Photo-Regulation of Kinesin Eg5 ATPase and Motor Activity using Novel Photochromic Inhibitor Composed of Spiropyran and Cysteine

The mitotic kinesin Eg5 has an important role in establishing the bipolar spindle which is directly involved in the cell division. Recently, several small molecules have been identified as potent specific inhibitor of Eg5. STLC is one of the inhibitor which does not compete with either ATP or microtubules but slows down ADP release. STLC binds to the pocket on Eg5 composed by a2, a3 and loop L5. STLC binding induces the downward swing of L5 to close the inhibitor binding pocket. Therefore, L5 is one of the key region to stabilize Eg5-STLC complex. Previously, we incorporated photochromic molecules into L5 to photo-control inhibitory activity of STLC. Successfully Eg5 mutant D130C modified with spiropyran derivative showed significant photo-reversible resistance to STLC.In this study, further application of photochromic molecules to regulate Eg5 activity was performed. We synthesized photochromic STLC analogue, IASP-L-Cys composed of spiropyran and L-cystein to regulate Eg5 ATPase and motor activity reversibly upon ultraviolet and visible lights irradiation. IASP-L-Cys exhibited merocyanine - spiro isomerization upon ultraviolet and visible light irradiations. Zwitterionic merocyanine isomer has a ring-opening structure and differs from the other isomer of spiro that is hydrophobic and ring-closing structure. Therefore, it is expected that photo-isomerization of IASP-L-Cys may alter its inhibitory activity for Eg5 significantly. Microtubule dependent ATPase activity of Eg5 was inhibited by IASP-L-Cys in a concentration dependent manner. And the inhibitory activity of IASP-L-Cys for Eg5 was drastically changed correlating to photo-isomerization. Merocyanine isomer showed much higher inhibition constant than that of spiro isomer (approximately 20 μM and 100 μM, respectively). We also examined the effect of phtoisomerization of IASP-L-Cys for the cell mitosis using HeLa cell.