Novel Screen Methodologies for Identification of New Microbial Metabolites
95
On further evaluation, WIN 64821 was found to inhibit SP induced calcium
release from astrocytoma cells, as measured by monitoring 45CaZ + etttux from
pre-treated U-373 MG cells, but the compound had no effect in the absence of
SP. In an NK-2 rat vas deferens assay, etedoisin-induced twitch potentiation
was inhibited by WIN 64821 in a concentration-dependent manner. Specificity
of WIN 64821 was demonstrated in a range of receptor binding and ion channel
assays.
Further microbial tachykinin receptor antagonists reported recently include
the NK-1/NK-2 receptor-active cyclic peptide WIN 66306 from an Aspergillus
sp. [-89], the benzodiazepine benzomalvins (the structure of one of which is
shown in Fig. 5) from a Penicillium sp. [90], and the fiscalin-related spiroquinazoline from Asperoillusflavipes [91].
3.4.2 GABAA-Benzodiazepine Receptor Binding Inhibitors
Benzodiazepines with tranquilising and anticonvulsant activity act by modulating the major inhibitory neuro-transmitter, gamma-amino butyric acid
(GABA). Activation of the GABAA receptor by GABA agonists causes the
chloride channel to open, and the resulting influx of chloride anions inhibits
neuronal firing by generating hyperpolarisation. The effect of benzodiazepines is
to increase the frequency of channel opening. Benzodiazepine therapy, although
effective, can lead to the development of dependence and has a number of side
effects. A search for alternatives to the benzodiazepines at Xenova utilized an
assay based on the binding of flunitrazepam to the GABAA benzodiazepine
receptor in an assay where the bound radioligand was separated from free
radioligand using filtration.
The use of this assay to screen microbial fermentation samples led to the
discovery of the xenovulenes, potent inhibitors of GABA-benzodiazepine binding produced by Acremonium stricture [92]. The xenovulenes contain oxygenated polyketide moieties fused to a sesquiterpenoid humulene ring. The
most potent member of the series is xenovulene A (Fig. 5), which inhibits
flunitrazepam binding to GABA-benzodiazepine receptors in a synaptosome
membrane preparation from ox cerebral cortex with an ICso of 40 nmol1-1
Pharmacological evaluation of xenovulene A has shown that it acts selectively
on a subset of receptors, giving the potential for a reduction in the side-effects
associated with the benzodiazepines (unpublished data; Xenova).
4 Enzyme Assays
As discussed in Sect. 1.3, a very wide variety of enzyme targets have been used in
the search for pharmacologically active compounds. Details of some of the
compounds isolated over the last seven years are included in this section.
95
On further evaluation, WIN 64821 was found to inhibit SP induced calcium
release from astrocytoma cells, as measured by monitoring 45CaZ + etttux from
pre-treated U-373 MG cells, but the compound had no effect in the absence of
SP. In an NK-2 rat vas deferens assay, etedoisin-induced twitch potentiation
was inhibited by WIN 64821 in a concentration-dependent manner. Specificity
of WIN 64821 was demonstrated in a range of receptor binding and ion channel
assays.
Further microbial tachykinin receptor antagonists reported recently include
the NK-1/NK-2 receptor-active cyclic peptide WIN 66306 from an Aspergillus
sp. [-89], the benzodiazepine benzomalvins (the structure of one of which is
shown in Fig. 5) from a Penicillium sp. [90], and the fiscalin-related spiroquinazoline from Asperoillusflavipes [91].
3.4.2 GABAA-Benzodiazepine Receptor Binding Inhibitors
Benzodiazepines with tranquilising and anticonvulsant activity act by modulating the major inhibitory neuro-transmitter, gamma-amino butyric acid
(GABA). Activation of the GABAA receptor by GABA agonists causes the
chloride channel to open, and the resulting influx of chloride anions inhibits
neuronal firing by generating hyperpolarisation. The effect of benzodiazepines is
to increase the frequency of channel opening. Benzodiazepine therapy, although
effective, can lead to the development of dependence and has a number of side
effects. A search for alternatives to the benzodiazepines at Xenova utilized an
assay based on the binding of flunitrazepam to the GABAA benzodiazepine
receptor in an assay where the bound radioligand was separated from free
radioligand using filtration.
The use of this assay to screen microbial fermentation samples led to the
discovery of the xenovulenes, potent inhibitors of GABA-benzodiazepine binding produced by Acremonium stricture [92]. The xenovulenes contain oxygenated polyketide moieties fused to a sesquiterpenoid humulene ring. The
most potent member of the series is xenovulene A (Fig. 5), which inhibits
flunitrazepam binding to GABA-benzodiazepine receptors in a synaptosome
membrane preparation from ox cerebral cortex with an ICso of 40 nmol1-1
Pharmacological evaluation of xenovulene A has shown that it acts selectively
on a subset of receptors, giving the potential for a reduction in the side-effects
associated with the benzodiazepines (unpublished data; Xenova).
4 Enzyme Assays
As discussed in Sect. 1.3, a very wide variety of enzyme targets have been used in
the search for pharmacologically active compounds. Details of some of the
compounds isolated over the last seven years are included in this section.