Protein kinases are central effector molecules of signal transduction, regulated through diverse mechanisms, making them prime targets for drug discovery. However, analyzing their physiological and mutation-driven pathological functions within intact cells remains challenging due to context-dependent interactions and unique scaffolding functions of respective enzyme entities. Serving as molecular switches, especially protein kinases control vital cellular functions spatiotemporally, and dysregulation of their activity promote diseases such as cancer.

In proof-of-concept studies we employed the Kinase Conformation (KinCon) reporter system to track conformational states of different disease-associated kinases. This allows for the precise assessment of drug-candidate-target engagements of allosteric and competitive small molecules in living cells and high-content format.

Our studies revealed unique sensitivities of the KinCon reporter system, particularly in tracking BRAF-kinase activity conformations upon melanoma drug binding. The effects of several FDA-approved BRAF inhibitors on kinase activity conformations were successfully validated and compared to next-generation BRAF blockers such as Plixorafenib. Moreover, we showed that common MEK1 patient mutations induced structural shifts toward an open, active kinase conformation, a process that was reversible upon MEK inhibitor binding, as evidenced by KinCon biosensor signals and supported by molecular dynamics simulations. Additionally, we applied the KinCon system to track vertical pathway inhibition of BRAF and MEK1, revealing that dual targeting with kinase inhibitors in BRAF-V600E contexts induces a closed, inactive MEK1 conformation more efficiently than single agents. Expanding this platform, we recently integrated challenging targets like the tumor suppressor p53 and enzymes of the E3 ligases family such as MDM2, enabling the analysis of small molecule interactions and functional validations across drug classes.

The results of this study underscore the importance of understanding conformational dynamics of deregulated enzymes in the context of disease and therapy. The KinCon reporter system has proven to be a valuable tool in identifying how kinases interact with small molecule inhibitors. Our findings suggest that this technology can predict cellular factors impacting drug efficacies, thereby aiding in the design of more effective therapeutic strategies. Recent expansion to challenging targets like MDM2, p53 and other E3 ligases demonstrates broad applicability for systematic drug candidate validation. This approach advances precision oncology by enabling direct tracking of target engagement in a cellular context to determine and anticipate drug candidate efficacies.