ROLE OF THE COLCHICINE RING A AND ITS METHOXY GROUPS IN THE BINDING TO TUBULIN AND MICROTUBULE INHIBITION

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elim ination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independe nt of the nature of ring C (or C'). Specifically, excision of the 2- o r 3-methoxy groups weakened binding by ca. 0.4 kcal mol(-1), while tha t of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol(-1). The effect on the inhibition of microtubule assembly, express ed as the equilibrium constant for the binding of the tubulin-drug com plex to the end of a microtubule, was more complex and strongly depend ent on the nature of ring C (or C'). This was attributed to the abilit ies of various groups on ring C' to overcome the wobbling in the tubul in-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the me chanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in t he interactions that key oxygen atoms of ring C of COL or C' of struct ural analogues establish with the protein. It is proposed that the 4-m ethoxy group of ring A serves as a key attachment point for immobiliza tion of the drugs on the protein.