Novel Screen Methodologies for Identification of New Microbial Metabolites
87
same time as effects seen on cAMP with adenylate cyclase modulators. It was
therefore concluded that lactacystin is potentially a new type of NGF.
In a second study, a rat pheochromocytoma cell line PC 12 was used to assess
neurite outgrowth. In this case, cells were seeded onto collagen-coated 24-well
plates and neurite growth scored microscopically after 48 h incubation with the
sample. This study led to the discovery of two novel potentiators of NGF:
NG-011 and NG-012 [54, 55]. These compounds are cyclic polylactones produced by Penicillium verruculosum. Optimum potentiation of NGF-induced
neurite outgrowth was only achieved at relatively high concentrations
(100 gg ml- 1) of NG-011 and NG-012.
In a similar screening programme using PC12R cells, test samples were
added to cells pretreated with NGF. After incubation, cells were fixed with
gluteraldehyde and stained with Harris hematoxylin solution. Neurite outgrowth
was monitored using a phase contrast microscope connected to a video camera
CRT system. BU-4514N, a metabolite of a Microtetraspora sp., was discovered
using this assay [56]. It has a complex structure similar to those of the
delaminomycins (Sect. 2.1.3), containing N-acyltetramic acid and decalin moieties and an additional aminosugar portion. Low micromolar concentrations of
BU-4514N significantly induced neurite outgrowths both in number and length.
3 Receptor Binding Assays
3.1 Inflammatory Disease
A number of ligand receptor interactions are known to be involved in mediation
of inflammatory responses, and these have been targeted in the search for new
pharmacologically active agents.
3.1.1 Leukotriene B4 (LTB4) Binding lnhibitors
Leukotriene B4 (LTB4) stimulates aggregation and degranulation of polymorphonuclear leucocytes (PMNLs), and promotes chemotaxis and chemokinesis.
LTB4 has been detected at sites of inflammation in sufficient concentration to
alter PMNL function, suggesting that it may serve as a mediator in inflammatory response. LTB4 antagonists may therefore be useful in treating diseases
associated with inflammatory cell infiltration.
In this screen, [3H]-LTB4 and a PMNL membrane suspension were incubated with test samples. After the incubation period, the bound [3H]-LTB4 was
separated from the free ligand using a glass fibre filter and a cell harvester. This
led to the discovery of WF 11605 (Fig. 3), a novel tetracyclic triterpene glucoside
produced by an unidentified fungal strain [57, 58].
87
same time as effects seen on cAMP with adenylate cyclase modulators. It was
therefore concluded that lactacystin is potentially a new type of NGF.
In a second study, a rat pheochromocytoma cell line PC 12 was used to assess
neurite outgrowth. In this case, cells were seeded onto collagen-coated 24-well
plates and neurite growth scored microscopically after 48 h incubation with the
sample. This study led to the discovery of two novel potentiators of NGF:
NG-011 and NG-012 [54, 55]. These compounds are cyclic polylactones produced by Penicillium verruculosum. Optimum potentiation of NGF-induced
neurite outgrowth was only achieved at relatively high concentrations
(100 gg ml- 1) of NG-011 and NG-012.
In a similar screening programme using PC12R cells, test samples were
added to cells pretreated with NGF. After incubation, cells were fixed with
gluteraldehyde and stained with Harris hematoxylin solution. Neurite outgrowth
was monitored using a phase contrast microscope connected to a video camera
CRT system. BU-4514N, a metabolite of a Microtetraspora sp., was discovered
using this assay [56]. It has a complex structure similar to those of the
delaminomycins (Sect. 2.1.3), containing N-acyltetramic acid and decalin moieties and an additional aminosugar portion. Low micromolar concentrations of
BU-4514N significantly induced neurite outgrowths both in number and length.
3 Receptor Binding Assays
3.1 Inflammatory Disease
A number of ligand receptor interactions are known to be involved in mediation
of inflammatory responses, and these have been targeted in the search for new
pharmacologically active agents.
3.1.1 Leukotriene B4 (LTB4) Binding lnhibitors
Leukotriene B4 (LTB4) stimulates aggregation and degranulation of polymorphonuclear leucocytes (PMNLs), and promotes chemotaxis and chemokinesis.
LTB4 has been detected at sites of inflammation in sufficient concentration to
alter PMNL function, suggesting that it may serve as a mediator in inflammatory response. LTB4 antagonists may therefore be useful in treating diseases
associated with inflammatory cell infiltration.
In this screen, [3H]-LTB4 and a PMNL membrane suspension were incubated with test samples. After the incubation period, the bound [3H]-LTB4 was
separated from the free ligand using a glass fibre filter and a cell harvester. This
led to the discovery of WF 11605 (Fig. 3), a novel tetracyclic triterpene glucoside
produced by an unidentified fungal strain [57, 58].