Characterising the Biochemical and Biophysical Interactions Between Inhibitory Affimer Reagents and the PAK1 Kinase Domain

The p21-activated kinase (PAK) family of serine/threonine kinases plays a fundamental role in cell survival, proliferation, and cytoskeletal regulation. Studies have revealed that PAK1 interacts with key oncogenic signalling pathways, including the MAPK pathway, and have also corroborated the dysregulation of PAK1 expression with a variety of neurological and oncogenic conditions. Although this has cemented the appeal of PAK1 as a therapeutic target, not a single therapeutic has been clinically approved as of 2020.

This project looks at characterising the interactions between Affimers and the active(K299R) and inactive(K299R/D389N) PAK1 kinase domains (KDs) in an attempt to better inform the drug discovery pipeline for anti-PAK1 therapeutics. From a screen of 31 Affimers isolated against both PAK1 KDs, Affimers P2, P11 and P13 inhibited the wildtype PAK1 with micromolar IC50 values. Affimer P2 had an IC50 (2.62 ± 0.27 μM) comparable to IPA-3, a well characterised allosteric PAK1 inhibitor. Further characterisation of the three Affimers through surface plasmon resonance (SPR) and NanoBRET™ revealed nanomolar affinities for the Affimer- PAK1 KD interactions as well as the ability for the Affimers to bind to both PAK1 KDs within the reducing environment of HEK293 cells, respectively. Furthermore, a fresh phage display screen against both PAK1 KDs generated an additional 112 unique Affimers. Further work will be required to better characterise the Affimer-PAK1 KD interactions through x-ray crystallography as well as biophysical and biochemical techniques to identify novel allosteric binding sites on the PAK1 KD. Finally, this project aims to highlight that, Affimers have the potential to expand our understanding of PAK1 function and provide a paradigm to catalyse the development of allosteric small molecule PAK1 inhibitors.