The mitotic motor protein Eg5 is essential for bipolar spindle formation and is a validated target for cancer therapy due to its overexpression in tumor cells. In this study, we developed a novel photochromic inhibitor, SP-SAB-SP, which combines azobenzene and two spiropyran moieties, enabling light-controlled regulation of Eg5 activity. Azobenzene undergoes cis-trans isomerization, while spiropyran undergoes spiro-merocyanine isomerization. Through combinations of these photochromic units, SP-SAB-SP can adopt multiple isomeric states depending on irradiation with specific wavelengths of light. We identified three states: MC-Cis-MC (UV), SP-Trans-SP (visible light), and MC-Trans-MC (dark). SP-SAB-SP was synthesized and characterized by structural and biochemical assays, confirming interaction with the Eg5 motor domain. Distinct inhibitory activities against basal and microtubule (MT)-stimulated ATPase were observed among the photoisomerization states. Eg5 inhibition was classified as weak (MC-Trans-MC), moderate (MC-Cis-MC), and strong (SP-Trans-SP). In motility assays with fluorescently labeled MTs, SP-SAB-SP also suppressed Eg5-driven motility with state-dependent potency. Furthermore, molecular docking simulations are underway to evaluate binding specificity and to complement the experimental findings. Comparison with a previously reported inhibitor containing one spiropyran and one azobenzene revealed that SP-SAB-SP achieves superior photo-regulated inhibitory efficiency.
Salwadi et al. (Sun,) studied this question.