anti-proliferative activity against 60 human cancer cell lines. Structure-activity relationships (SAR) study revealed that the test compounds with side chains two carbon spacer between amido and amine are important structural moiety for telomerase inhibition. Although the exact mechanism of how this amine group contributes to its activity is still unclear, however, the amine group in the extended arm of the bis-substituted anthraquinone might contribute to proper binding to the residues within the grove of G-quadruplex structure. Our results indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase inhibitors that have the potential to be further developed into novel anticancer chemotherapeutic agents.