Inhibition of Human Telomerase by Rubromycins:  Implication of Spiroketal System of the Compounds as an Active Moiety

Ueno, Takamasa; Takahashi, Hirotada; Oda, Masako; Mizunuma, Makiko; Yokoyama, Akihisa; Goto, Yuhei; Mizushina, Yoshiyuki; Sakaguchi, Kengo; Hayashi, Hideya

doi:10.1021/bi992661i

Cited by 143 publications

(143 citation statements)

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“…They exhibit activity against the reverse transcriptase of human immunodeficiency virus-1, [2] and their inhibition of human telomerase, reported in 2000, [3] has made these compounds attractive lead compounds for the development of new types of anticancer drugs. The spiroketal unit is essential for their inhibitory activity towards telomerase: The related compound a-rubromycin (3), which lacks the spiroketal moiety, exhibits decreased activity.…”

mentioning

confidence: 99%

Total Synthesis of (±)‐γ‐Rubromycin on the Basis of Two Aromatic Pummerer‐Type Reactions

et al. 2007

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mentioning

confidence: 99%

Total Synthesis of (±)‐γ‐Rubromycin on the Basis of Two Aromatic Pummerer‐Type Reactions

et al. 2007

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“…Telomerase activity was determined using the PCR-based telomeric repeat amplification protocol as described previously (40) with some modifications (41).…”

Section: Methodsmentioning

confidence: 99%

A Plant Phytotoxin, Solanapyrone A, Is an Inhibitor of DNA Polymerase β and λ

Mizushina¹,

Kamisuki²,

Kasai³

et al. 2002

Journal of Biological Chemistry

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Eukaryotic cells reportedly contain three replicative DNAdirected DNA polymerases (EC 2.7.7.7) (pol ␣, ␦, and ⑀), 1 mitochondrial DNA polymerase (pol ␥), and at least nine repair types of DNA polymerase (pol ␤, ␦, ⑀, , , , , , and ) (1-8), and we have screened for natural compounds that selectively inhibit each of these eukaryotic DNA polymerases and found several inhibitors (9 -15). The purpose was to use the compounds as tools and molecular probes to distinguish DNA polymerases and to clarify their biological and in vivo functions. In this study, we found an interesting fungus-produced compound that selectively inhibits the activities of pol ␤ and pol . Interestingly, the compound was found to be a plant phytotoxin, solanapyrone A, isolated from the causal fungus of early blight disease in potato. To our knowledge, such specific natural compounds have not been reported with the exception of the pol ␤-inhibitor, prunasin, which we reported previously (13). The compound was a stronger pol ␤-inhibitor than prunasin, and no pol -inhibitors have been reported.Pol ␤ is the lowest molecular mass (39 kDa) DNA polymerase lacking such intrinsic accessory activities as 3Ј-or 5Ј-exonuclease, endonuclease, dNMP turnover, or pyrophosphorolysis (16). Pol ␤ consists of an independently folded N-terminal 8-kDa domain and C-terminal 31-kDa domain (17,18). The N-terminal domain was originally characterized as a single-stranded DNA (ssDNA)-binding domain. It was then found to possess binding specificity for the 5Ј-phosphate in gapped DNA (19 -21) and a helix-hairpin-helix motif found in several other DNA repair enzymes (22,23). Recently, Matsumoto and Kim (24) demonstrated that pol ␤ catalyzes the removal of dRP from the AP endonuclease-incised AP site via ␤-elimination as opposed to hydrolysis, and that this dRP lyase activity resides in the 8-kDa domain of pol ␤. According to recent studies (7, 25), moreover, pol ␤ is known to have another family polymerase, pol . Pol ␤ has been present in all tissues examined and is generally expressed at a low level as are a number of other so-called constitutive "house-keeping" enzymes. Although the in vivo function of the family enzyme, pol , is unclear as yet, pol appears to work in a similar manner to pol ␤ (7). As to why a plant phytotoxin is a pol ␤-family-specific inhibitor, we presently are analyzing the structure and function of pol ␤ and pol using the inhibitor from two different view-points to understand the precise role of each of the polymerases in vivo and to develop a drug design strategy for cancer chemotherapy agents. We previously reported the three-dimensional structure of pol ␤ and the N-terminal 8-kDa domain (the DNA template-binding domain) with or without long chain fatty acids to further clarify the structure and function of pol ␤ (26). Because at least pol ␤ is an essential enzyme for nucleotide excision repair (1, 2), the plant phytotoxin may lead to blockage of the DNA repair systems of rescue cancer cells under clinical radiation-therapy or chemotherapy. The plan...

