Interference with protein-protein interactions
A consortium was formed which comprised some of Europe's leading research groups in the field of signal transduction pathways, structural biology and bioinformatics. The group took a multidisciplinary approached to identifying possible new pathways involved in cancer and conducted screening for potential new anti-cancer compounds. One of the aims of the TARGETS FOR CANCER T project was to identify small candidate molecules, typically peptides. Researchers focussed on those molecules which possessed the desired inhibitory function, such as that found in some kinases or in particular protein-protein interactions. The interaction of activated Ras protein with Raf initiates signalling cascades that lead to a significant percentage of human tumours. An assay was therefore developed based on the immobilised protein GST-Ras G12V and the Ras binding domain of Raf kinase, which was labelled with Alexa dye. Furthermore, the assay proved suitable for the potential identification of inducers of Ras GTPase activity, as well as the detection of altered interaction between Ras and Raf compounds. Using the assay, researchers from the Max Planck Institute, Dortmund, in collaboration with the commercial partner Evotec OAI AG, screened a library containing 60,000 compounds. The successful collaboration between the Max Planck Institute and Evotec OAI AG was maintained. The two project partners used the assay to screen more than 70,000 compounds for their ability to inhibit Ras/Raf interaction or to induce GTPase activity of Ras G12V. The work resulted in a patent, which can identify reversible protein-protein interactions between GTPase negative forms of GTP-binding proteins, and their effector's proteins. The system can also pinpoint those chemical compounds which inhibit the above interactions in the living cell and can detect the rescue of blocked GTPase activity. One considerable advantage is that the assay can be used with any of the protein-protein interactions that possess medium or high affinity.
