STOICHIOMETRIC AND SUBSTOICHIOMETRIC INHIBITION OF TUBULIN SELF-ASSEMBLY BY COLCHICINE ANALOGS

The mechanism of the stoichiometric and substoichiometric inhibitions of tubulin self-assembly by several structural analogues of colchicine (COL) was investigated. The inhibition data were analyzed in terms of a simple model that takes into consideration K-g, the normal microtub ule growth constant, equal to Cr-1 (Cr is the critical concentration f or microtubule formation), and K-b, the binding constant of the drug t o tubulin. In this manner, the value of the microtubule inhibition con stant (K-i), which is the binding constant of the tubulin-drug complex to the end of a growing microtubule (which stops the microtubule grow th), was determined. The results of the analysis of microtubule inhibi tion by the various colchicine analogues show that all the inhibitions can be expressed reasonably by this model. The strongest inhibitors f ound were colchicine (COL), allocolchicine (ALLO), and the biphenyl ke to analogue 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB), which had essentially identical values of K-i= (2.1 +/- 0.3) x 10(6) M(-1). MTC, the two-ring analogue of colchicine, was weaker (K-i = 5.6 x 10(5) M( -1)). A most striking result was that tropolone methyl ether (TME), wh ich is ring C of COL, and which binds very weakly to tubulin (K-b = 3. 5 x 10(2) M(-1)), is a substoichiometric inhibitor. Its K-i value of 8 .7 x 10(5) M(-1) makes it identical in strength to MTC, suggesting tha t ring A makes little or no contribution to the induction of assembly inhibition. The three biphenyls, which bind to tubulin with similar af finity, spanned the spectrum from strong substoichiometric inhibition (TKB) to stoichiometric inhibition for 2,3,4-trimethoxy-4'-carbomethox y-1,1'-biphenyl (TCB) and an intermediate mode for the methoxy derivat ive 2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB). The extent of tubulin b ound to drugs at 50% inhibition (r) was ca. 2% for TKB, ALLO, and COL, i.e. one liganded tubulin for every 40-50 molecules of free protein ( substoichiometric). This ratio was 1:1.5 for TCB (stoichiometric) and 1:6 for TMB (intermediate). For TME, which is a single ring compound, it was 1:25. The progression of the stoichiometries varied directly wi th K-i and was totally unrelated to the values of K-b, which indicated the control of the stoichiometry by K-i and the close thermodynamic l inkage between r and K-i. Comparison of the inhibitory capabilities of the various drugs identified the need for strong substoichiometric in hibition of a carbonyl group on ring C or C'. Furthermore, this group must be properly oriented by interaction with the protein or by the st ructural rigidity imparted by ring B, as in ALLO. The simple linked eq uilibrium model developed in this paper permits the alignment of drugs along a continuum that ranges from stoichiometric to strong substoich iometric modes of microtubule inhibition. Furthermore, it shows that t he previously identified two classes are the two ends of a monotonousl y progressing spectrum described by a single mechanism of action.