Targeting RASopathic fibrosis signalling pathways using monobodies
The project ambitions were to develop novel anti-fibrotic therapeutics by identifying allosteric protein interfaces in the Ras-Erk pathway, with the long-term goal of using these as therapeutic targets, and developing anti-fibrotic drugs. Following award of this project in 2014, and initial development of the yeast screening technology, we had a major breakthrough where we identified a method to identify novel anti-inflammatory proteins (evasins) from tick saliva. As such anti-inflammatory compounds would have anti-fibrotic effects, and moreover could be used directly as drugs. With permission from the CRM director Professor Paul Riley, we used the funds awarded to create a library of tick evasins using gene synthesis. These libraries were expressed in yeast and screened with fluorescent chemokines using FACS, and high-affinity evasins targeting both CC and CXC class chemokines were identified. Importantly individual evasins bind chemokines in a unique "one-to-many" mechanism of action, overcoming the robustness of the chemokine network. We developed methods to express and purify the evasin proteins in mammalian cells, and characterized them using biophysical assays such as biolayer interferometry, and cell migration assays.
1. Singh, K., Davies, G., Alenazi, Y., Eaton, J.R.O., Kawamura, A. & Bhattacharya, S. Yeast surface display identifies a family of evasins from ticks with novel polyvalent CC chemokine-binding activities. Sci Rep 7, 4267 (2017).
2. Eaton, J.R.O., Alenazi, Y., Singh, K., Davies, G., Geis-Asteggiante, L., Kessler, B., Robinson, C.V., Kawamura, A. & Bhattacharya, S. The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins. J Biol Chem 293, 6134-6146 (2018).
3. Alenazi, Y., Singh, K., Davies, G., Eaton, J.R.O., Elders, P., Kawamura, A. & Bhattacharya, S. Genetically engineered two-warhead evasins provide a method to achieve precision targeting of disease-relevant chemokine subsets. Sci Rep 8, 6333 (2018).