RO-09-2210 EXHIBITS POTENT ANTIPROLIFERATIVE EFFECTS ON ACTIVATED T-CELLS BY SELECTIVELY BLOCKING MKK ACTIVITY

By using high throughput screening of microbial broths, we have identi fied a compound, designated Ro 09-2210, which is able to block anti-CD 3 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 s timulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cel ls using a variety of reporter gene constructs and showed effective in hibition 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 API with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter construc ts (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 a ctivation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibitin g an activator of AP-1 which was upstream of c-jun and downstream of r as signaling. To investigate further, we then purified a number of dif ferent kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), an d showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (I C50 = 59 nM).