100
D.C. Hill et al.
using an assay involving rat brain PLC, and monitoring [3H]-phosphatidyl
inositol metabolism [114, 115].
Hispidospermidin inhibited rat brain PLC in a dose-dependent manner and
was cytoxic to HeLa $3 cells. Another microbial metabolite discovered during
the same screening programme was vinaxanthone, a chromone-substituted
xanthone from Penicillium vinaceum [116], which has also been characterised as
a CD4-binding inhibitor (Sect. 3.1.2).
4.2.2 Compounds Inhibiting DNA-Dependent Enzymes
Enzymes responsible for altering topological conformation of DNA have also
been chosen as targets for dicovery of compounds which may be useful in cancer
therapy.
DNA topoisomerases are nuclear enzymes which catalyse breaking and
rejoining of DNA strands, and therefore control topological states of DNA.
Anti-tumour drugs such as camptothecin and its derivatives have been shown to
interfere with single strand breakage-rejoining of DNA catalysed by
topoisomerase I (Topo I), by stabilising a tight Topo I-DNA complex termed
a "cleavable complex" and preventing the final rejoining step of the
reaction.
An assay based on Topo I mediated cleavage of plasmid pUL402 DNA by
calf thymus Topo I was used to screen microbial samples and resulted in the
discovery of UCE6 (Fig. 7), a naphthacene-5,12-dione produced by an actinomycete culture [117]. UCE6 activity was dose-dependent and comparable to
that of camptothecin. The compound did not interfere with the breakagerejoining reaction of calf thymus topoisomerase II (Topo II), and did not inhibit
other DNA active enzymes such as DNA ligase. UCE6 did not induce changes
in the supercoiled structure of pUL402 DNA in the absence of Topo I and had
cytotoxic activity against HeLaS3 cells.
A second approach to the detection of Topo I inhibitors involved an assay
for relaxation of supercoiled pBR322 plasmid DNA by calf thymus Topo I.
Metabolites identified because of their inhibitory activity in this assay include
naphthacenecarboxamides TAN-1518 A and B from a Streptomyces sp. [118],
and the epioligothiadiketopiperazines TAN-1496-A, C and E from a Microsphaeropsis sp. [119]. The structure of TAN-1518 A is shown in Fig. 7. Both sets
of metabolites strongly suppressed the growth of various murine and human
tumour cells and induced apoptosis.
A comparable assay was used to discover the Topo II inhibitor UCT4B
[120, 121]. The assay monitored the relaxation of supercoiled pUL406 DNA by
calf thymus Topo II. UCT 4B (Fig. 7) is a diterpene closely related to the known
antitumour antibiotic terpentecin, with which it was co-produced by a Streptomyces sp. UCT4B had no effect on pUL406 DNA in the absence of Topo II. The
compound prolonged the life span of mice inoculated with P388 lymphocytic
leukemia cells.
D.C. Hill et al.
using an assay involving rat brain PLC, and monitoring [3H]-phosphatidyl
inositol metabolism [114, 115].
Hispidospermidin inhibited rat brain PLC in a dose-dependent manner and
was cytoxic to HeLa $3 cells. Another microbial metabolite discovered during
the same screening programme was vinaxanthone, a chromone-substituted
xanthone from Penicillium vinaceum [116], which has also been characterised as
a CD4-binding inhibitor (Sect. 3.1.2).
4.2.2 Compounds Inhibiting DNA-Dependent Enzymes
Enzymes responsible for altering topological conformation of DNA have also
been chosen as targets for dicovery of compounds which may be useful in cancer
therapy.
DNA topoisomerases are nuclear enzymes which catalyse breaking and
rejoining of DNA strands, and therefore control topological states of DNA.
Anti-tumour drugs such as camptothecin and its derivatives have been shown to
interfere with single strand breakage-rejoining of DNA catalysed by
topoisomerase I (Topo I), by stabilising a tight Topo I-DNA complex termed
a "cleavable complex" and preventing the final rejoining step of the
reaction.
An assay based on Topo I mediated cleavage of plasmid pUL402 DNA by
calf thymus Topo I was used to screen microbial samples and resulted in the
discovery of UCE6 (Fig. 7), a naphthacene-5,12-dione produced by an actinomycete culture [117]. UCE6 activity was dose-dependent and comparable to
that of camptothecin. The compound did not interfere with the breakagerejoining reaction of calf thymus topoisomerase II (Topo II), and did not inhibit
other DNA active enzymes such as DNA ligase. UCE6 did not induce changes
in the supercoiled structure of pUL402 DNA in the absence of Topo I and had
cytotoxic activity against HeLaS3 cells.
A second approach to the detection of Topo I inhibitors involved an assay
for relaxation of supercoiled pBR322 plasmid DNA by calf thymus Topo I.
Metabolites identified because of their inhibitory activity in this assay include
naphthacenecarboxamides TAN-1518 A and B from a Streptomyces sp. [118],
and the epioligothiadiketopiperazines TAN-1496-A, C and E from a Microsphaeropsis sp. [119]. The structure of TAN-1518 A is shown in Fig. 7. Both sets
of metabolites strongly suppressed the growth of various murine and human
tumour cells and induced apoptosis.
A comparable assay was used to discover the Topo II inhibitor UCT4B
[120, 121]. The assay monitored the relaxation of supercoiled pUL406 DNA by
calf thymus Topo II. UCT 4B (Fig. 7) is a diterpene closely related to the known
antitumour antibiotic terpentecin, with which it was co-produced by a Streptomyces sp. UCT4B had no effect on pUL406 DNA in the absence of Topo II. The
compound prolonged the life span of mice inoculated with P388 lymphocytic
leukemia cells.