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The work presented here has con tributed to a better understanding of the mechanism of action of putative Ras Raf interaction inhibitors based on the MCP110 pharmacophore. In addition to support ing previous conclusions that MCP110 significantly inhi bits the signals caused by activated Ras in vitro and in vivo, we have been able to narrow the requirements for its activity by successfully using it to disrupt the Ras Raf RBD interaction.<br />Given these results, it will be inter esting to see if more focused screens involving Ras and the Raf RBD can identify additional potent and selective Ras Raf interaction inhibitors. Methods C. elegans strain maintenance and culturing conditions Strain maintenance and nomenclature are as described, Strains were cultured on 2% NG agar plates seeded with E. coli strain OP50. SD418 gaIs37 mpk 1 strain was maintained at 15 C and switched to 25 C to induce its conditional hyper induced phenotype. Briefly, activated Ras results from an in situ mutation in LET 60 Ras that causes a G13E change equivalent to G13E in human Ras, which is functionally similar to the well known G12V activating mutation, Activation of Raf is a multistep process in which several regulatory residues are modified to regulate its kinase activity. Transgenic alteration of the conserved Akt negative reg ulatory sites from serine to alanine at residues 312 and 453 in Raf leads to a hyper induced phenotype comparable to that conferred by activated Ras, Activated MEK ERK results from transgenic expression of both activated Drosophila MEK and activated C. elegans ERK, all driven by a heat shock promoter. Consequently, the MEK ERK Muv phenotype is temperature sensitive, such that animals are grown at 25 C to induce a Muv phenotype, but are wild type at 15 C, Finally, Ets refers to loss of the LIN 1 Ets transcription factor function. LIN 1 inhibits vulval fate, so LIN 1 loss results in hyper induction, Drug assays and quantification of the multivulva phenotype We have described in detail the experimental proce dures for C. elegans drug treatments and phenotype quantification, Briefly, experiments were performed in 6 well tissue culture plates in which only the four corner wells were filled with 3 ml of 2% NG agar. Either vehicle alone or vehicle plus experimental drug was diluted in M9 buffer and applied in a defined volume to the agar in each well to achieve the final dose. Plates absorbed the drug overnight, then were seeded with 90 ul of OP50 overnight culture and allowed to grow for 24 hours to ensure a suitable bacterial lawn. To obtain a population of treated animals that was developmentally synchronous, we harvested embryos during a narrow time frame.<br />For each strain and drug, 12 15 adult hermaphrodites laid eggs for 3 hours, after which the parents were removed. Animals to be assayed were exposed to drug through out development. Animals harboring activated Ras, Raf and MEK MAPK were scored as early adults using the dissecting microscope. Animals harboring activation of the Ets like transcription factor were scored at the 4th larval stage because adult pseudovulvae were too distorted to quantify clearly, For DIC microscopy, animals were mounted on slides in M9 buffer containing 5 mM sodium azide.