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2 Cell-Based Screens
D.C. Hill et al.
2.1 Inflammatory Disease
The cellular nature and complexity of interactions involved in the immune
response and inflamatory disease has often driven the search for new active
metabolites into celI-based screen formats.
2.1.1 Immunomodulation
Compounds with immunomodulatory activity, such as kifunesine (FR-900494)
and conagenin, have been isolated and characterised using relatively simple
cell-based assays to monitor the incorporation of [3H]-thymidine into mouse
spleen cells and T cells. Kifunesine is a cyclic oxamide derivative of 1-aminosubstituted mannojirimycin which was isolated from the actinomycete
Kitasatosporia kifunense [11, 12]. Conagenin is a 2-methylserine derivative
isolated from Streptomyces reseosporus [13].
Both compounds demonstrated activity in more complex functional assay
systems. Kifunesine concentrations of 0.1-1 ~tgm1-1 restored the ability of
immuno-deficient mice to produce antibody to sheep red blood cells (SRBC),
and conagenin was found to modulate T cell activity in vivo [14, 15]. Kifunesine
is also a weak inhibitor of a-mannosidase.
2.1.2 Immunosuppression
Assays based on the mixed lymphocyte reaction (MLR) have been used in
screening programmes for novel compounds with potential immunosuppressive
activity, as well as activity in the suppression of allograft rejection in transplantations, therapy of autoimmune disease and hypersensitivity.
A productive programme in terms of compounds reported has identified
ISP-1 (myriocin), a number of synthetic myriocin derivatives including
14-deoxymyriocin, and the related fungal metabolites, the mycestericins, as
immunosuppressive agents. In the assay used to discover these compounds,
responder cells (mice splenocytes) and stimulator cells (co-irradiated splenocytes
treated with mitomycin C) were mixed in 96-well plates and cell proliferation
was measured using [3H]-thymidine incorporation. Screening was focused on
Trichoderma sp. and Isaria sp. as these fungi are known producers of cyclosporins and immunosuppressive cyclic depsipeptides. It is interesting that the
compounds detected were all related and form a new class of immunosuppressive agents.
Myriocin (Fig. 1), an unusual lipophilic amino acid, was found to be five to
ten times more potent than cyclosporin A (CsA) in inhibiting the MLR [16].
2 Cell-Based Screens
D.C. Hill et al.
2.1 Inflammatory Disease
The cellular nature and complexity of interactions involved in the immune
response and inflamatory disease has often driven the search for new active
metabolites into celI-based screen formats.
2.1.1 Immunomodulation
Compounds with immunomodulatory activity, such as kifunesine (FR-900494)
and conagenin, have been isolated and characterised using relatively simple
cell-based assays to monitor the incorporation of [3H]-thymidine into mouse
spleen cells and T cells. Kifunesine is a cyclic oxamide derivative of 1-aminosubstituted mannojirimycin which was isolated from the actinomycete
Kitasatosporia kifunense [11, 12]. Conagenin is a 2-methylserine derivative
isolated from Streptomyces reseosporus [13].
Both compounds demonstrated activity in more complex functional assay
systems. Kifunesine concentrations of 0.1-1 ~tgm1-1 restored the ability of
immuno-deficient mice to produce antibody to sheep red blood cells (SRBC),
and conagenin was found to modulate T cell activity in vivo [14, 15]. Kifunesine
is also a weak inhibitor of a-mannosidase.
2.1.2 Immunosuppression
Assays based on the mixed lymphocyte reaction (MLR) have been used in
screening programmes for novel compounds with potential immunosuppressive
activity, as well as activity in the suppression of allograft rejection in transplantations, therapy of autoimmune disease and hypersensitivity.
A productive programme in terms of compounds reported has identified
ISP-1 (myriocin), a number of synthetic myriocin derivatives including
14-deoxymyriocin, and the related fungal metabolites, the mycestericins, as
immunosuppressive agents. In the assay used to discover these compounds,
responder cells (mice splenocytes) and stimulator cells (co-irradiated splenocytes
treated with mitomycin C) were mixed in 96-well plates and cell proliferation
was measured using [3H]-thymidine incorporation. Screening was focused on
Trichoderma sp. and Isaria sp. as these fungi are known producers of cyclosporins and immunosuppressive cyclic depsipeptides. It is interesting that the
compounds detected were all related and form a new class of immunosuppressive agents.
Myriocin (Fig. 1), an unusual lipophilic amino acid, was found to be five to
ten times more potent than cyclosporin A (CsA) in inhibiting the MLR [16].