Telomestatin, a Novel Telomerase Inhibitor from Streptomyces anulatus

K Shin-ya, K Wierzba, K Matsuo, T Ohtani… - Journal of the …, 2001 - ACS Publications
K Shin-ya, K Wierzba, K Matsuo, T Ohtani, Y Yamada, K Furihata, Y Hayakawa, H Seto
Journal of the American Chemical Society, 2001ACS Publications
Telomeres are the guanine-rich, simple repeat sequences of TTAGGG that constitute the
physical termini of eukaryotic chromosomes. Maintenance of telomeres, in which the
specialized ribonucleoprotein complex known as telomerase mediates, is significant for
immortalization in cancer cells. 1 Since the correlation between telomerase activity and
tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and
that telomerase inhibitors represent a class of chemotherapeutic agents, we attempted to …
Telomeres are the guanine-rich, simple repeat sequences of TTAGGG that constitute the physical termini of eukaryotic chromosomes. Maintenance of telomeres, in which the specialized ribonucleoprotein complex known as telomerase mediates, is significant for immortalization in cancer cells. 1 Since the correlation between telomerase activity and tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents, we attempted to screen telomerase inhibitors from the metabolites of the microorganism. A wide range of screening resulted in the isolation of a potent specific telomerase inhibitor designated as telomestatin (1) from Streptomyces anulatus 3533-SV4. Although telomerase consists of several components with DNA polymerase or reverse transcriptase activity in addition to its intrinsic telomerase component, 1 specifically inhibited telomerase without affecting DNA polymerases and reverse transcriptases (RT) such as Taq polymerase and HIV-RT. Here, we describe the structure and biological properties of 1. The telomestatin-producing organism, Streptomyces anulatus 3533-SV4, was cultivated in a production medium consisting of 2% glycerol, 1.0% molasses, 0.5% casein, 0.1% polypepton, and 0.4% CaCO3 for 3 days in a jar fermenter. The whole culture broth was centrifuged and the collected mycelium was extracted with the same volume of acetone as that of the culture broth. After concentration in vacuo, the residual aqueous layer was partitioned between EtOAc and H2O. The concentrated organic layer was applied to a silica gel column and eluted with CHCl3-MeOH (20: 1 to 10: 1). The active eluate was chromatographed on a silica gel column with CHCl3-MeOH-NH4OH (700: 100: 1) as the solvent system. Finally, a pure sample of 1 was obtained as a white yellowish powder by HPLC using a PEGASIL ODS column developed with 70% CH3CN containing 0.1% trifluoroacetic acid. A high-resolution FAB-MS of 1 (m-nitrobenzyl alcohol)[R] D-9.4 (c 0.13, MeOH)[mp 134-143 C dec] established the molecular formula of 1 as C26H14N8O7S [(M+ H)+, m/z 583.0790 (calcd 583.0784)]. The 1H and 13C NMR spectral data together with direct 13C-1H correlation established by an HMQC experiment for 1 are summarized. 2
The 1H NMR spectrum of 1 showed five isolated aromatic proton signals 12-H (δH 8.12), 15-H (δH 8.24), 18-H (δH 8.13), 21-H (δH 8.34), and 24-H (δH 8.12), which were connected to carbon signals appearing at δC 137.5-141.2 by HMQC correlations. Long-range couplings from these aromatic protons to quaternary aromatic carbons at δC 130.4-136.7 and at δC 156.2-157.3 proved the presence of five oxazole moieties (rings D to
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