LIM kinase/inhibitor binding study in cell lysates using microscale thermophoresis in the red spectrum

Background: Cellular function depends on complex molecular interactions that activate signaling pathways - central to drug discovery projects. LIM kinases (LIMKs) are attractive therapeutic targets implicated in various diseases, yet no clinically approved LIMK inhibitors exist, likely due to limited understanding of their molecular behavior under near-physiological conditions. This breach stems in part from the difficulty in purifying these proteins. To address this, there is a crucial need for a simple and rapid method to determine LIMK-inhibitor binding affinities (K_d values) directly in cell lysates, bypassing challenging purification and immobilization steps.

Results: The monomeric near-infrared fluorescent protein miRFP670 (excitation/emission: 640/680 nm) is an advantageous fluorescent tag for microscale thermophoresis (MST) assays in cell lysates. The miRFP670-tagged LIMKs were successfully overexpressed in HEK293 cells, characterized, and validated by western blotting. MST assay conditions - including MST buffer composition and target storage - were carefully optimized and negative controls were consistently used to ensure data reproducibility as well as assay specificity. The MST conditions were as follows: miRFP670-kinases at a fixed concentration of a few tens of nM in cell lysate supplemented with 30 % (v/v) glycerol for convenient storage; 0.005 % (v/v) DMSO in the MST buffer; 37 °C; 20 % LED power; medium MST power; and standard capillaries. The presence of 0.1 % Triton X-100 in the MST buffer was essential to prevent protein aggregation, as evidenced by smooth MST time traces. The reproducibility and stability of signals across all capillaries attest to the optimality of the incubation conditions and the attainment of a stable binding equilibrium. This consistency also validates the robustness of the assay. MST analysis of cell lysates yielded K_d values for full-length and kinase domains and show strong agreement with literature where available. Notably, sub-micromolar K_d values were confirmed for the reference inhibitors LX7101, BMS-5, and TH-257, with slight differences observed between full-length LIMKs and their kinase domains - differences often overlooked in the literature.

Significance and novelty: This work establishes the first detailed production, characterization, and use of miRFP670 in MST-based evaluation of biomolecular interactions under near-native conditions. miRFP670 operates in a spectral region (red) with minimal cellular autofluorescence, enhancing signal specificity and sensitivity. This approach offers a straightforward and broadly applicable, purification-free platform for early-stage drug discovery.

Binding affinity; Cell lysates; LIM kinases; Microscale thermophoresis; Molecular interactions; Red fluorescent miRFP670 protein.