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“…Telomerase activity was determined using the polymerase chain reaction-based telomeric repeat amplification protocol as described (26) with some modifications (27).…”

Section: Methodsmentioning

confidence: 99%

Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

Mizushina¹,

Kamisuki²,

Mizuno³

et al. 2000

Journal of Biological Chemistry

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Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase ␣ (pol ␣) in vitro. Surprisingly, among the polymerases and DNA metabolic enzymes tested, dehydroaltenusin inhibited only mammalian pol ␣. Dehydroaltenusin did not influence the activities of the other replicative DNA polymerases, such as ␦ and ⑀; it also showed no effect even on the pol ␣ activity from another vertebrate (fish) or plant species. The inhibitory effect of dehydroaltenusin on mammalian pol ␣ was dose-dependent, and 50% inhibition was observed at a concentration of 0.5 M. Dehydroaltenusin-induced inhibition of mammalian pol ␣ activity was competitive with the template-primer and non-competitive with the dNTP substrate. BIAcore analysis demonstrated that dehydroaltenusin bound to the core domain of the largest subunit, p180, of mouse pol ␣, which has catalytic activity, but did not bind to the smallest subunit or the DNA primase p46 of mouse pol ␣. These results suggest that the dehydroaltenusin molecule competes with the template-primer molecule on its binding site of the catalytic domain of mammalian pol ␣, binds to the site, and simultaneously disturbs dNTP substrate incorporation into the template-primer.Recent investigations have revealed that eukaryotic cells contain at least eight types of DNA polymerase (pol ␣, ␤, ␥, ␦, ⑀, , , and ) (1-4). We screened for natural compounds that selectively inhibit one of the eukaryotic DNA polymerases and found several inhibitors (5-8). Selective inhibitors of DNA polymerases are useful tools and molecular probes to distinguish DNA polymerases and clarify their biological and in vivo functions (9). For example, aphidicolin is a selective inhibitor of both DNA polymerase (pol) 1 ␣ and eukaryotic DNA replicative polymerase, indicating that this polymerase is essential for DNA replication (10). Aphidicolin inhibitor has been very useful for studying the DNA replication system (11); however, there have been no previous reports of inhibitors capable of distinguishing among pol ␣, ␦, and ⑀.We found an interesting inhibitor that influenced only the activity of mammalian pol ␣. The agent, which was determined to be dehydroaltenusin, has been reported to be the inhibitor of myosin light chain kinase (12, 13). Dehydroaltenusin did not affect the activities of replicative DNA polymerases, such as ␦ and ⑀, from calf thymus and HeLa cells, respectively; it also showed no effect on pol ␣ activity even from another vertebrate, i.e. fish, although the amino acid sequences of both DNA polymerases are highly homologous. The agent must be able to determine small structural differences between mammalian and fish pol ␣. Therefore, dehydroaltenusin could be a useful tool with which to study the biochemical functions of pol ␣ and a molecular probe to distinguish the structure of pol ␣. EXPERIMENTAL PROCEDURESMaterials-Nucleotides and chemically synthesized template-primers, such as poly(dA) and oligo(dT) 12-18 , were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). [ 3 H]dTTP (43 Ci/mmol) was pur...

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Inhibition of Human Telomerase by Rubromycins: Implication of Spiroketal System of the Compounds as an Active Moiety

Cited by 143 publications

References 40 publications

Total Synthesis of (±)‐γ‐Rubromycin on the Basis of Two Aromatic Pummerer‐Type Reactions

Total Synthesis of (±)‐γ‐Rubromycin on the Basis of Two Aromatic Pummerer‐Type Reactions

A Plant Phytotoxin, Solanapyrone A, Is an Inhibitor of DNA Polymerase β and λ

Dehydroaltenusin, a Mammalian DNA Polymerase α Inhibitor

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