The choice of small-scale fermentation systems contributes significantly to a successful scale-up. Creasing of flasks and the chosen shaker parameters influence the production of secondary metabolites in a strain- and even compound-specific manner. Using actinomycetes and fungi as model organisms the influence of the small-scale fermentation system on the production of various secondary metabolites is described and the effects on screening success and scale-up are considered.

Inhibition of CD28 signalling after an immune response impedes T cell activation and can lead to immunosuppression. To identify inhibitors of anti-CD28 induced IL-2 production, a library of fungal metabolites was screened in a cell-based, high throughput assay. A reduced novel benzofluoranthene, tentatively named as (6bS, 7R, 8S)-7-methoxy-4, 8, 9-trihydroxy-1, 6b, 7, 8-tetrahydro-2H-benzo[j] fluoranthen-3-one (XR774), from Cladosporium cf. cladosporioides, was isolated. XR774 inhibited IL-2 mRNA and protein expression induced by anti-CD28 and anti-CD3 but had no effect on IL-2 induction by PMA and ionomycin. Moreover, XR774 inhibited the activity of the tyrosine kinases, Fyn, Lck, Abl and epidermal growth factor receptor (EGFR) with nanomolar activity, whereas micromolar concentrations of XR774 were ineffective on the serine–threonine kinase, PKA. Kinetic analysis of Fyn kinase inhibition was consistent with XR774 as a competitive inhibitor with respect to ATP.In peripheral blood mononuclear cells (PBMC), XR774 inhibited anti-CD3 and anti-CD28 induced IL-2 and IL-2R α chain (CD25) expression but was consistently less active for inhibition of IFN-γ production. On stimulation with PMA and anti-CD28, XR774 inhibited IL-2 production but had no effect on CD25 expression and enhanced IFN-γ production. In contrast, the ansamycin, geldanamycin, inhibited both IL-2 and IFN-γ production induced by anti-CD3 and anti-CD28 or PMA and anti-CD28. No significant associated cytotoxicity or inhibition of protein synthesis was observed at concentrations up to 14 μM. Thus, XR774 represents a novel class of pharmacological agent with selective biological activities that distinguish it from other natural product inhibitors, such as the ansamycins.

Resorcylic acid lactones are fungal metabolites that exhibit a wide range of biological properties which includes oestrogenic, antifungal, phytotoxic and anti-inflammatory activity. The capacity of 5Z-7-oxo-zeaenol, a resorcylic lactone of fungal origin and six naturally occurring analogues to inhibit lipopolysaccharide (LPS)-induced cytokine production in phorbol 12-myristate-13-acetate (PMA)-treated cultured myelomonocytic cells (U937) was compared. The activity of the natural analogues in the U937 assay varied over 10(4)-fold, with 5Z-7-oxo-zeaenol the most potent of those tested inhibiting tumour necrosis factor-alpha (TNF alpha) production in these cells with IC50 of 6 nM. The isomeric 7-oxo-zeaenol and structurally more distant monorden (radicicol) were the next most active compounds with IC50 similar to 500 nM, and zearalenone, the least active with IC50 > 400 mu M. 5Z-7-oxo-zeaenol retained activity in LPS-stimulated peripheral blood mononuclear cells with an IC50 of 10-25 nM. This compound also inhibited LPS-induced TNF alpha production in whole blood experiments (IC50 100-1000 nM) and lowered serum levels of TNF alpha in mice when administered prior to LPS. 5Z-7-oxo-zeaenol was shown to inhibit the phosphorylation and activation of mitogen-activated protein kinase (MAPK) induced by LPS. These data are consistent with a mechanism of action at or upstream of MAPK with resultant downstream effects. This series of naturally occurring analogues represents an interesting group of compounds with diverse biological properties. Of this series, 5Z-7-oxo-zeanenol has exceptionally potent antiinflammatory properties exhibited by its strong inhibition of cytokine production. (C) 1999 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved.

CD4, a cell‐surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus‐1 (HIV‐1). Screening of microbial metabolites for CD4‐binding activity using an enzyme‐linked immunosorbent assay based on the binding of the CD4‐specific monoclonal antibody (mAb), anti‐Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti‐Leu 3a and that of several other D1/D2 epitope‐specific mAb with CD4, but only weakly inhibited the binding of HIV‐1 gp120. Binding of a representative MHC class II molecule, HLA‐DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 μM). In functional assays 411F inhibited antigen‐induced CD4‐dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide‐induced CD4+ T cell proliferative responses but not alloantigen‐induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4‐dependent functions by binding over a broad area of the positively charged amino‐terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.

The interaction of antibodies with protein antigens is accepted as a paradigm of protein-protein interactions. In searching for a new generation of immunomodulatory compounds based on the interaction of the T-cell surface glycoprotein CD4 with MHC class II antigens, a model assay has been developed in which MHC molecules have been substituted by a monoclonal antibody (anti-Leu3a) to the CD4 amino-terminal domain-specific epitope, Leu3a. This assay can detect diverse classes of molecules including proteins such as HIV envelope glycoprotein gp120 and low molecular weight compounds such as aurin tricarboxylic acid, dextran sulphate and Evans blue. The interaction of these molecules with CD4 in the assay appears to be identical to their interaction with native CD4 on intact cells. Other protein-antibody pairs could be substituted for CD4-anti-Leu3a enabling this assay format to be used for the detection of proteins or small organic compounds which interfere with a wide range of therapeutically-relevant macromolecular interactions.