title: Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity.
abstract: By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).
authors.affiliation: Roche Research Centre, Herts, United Kingdom.
authors.forename: D H
authors.initials: DH
authors.lastname: Williams
authors.forename: S E
authors.initials: SE
authors.lastname: Wilkinson
authors.forename: T
authors.initials: T
authors.lastname: Purton
authors.forename: A
authors.initials: A
authors.lastname: Lamont
authors.forename: H
authors.initials: H
authors.lastname: Flotow
authors.forename: E J
authors.initials: EJ
authors.lastname: Murray
mesh_terms: D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000187:drug effects*; D048369:MAP Kinase Kinase 1; D048728:MAP Kinase Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012459:Salicylates / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology* / Q000276:immunology; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D018631:ras Proteins / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; C113152:Ro 09-2210; D012459:Salicylates; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins