Novel Screen Methodologies for Identification of New Microbial Metabolites
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2.2.2 Cell Cycle Inhibitors
Events in the eukaryotic cell cycle are also potential targets for discovery of new
anti-cancer drugs. The eukaryotic cell cycle consists of two major events: DNA
replication in S phase and mitotic cell division in M phase. These are separated
by two intervening (gap) phases, G1 and G2, during which many protooncogene functions are in operation. Cancer can be characterised as a failure of
cell cycle regulation resulting in uncontrolled proliferation. Hence inhibition of
the cell cycle offers opportunities to combat cancer.
A screening programme has been based on monitoring effects of microbial
metabolites on an early S phase synchronous culture of rat normal 3Y1 fibroblasts by flow cytometry. The distribution of DNA content in naked nuclei of
3Y1 cells was determined using propidium iodide staining. The stage of cell
arrest was estimated by comparison with data generated using known
compounds (vinblastine-M phase, trichostatin A-G2 phase, leptomycin
B-G1 and G2 phases). This screen was used to identify leptolstatin, a new
gap phase specific inhibitor [34,35]. Leptolstatin is an unsaturated 0tpyrone derivative which is closely related to the leptomycins produced by a
Streptomyces sp.
Leptolstatin had a strong reversible inhibitory effect on 3Y1 cells. Removal
of leptolstatin from the arrested culture induced additional DNA synthesis in
G2-arrested cells. This suggested that diploid G2 cells were converted to tetraploid G1 cells during G2 arrest. Compound removal induced formation of
proliferative tetraploid cells.
2.2.3 Other Screen Methodologies and Targets
In cancer cells various steps of such signal transduction pathways are activated,
or short-circuited, by oncogenes and this leads to less regulated growth. Therefore these events are a possible target for discovery of selective anti-tumour
agents.
Differences in cell phenotypes resulting from aberrant expression of the
signal transduction molecule ras have been used to detect compounds active in
cancer targets. A screen using transformation of ras § cell morphology to the ras
phenotype led to the discovery of reductoleptomycin A from a Streptomyces sp.
1-36]. Reductoleptomycin A is structurally very similar to leptolstatin
(Sect. 2.2.2). It is noteworthy that the dorrigocins, a series of glutarimide
metabolites from Streptomyces platensis subsp, rosaceus, recently reported to
cause a change in the morphology of ras § NIH/3T3 cells to that of normal
cells by a process involving inhibition of carboxyl methylation, appear to have
been discovered during the course of screening for antifungal antibiotics
[37-39].
Angelmicins A and B from a Microbispora sp. were isolated as selective
inhibitors of growth of src-transformed cells in a separate screen which used
83
2.2.2 Cell Cycle Inhibitors
Events in the eukaryotic cell cycle are also potential targets for discovery of new
anti-cancer drugs. The eukaryotic cell cycle consists of two major events: DNA
replication in S phase and mitotic cell division in M phase. These are separated
by two intervening (gap) phases, G1 and G2, during which many protooncogene functions are in operation. Cancer can be characterised as a failure of
cell cycle regulation resulting in uncontrolled proliferation. Hence inhibition of
the cell cycle offers opportunities to combat cancer.
A screening programme has been based on monitoring effects of microbial
metabolites on an early S phase synchronous culture of rat normal 3Y1 fibroblasts by flow cytometry. The distribution of DNA content in naked nuclei of
3Y1 cells was determined using propidium iodide staining. The stage of cell
arrest was estimated by comparison with data generated using known
compounds (vinblastine-M phase, trichostatin A-G2 phase, leptomycin
B-G1 and G2 phases). This screen was used to identify leptolstatin, a new
gap phase specific inhibitor [34,35]. Leptolstatin is an unsaturated 0tpyrone derivative which is closely related to the leptomycins produced by a
Streptomyces sp.
Leptolstatin had a strong reversible inhibitory effect on 3Y1 cells. Removal
of leptolstatin from the arrested culture induced additional DNA synthesis in
G2-arrested cells. This suggested that diploid G2 cells were converted to tetraploid G1 cells during G2 arrest. Compound removal induced formation of
proliferative tetraploid cells.
2.2.3 Other Screen Methodologies and Targets
In cancer cells various steps of such signal transduction pathways are activated,
or short-circuited, by oncogenes and this leads to less regulated growth. Therefore these events are a possible target for discovery of selective anti-tumour
agents.
Differences in cell phenotypes resulting from aberrant expression of the
signal transduction molecule ras have been used to detect compounds active in
cancer targets. A screen using transformation of ras § cell morphology to the ras
phenotype led to the discovery of reductoleptomycin A from a Streptomyces sp.
1-36]. Reductoleptomycin A is structurally very similar to leptolstatin
(Sect. 2.2.2). It is noteworthy that the dorrigocins, a series of glutarimide
metabolites from Streptomyces platensis subsp, rosaceus, recently reported to
cause a change in the morphology of ras § NIH/3T3 cells to that of normal
cells by a process involving inhibition of carboxyl methylation, appear to have
been discovered during the course of screening for antifungal antibiotics
[37-39].
Angelmicins A and B from a Microbispora sp. were isolated as selective
inhibitors of growth of src-transformed cells in a separate screen which used