Novel Screen Methodologies for Identification of New Microbial Metabolites
93
(Fig. 4) from an Aspergillus sp. [77], the aselacins from an Acremonium sp.
[78, 79], the haloemodins from Fusarium aquaeductum [80], and new azaphilones from Penicillium sclerotiorum (the structure of one of which is shown in
Fig. 4) which were discovered at Xenova in a partnership programme with
Parke-Davis Pharmaceutical Research [81]. The ETA/ETB receptor selectivity
of these compounds is varied. RES-701-1 and the haloemodins are reported to
be ETB-specific.
3.4 CNS Disease
Receptor binding assays have also been used to screen for inhibitors which may
be useful in the treatment of CNS disease.
3.4.1 Inhibition of Tachykinin Binding
Tachykinins may play an important role in the pathophysiology of airway
diseases, especially asthma. There are at least three related peptide tachykinins:
substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), and three
different receptor types NK-1, NK-2 and NK-3. It is thought that the mechanical and chemical stimulation of the sensory airway nerve endings causes release
of tachykinins, such as SP and NK, from axon collaterals leading to smooth
muscle contraction, mucus secretion, vasodilation and protein extravasation.
Using [3H]-SP binding to guinea-pig lung membranes and separating the
bound from the free ligand by filtration, WS9326A, a novel acylated macrocyclic
hexapeptide lactone, was discovered [82, 83]. The compound is a tachykinin
receptor-active metabolite of Streptomyces violaceusniger, which was found to
be a dual NK1, NK2 antagonist on further evaluation.
Anthrotainin (Fig. 5) was discovered using an assay based on the binding of
[125I]-SP to NK-1 receptor rich rat forebrain membranes [84]. Anthrotainin is
a novel tetracyclic metabolite of Gliocladium catenulatus which is related to
viridicatumtoxin. Treatment of anthrotainin with diazomethane produced
a more stable but unusual adduct in which the amide moiety was methylated.
Anthrotainin inhibited [125I]-SP binds to rat forebrain membranes in a concentration dependent manner. However, the more stable methylated product was
inactive. Studies on the dissociation rate of [~25I]-SP in the presence and
absence of anthrotainin indicated that interaction of the compound with the
receptor is not strictly competitive with SP binding.
Fiscalins A, B and C were discovered as new inhibitors of the binding of SP
to human NK-1 receptors from U373 MG cells [85]. The structure of fiscalin
A is shown in Fig. 5. In the binding assay, [~25I]-Bolton-Hunter-SP (BH-SP)
and test samples were added to cells grown to confluence in 96-well plates. After
incubation and washing, cells were detached from the plates with detergent
and radioactivity was measured. The fiscalins are metabolites of the fungus
93
(Fig. 4) from an Aspergillus sp. [77], the aselacins from an Acremonium sp.
[78, 79], the haloemodins from Fusarium aquaeductum [80], and new azaphilones from Penicillium sclerotiorum (the structure of one of which is shown in
Fig. 4) which were discovered at Xenova in a partnership programme with
Parke-Davis Pharmaceutical Research [81]. The ETA/ETB receptor selectivity
of these compounds is varied. RES-701-1 and the haloemodins are reported to
be ETB-specific.
3.4 CNS Disease
Receptor binding assays have also been used to screen for inhibitors which may
be useful in the treatment of CNS disease.
3.4.1 Inhibition of Tachykinin Binding
Tachykinins may play an important role in the pathophysiology of airway
diseases, especially asthma. There are at least three related peptide tachykinins:
substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), and three
different receptor types NK-1, NK-2 and NK-3. It is thought that the mechanical and chemical stimulation of the sensory airway nerve endings causes release
of tachykinins, such as SP and NK, from axon collaterals leading to smooth
muscle contraction, mucus secretion, vasodilation and protein extravasation.
Using [3H]-SP binding to guinea-pig lung membranes and separating the
bound from the free ligand by filtration, WS9326A, a novel acylated macrocyclic
hexapeptide lactone, was discovered [82, 83]. The compound is a tachykinin
receptor-active metabolite of Streptomyces violaceusniger, which was found to
be a dual NK1, NK2 antagonist on further evaluation.
Anthrotainin (Fig. 5) was discovered using an assay based on the binding of
[125I]-SP to NK-1 receptor rich rat forebrain membranes [84]. Anthrotainin is
a novel tetracyclic metabolite of Gliocladium catenulatus which is related to
viridicatumtoxin. Treatment of anthrotainin with diazomethane produced
a more stable but unusual adduct in which the amide moiety was methylated.
Anthrotainin inhibited [125I]-SP binds to rat forebrain membranes in a concentration dependent manner. However, the more stable methylated product was
inactive. Studies on the dissociation rate of [~25I]-SP in the presence and
absence of anthrotainin indicated that interaction of the compound with the
receptor is not strictly competitive with SP binding.
Fiscalins A, B and C were discovered as new inhibitors of the binding of SP
to human NK-1 receptors from U373 MG cells [85]. The structure of fiscalin
A is shown in Fig. 5. In the binding assay, [~25I]-Bolton-Hunter-SP (BH-SP)
and test samples were added to cells grown to confluence in 96-well plates. After
incubation and washing, cells were detached from the plates with detergent
and radioactivity was measured. The fiscalins are metabolites of the fungus