Using CE to confirm the activity of fluorescent miRFP670-LIMK1 protein produced for MST assays directly in cell...

LIMK1 is a promising therapeutic target for multiple pathologies. Its holistic study seems crucial, as none of its inhibitors has passed clinical trials. The need for new fluorescent probes for the study of enzyme/ligand interactions is always increasing with the development of new analytical tools. A method for the assessment of LIMK activity directly in cell lysate is needed to ensure that interaction studies can be performed close to native conditions and by using active enzymes. Here, we introduce a study of LIM Kinase activity and inhibition monitored directly in cell lysate by capillary electrophoresis (CE). The miRFP670-LIMK1 was prepared because it is a fluorescent fusion protein emitting in the red spectrum and is thus suitable for microscale thermophoresis (MST) affinity assays. A PDADMAC-coated capillary was mandatory to avoid adsorption issues. The results directly confirm, for the first time, that miRFP670-LIMK1 is still active in the cell lysate even when the miRFP670 fluorescent tag is attached to its C-terminal. The inhibition of miRFP670-LIMK1 activity in the presence of TH-257 was also confirmed by CE. The dissociation constant, K_d, of the interaction miRFP670-LIMK1/TH-257 was then evaluated by MST, again directly in the cell lysate without any purification step. Submicromolar cellular on-target activity was revealed (K_d = 9 nM), confirming that TH-257 is suitable as a chemical probe for LIMK1. This study presents an innovative application of CE as a direct approach to verify, in a straightforward and simple manner, without purification or radioactivity, if an enzymatic target maintains its functionality after labeling. This work also highlights the complementarity of CE and MST in advancing pharmaceutical development under conditions that closely mimic those in vivo.