LINKAGES IN TUBULIN-COLCHICINE FUNCTIONS - THE ROLE OF THE RING-C (C') OXYGENS AND RING-B IN THE CONTROLS

Linkages between structural components uf colchicine (COL) and its bip henyl analogues (allocolchicine, ALLO, and its analogues) in the bindi ng to tubulin and its functional consequences were scrutinized. Three ring ALLO analogues with the carbomethoxyl in position 3' of ring C' r eplaced by a carbomethyl (KAC) and methoxy (MAC) groups were synthesiz ed. The binding properties and consequences of binding (microtubule in hibition, abnormal polymerization, and induction of GTPase activity) w ere compared within the series of three ring and two ring compounds, a s well as between pairs consisting of a two ring and a three ring comp ound with identical groups in position 4'. Binding measurements showed that the binding of KAC to the COL binding site proceeded with simila r chemical characteristics as that of its two ring analogue (TKB), but with the kinetic characteristics of ALLO. The binding constant of KAC was found to be 1.9 x 10(6) M-1 and that of MAC was 4.6 x 10(5) M-1. The binding strength of the three ring analogues in descending order w as KAC > ALLO > MAC, with increments similar to the biphenyl compounds , TKB > TCB > TMB. The difference in binding affinities between the pa irs of three ring and two ring molecules was invariant (delta Delta G degrees = -1.3 +/- 0.2 kcal/mol(-1)), showing that in all cases ring B makes only an entropic contribution by suppressing free rotation abou t the biaryl bond. In the case of microtubule inhibition, all three ri ng compounds inhibited strongly with similar potencies, even though th e spread ill inhibition strength between the corresponding two ring mo lecules was >3.3 kcal mol(-1) of free energy. This difference was inte rpreted in terms of the ability of the various molecules to maintain t ubulin in the proper conformation for binding in abnormal geometry to the growth end of a microtubule. This ability attains st maximal plate au value for three ring compounds, independently of the oxygen-contain ing group in ring C' (or C) and is maintained for the methyl ketone wh ether in a two or three ring compound. The induction of the GTPase act ivity was found to follow in general the binding affinity, with the ex ception that molecules that contained a methyl ketone were stronger GT Pase inducers than expected from their alignment according to binding affinity. The finding that the binding of tropolone methyl ether (ring C of COL) induced a GTPase activity shows that ring C contains the ab ility to induce both substoichiometric microtubule inhibition and GTPa se activity. Rings A and B act only as anchors in the binding, with ri ng A making an energetic contribution, while the effect of ring B is o nly entropic. It was concluded that both microtubule assembly inhibiti on and induction of GTPase activity were modulated by the same postbin ding conformational change in tubulin. The difference between the stre ngths of these activities induced by ligands reflects the difference b etween a narrow allosteric effect between two well-defined sites in th e case of GTPase activity and a broad effect aimed at the multiple sit es involved in the incorporation of a tubulin protomer into the microt ubule structure. Thus. there seems to be a loose thermodynamic linkage between binding and GTPase activity, while there is none between bind ing and microtubule inhibition, the two phenomena being Linked only ki netically.