Novel Screen Methodologies for Identification of New Microbial Metabolites
81
N-acyltetramic acids [23]. Delaminomycins A, B and C inhibited cell adhesion
to fibronectin, laminin and type IV collagen. In addition A and B inhibited
adhesion of B16 melanoma cells to all ECM components but C did not inhibit
adhesion to fibronectin. Delaminomycin A had the most potent activity. All
three compounds inhibited the MLR, as determined by [3H]-thymidine incorporation into rat spleen cells, in a dose-dependent manner. Delaminomycin
A did not suppress SRBC induced antibody formation and strongly suppressed
the DTH response in mice. The compound had low toxicity but did not inhibit
tumour metastasis in vivo.
Cytostatin (Fig. 1), a novel phosphorylated a-pyrone derivative produced by
a Streptomyces sp., was identified as an inhibitor of cell adhesion to ECM
components during the screening programme in which the delaminomycins
were discovered [24, 25]. It did inhibit turnout metastasis in vivo, exhibiting
antimetastatic activity on B16 melanoma cells in mice.
2.1.4 Macrophage Activation
Macrophages act as regulatory and effector cells in the host defence system and
are potentially destructive to invading micro-organisms and neoplastic cells.
Macrophages reach target sites and act on target cells by a variety of mechanisms including production of oxygen radicals and release of lysosomal enzymes.
Two novel staurosporine analogues were characterised as microbial promoters of macrophage activation using an assay which involved observation of
the induction of spreading and elongation of Mml cells by scanning electron
microscopy [26, 27].
TAN-999 (Fig. 1) and TAN-1030, new related indolocarbozole alkaloids
isolated from Nocardiopsis dassonvillei and a Streptomyces sp. respectively,
induced Mml cell elongation similar to that seen in activated macrophages when
tested in this system.
Using fluorescein isothiocynate (FITC)-labelled latex particles, these metabolites were shown to augment phagocytic activity of Mml and a highly phagocytic murine macrophage cell line J774A.1. Fcy receptors on the cell lines were
upregulated, 13-glucuronidase (and hence lysosomal) activity was increased, and
phagocytosis-dependent respiratory burst in mouse peritoneal macrophages
was greatly enhanced both in vitro and in vivo. It was concluded that both
TAN-999 and TAN-1030A were capable of activating macrophages. In all cases
TAN-999 was the most active metabolite.
Because of their similarity to staurosporine, a potent protein kinase inhibitor
which is active particularly against PKC, TAN-999 and TAN-1030 were evaluated further. TAN-999 was also found to inhibit PKC, and genistein, a tyrosine
kinase inhibitor, induced Mml cell spreading and enhanced peritoneal mouse
macrophage respiratory burst. This implied that macrophage activation is
protein kinase regulated, confirming that protein kinases are an important
81
N-acyltetramic acids [23]. Delaminomycins A, B and C inhibited cell adhesion
to fibronectin, laminin and type IV collagen. In addition A and B inhibited
adhesion of B16 melanoma cells to all ECM components but C did not inhibit
adhesion to fibronectin. Delaminomycin A had the most potent activity. All
three compounds inhibited the MLR, as determined by [3H]-thymidine incorporation into rat spleen cells, in a dose-dependent manner. Delaminomycin
A did not suppress SRBC induced antibody formation and strongly suppressed
the DTH response in mice. The compound had low toxicity but did not inhibit
tumour metastasis in vivo.
Cytostatin (Fig. 1), a novel phosphorylated a-pyrone derivative produced by
a Streptomyces sp., was identified as an inhibitor of cell adhesion to ECM
components during the screening programme in which the delaminomycins
were discovered [24, 25]. It did inhibit turnout metastasis in vivo, exhibiting
antimetastatic activity on B16 melanoma cells in mice.
2.1.4 Macrophage Activation
Macrophages act as regulatory and effector cells in the host defence system and
are potentially destructive to invading micro-organisms and neoplastic cells.
Macrophages reach target sites and act on target cells by a variety of mechanisms including production of oxygen radicals and release of lysosomal enzymes.
Two novel staurosporine analogues were characterised as microbial promoters of macrophage activation using an assay which involved observation of
the induction of spreading and elongation of Mml cells by scanning electron
microscopy [26, 27].
TAN-999 (Fig. 1) and TAN-1030, new related indolocarbozole alkaloids
isolated from Nocardiopsis dassonvillei and a Streptomyces sp. respectively,
induced Mml cell elongation similar to that seen in activated macrophages when
tested in this system.
Using fluorescein isothiocynate (FITC)-labelled latex particles, these metabolites were shown to augment phagocytic activity of Mml and a highly phagocytic murine macrophage cell line J774A.1. Fcy receptors on the cell lines were
upregulated, 13-glucuronidase (and hence lysosomal) activity was increased, and
phagocytosis-dependent respiratory burst in mouse peritoneal macrophages
was greatly enhanced both in vitro and in vivo. It was concluded that both
TAN-999 and TAN-1030A were capable of activating macrophages. In all cases
TAN-999 was the most active metabolite.
Because of their similarity to staurosporine, a potent protein kinase inhibitor
which is active particularly against PKC, TAN-999 and TAN-1030 were evaluated further. TAN-999 was also found to inhibit PKC, and genistein, a tyrosine
kinase inhibitor, induced Mml cell spreading and enhanced peritoneal mouse
macrophage respiratory burst. This implied that macrophage activation is
protein kinase regulated, confirming that protein kinases are an important