Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.

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Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes.

Inhibition of telomerase activity by telomerase inhibitors induces a gradual loss of telomeres, and this in turn causes cancer cells to enter to a crisis stage. Here, we report the telomerase inhibitor telomestatin, which is known to stabilize G-quadruplex structures at 3 0 single-stranded telomeric overhangs (G-tails), rapidly dissociates TRF2 from telomeres in cancer cells within a week, when given at a concentration that does not cause normal cells to die. The G-tails were dramatically reduced upon short-term treatment with the drug in cancer cell lines, but not in normal fibroblasts and epithelial cells. In addition, telomestatin also induced anaphase bridge formation in cancer cell lines. These effects of telomestatin were similar to those of dominant negative TRF2, which also causes a prompt loss of the telomeric G-tails and induces an anaphase bridge. These results indicate that telomestatin exerts its anticancer effect not only through inhibiting telomere elongation, but also by rapidly disrupting the capping function at the very ends of telomeres. Unlike conventional telomerase inhibitors that require long-term treatments, the G-quadruplex stabilizer telomestatin induced prompt cell death, and it was selectively effective in cancer cells. This study also identifies the TRF2 protein as a therapeutic target for treating many types of cancer which have the TRF2 protein at caps of the telomere DNA of each chromosome.

Disruption of the UPR may provide a novel therapeutic approach to targeting glucose-deprived solid tumors.

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. In order to determine whether G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), might have effects on telomere dynamics and to evaluate the clinical utility, we assessed the effects of telomestatin on BCR-ABL-positive human leukemia cells. We found that treatment with telomestatin reproducibly inhibited telomerase activity in the BCR-ABLpositive leukemic cell lines OM9;22 and K562, resulting in telomere shortening. Inhibition of telomerase activity by telomestatin disrupts telomere maintenance and ultimately results in telomere dysfunction. Telomestatin completely suppressed the plating efficiency of K562 cells at 1 lm; however, telomestatin had less effects on BFU-Es and CFU-GMs colony formation from normal bone marrow CD34-positive cells. Enhanced chemosensitivity toward imatinib and chemotherapeutic agents was also observed in telomestatin-treated K562 cells. Further, the combination of telomestatin plus imatinib more effectively inhibited hematopoietic colony formation by primary human chronic myelogenous leukemia cells. Last, telomestatin induced the activation of ATM and Chk2, and subsequently increased the expression of p21 CIP1 and p27 KIP1 . These results demonstrate that telomere dysfunction induced by telomestatin activates the ATM-dependent DNA damage response. We conclude that telomerase inhibitors combined with the use of imatinib and other chemotherapeutic agents may be very useful for the treatment of human leukemia.

Telomestatin is a potent G-quadruplex ligand that interacts with the 3 telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the doublestranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the ␥-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.

Recent studies described several different routes that facilitate nitrogen-nitrogen bond formation in natural product biosynthesis. We report herein the identification of unprecedented machinery for hydrazine formation involved in the biosynthesis of s56-p1, a dipeptide natural product with a unique hydrazone unit. The gene cassette comprising this machinery is widespread across several bacterial phyla, highlighting the overlooked potential of bacteria to synthesize hydrazine.