title: Differential regulation of CD3- and CD28-induced IL-2 and IFN-gamma production by a novel tyrosine kinase inhibitor XR774 from Cladosporium cf. cladosporioides.
abstract: 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 alpha chain (CD25) expression but was consistently less active for inhibition of IFN-gamma production. On stimulation with PMA and anti-CD28, XR774 inhibited IL-2 production but had no effect on CD25 expression and enhanced IFN-gamma production. In contrast, the ansamycin, geldanamycin, inhibited both IL-2 and IFN-gamma 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 microM. 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.
authors.affiliation: Xenova Ltd., 240 Bath Road, Slough, Berkshire, SL1 4EF, UK.
authors.forename: R
authors.initials: R
authors.lastname: Sadeghi
authors.forename: P
authors.initials: P
authors.lastname: Depledge
authors.forename: P
authors.initials: P
authors.lastname: Rawlins
authors.forename: N
authors.initials: N
authors.lastname: Dhanjal
authors.forename: A
authors.initials: A
authors.lastname: Manic
authors.forename: S
authors.initials: S
authors.lastname: Wrigley
authors.forename: B
authors.initials: B
authors.lastname: Foxwell
authors.forename: M
authors.initials: M
authors.lastname: Moore
mesh_terms: D016227:Benzoquinones; D018106:CD28 Antigens / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism*; D002963:Cladosporium / Q000737:chemistry; D016572:Cyclosporine / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D005449:Fluorenes / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D007376:Interleukin-2 / Q000096:biosynthesis*; D015759:Ionomycin / Q000494:pharmacology; D019169:Jurkat Cells; D047029:Lactams, Macrocyclic; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011809:Quinones / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D016227:Benzoquinones; D018106:CD28 Antigens; D017252:CD3 Complex; D004791:Enzyme Inhibitors; D005449:Fluorenes; D007376:Interleukin-2; D047029:Lactams, Macrocyclic; D011809:Quinones; C429612:XR774; D015759:Ionomycin; D007371:Interferon-gamma; D016572:Cyclosporine; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate; C001277:geldanamycin