Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.19.211177v1?rss=1 Authors: Nguyen, D., Lin, L. Y., Zhou, J., Kibby, E., Sia, T., Tillis, T., Vapuryan, N., Xu, M.-R., Potluri, R., Shin, Y., Erler, E., Bronkema, N., Boehmler, D., Chung, C., Burkhard, C., Grasso, M., Acevedo, L. A., Marmorstein, R., Fera, D. Abstract: In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas. Copy rights belong to original authors. Visit the link for more info