Novel Screen Methodologies for Identification of New Microbial Metabolites
99
consists of a conserved cysteine (C), two aliphatic amino acids (AA) and
a carboxy-terminal residue (X), which is a pre-requisite for post-translational
processing and subsequent malignant transformation. One of the key steps
here is farnesylation of the conserved cys residue by farnesyl protein transferase (FPT), an enzyme which has been targeted in an inhibitor screening
programme.
In the screening assay, FPT from human THP-1 cell cytosol was incubated
with sample, recombinant p21 protein and [3H]-farnesyl pyrophosphate, (FPP).
After incubation, the reaction was stopped, the mixture filtered through a glass
fibre filter, and the activity of the test samples determined with respect to
controls. This assay was used to discover the pepticinnamins, a family of
N-acylpeptides produced by a Streptomyces sp. [108, 109].
Protein tyrosine phosphatase (PTPase) is another enzyme which is thought
to be important in the regulation of intracellular signal transduction. Tyrosine
kinases associated with src-family protooncogene products are inactivated by
phosphorylation of a tyrosine residue near their carboxyl terminus.
In the assay involved in the discovery of dephostatin, Jurkat cell membranes
were used as a source of PTPase activity, and were assayed using o-phospho-Ltyrosine as a substrate and measuring liberated inorganic phosphate [110].
Dephostatin is 2-(N-methyl-N-nitroso)-hydroquinone, a novel metabolite of
a Streptomyces sp. [111]. Dephostatin is a competitive PTPase inhibitor, has
similar toxicity to oncogene-expressing cells as to normal parent cells, and is of
low toxicity to mice.
A number of oncogene products are tyrosine kinases which have also been
used in enzyme inhibitor screening programmes. Aberrant activation of such
enzymes has been implicated in human clinical cancers.
The target in one such screening programme was the EGF receptor tyrosine
kinase (EGF rTK). The incorporation of (~,32p) from (732p)-ATP was measured
using human recombinant EGF as a substrate. The reaction was terminated
with tricholoroacetic acid and incorporated radioactivity measured after separation through filter mats. Use of this assay resulted in the detection and
discovery of BE-23372M, a substituted butenolide produced by Rhizoctonia
solani [112, 113]. BE-23372M is a potent selective inhibitor ofEGF rTK, having
poor activity against mouse brain PKC and cAMP-dependent protein kinase.
The compound also inhibited proliferation of A431 cells (which overexpress
erb-B1, the EGF receptor) and MKN-7 cells (reported to express erb-B2 or
HER-2, a truncated and mutated form of the EGF receptor that lacks the
external EGF binding domain of the receptor and is activated in the absence of
ligand binding, in human cells). BE-23372M did not block binding of EGF to its
receptor.
Phospholipase C (PLC) is another enzyme which is important in signal
transduction, having a key role in phosphoinositol metabolism, and thereby
controlling various cellular responses to actions of hormones and peptide
growth factors. Hispidospermidin, a novel cage compound with a trimethylspermidine side chain produced by Chaetosphaeronema hispidulum, was discovered
99
consists of a conserved cysteine (C), two aliphatic amino acids (AA) and
a carboxy-terminal residue (X), which is a pre-requisite for post-translational
processing and subsequent malignant transformation. One of the key steps
here is farnesylation of the conserved cys residue by farnesyl protein transferase (FPT), an enzyme which has been targeted in an inhibitor screening
programme.
In the screening assay, FPT from human THP-1 cell cytosol was incubated
with sample, recombinant p21 protein and [3H]-farnesyl pyrophosphate, (FPP).
After incubation, the reaction was stopped, the mixture filtered through a glass
fibre filter, and the activity of the test samples determined with respect to
controls. This assay was used to discover the pepticinnamins, a family of
N-acylpeptides produced by a Streptomyces sp. [108, 109].
Protein tyrosine phosphatase (PTPase) is another enzyme which is thought
to be important in the regulation of intracellular signal transduction. Tyrosine
kinases associated with src-family protooncogene products are inactivated by
phosphorylation of a tyrosine residue near their carboxyl terminus.
In the assay involved in the discovery of dephostatin, Jurkat cell membranes
were used as a source of PTPase activity, and were assayed using o-phospho-Ltyrosine as a substrate and measuring liberated inorganic phosphate [110].
Dephostatin is 2-(N-methyl-N-nitroso)-hydroquinone, a novel metabolite of
a Streptomyces sp. [111]. Dephostatin is a competitive PTPase inhibitor, has
similar toxicity to oncogene-expressing cells as to normal parent cells, and is of
low toxicity to mice.
A number of oncogene products are tyrosine kinases which have also been
used in enzyme inhibitor screening programmes. Aberrant activation of such
enzymes has been implicated in human clinical cancers.
The target in one such screening programme was the EGF receptor tyrosine
kinase (EGF rTK). The incorporation of (~,32p) from (732p)-ATP was measured
using human recombinant EGF as a substrate. The reaction was terminated
with tricholoroacetic acid and incorporated radioactivity measured after separation through filter mats. Use of this assay resulted in the detection and
discovery of BE-23372M, a substituted butenolide produced by Rhizoctonia
solani [112, 113]. BE-23372M is a potent selective inhibitor ofEGF rTK, having
poor activity against mouse brain PKC and cAMP-dependent protein kinase.
The compound also inhibited proliferation of A431 cells (which overexpress
erb-B1, the EGF receptor) and MKN-7 cells (reported to express erb-B2 or
HER-2, a truncated and mutated form of the EGF receptor that lacks the
external EGF binding domain of the receptor and is activated in the absence of
ligand binding, in human cells). BE-23372M did not block binding of EGF to its
receptor.
Phospholipase C (PLC) is another enzyme which is important in signal
transduction, having a key role in phosphoinositol metabolism, and thereby
controlling various cellular responses to actions of hormones and peptide
growth factors. Hispidospermidin, a novel cage compound with a trimethylspermidine side chain produced by Chaetosphaeronema hispidulum, was discovered