No 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).

Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II} is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and TIB, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but TIA appears to represent a different class of termination site. TIA is situated -10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the ~-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factorl, whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.

p56lck, a member of the src family of non-receptor protein receptor kinases, is required for normal signal transduction through the T cell receptor. Inappropriate T cell activation and proliferation has been identified as an early event in auto-immune disease-agents which control T cell activation through modulation of p56lck kinase activity could therefore be potential therapeutic agents for a range of pathological conditions. To identify p56lck inhibitors, we have established an assay system suitable for the high throughput screening of compound libraries. The assay uses enzyme purified from baculovirus infected SF9 cells, and a novel peptidic substrate identified by L. Cantley from a degenerate combinatorial peptide library. We have used this assay system to screen a number of different compounds as potential inhibitors of p56lck. In addition, peptides based on the substrate sequence were also tested to identify a sequence that could be used in the rational design of peptide inhibitors of p56lck.