Molecular action mode of Hippospongic acid A, an inhibitor of gastrulation of starfish embryos.
Hippospongic acid A (HA-A) is a novel natural triterpene metabolite that exhibits inhibitory activity against the gastrulation of starfish embryos isolated from a marine sponge, Hippospongia sp. We succeeded in chemically synthesizing the natural enantiomer and the racemate HA-A. In this study, we examined its action mode in vitro. HA-A was a rare compound that could selectively but uniformly inhibit the activities of all the vertebrate DNA polymerases tested such as alpha, beta, delta, epsilon, eta, kappa, and lambda, in the IC(50) range of 5.9-17.6 microM, and interestingly also those of human DNA topoisomerases I and II (IC(50) = 15-25 microM). HA-A exhibited no inhibitory effect on DNA polymerases from insects, plants and prokaryotes, or on many other DNA metabolic enzymes. HA-A was an inhibitor specific to DNA polymerases and DNA topoisomerases from vertebrates, but not selective as to a subclass species among the enzymes. Since DNA polymerase beta is the smallest, we used it to analyze the biochemical relationship with HA-A. Biochemical, BIAcore and computer modeling analyses demonstrated that HA-A bound selectively to the N-terminal 8 kDa DNA template-binding domain of DNA polymerase beta, and HA-A inhibited the ssDNA binding activity. HA-A could prevent the growth of NUGC-3 cancer cells at both the G1 and G2/M phases, and induce apoptosis in the cells. The LD(50) value was 9.5 microM, i.e. in the same range as for the enzyme inhibition. Therefore, we concluded that one molecular basis of the gastrulation of starfish embryos is a process that requires DNA polymerases and DNA topoisomerases, and subsequently the gastrulation was inhibited by HA-A. We also discussed the in vivo role of HA-A.
Mizushina
Yoshiyuki
Y
Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180. mizushin@nutr.kobegakuin.ac.jp
Murakami
Chikako
C
Takikawa
Hirosato
H
Kasai
Nobuyuki
N
Xu
Xianai
X
Mori
Kenji
K
Oshige
Masahiko
M
Yamaguchi
Toyofumi
T
Saneyoshi
Mineo
M
Shimazaki
Noriko
N
Koiwai
Osamu
O
Yoshida
Hiromi
H
Sugawara
Fumio
F
Sakaguchi
Kengo
K
D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D026942:DNA Topoisomerases / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005775:Gastrula / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D019384:Nucleic Acid Synthesis Inhibitors; D011161:Porifera / Q000378:metabolism; D013215:Starfish / Q000196:embryology* / Q000378:metabolism; D013237:Stereoisomerism; D059003:Topoisomerase Inhibitors; D014315:Triterpenes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D059003:Topoisomerase Inhibitors; D014315:Triterpenes; C459583:hippospongic acid A; D004259:DNA-Directed DNA Polymerase; D026942:DNA Topoisomerases