Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues.
Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues.
复制标题
秋水仙碱类似物对微管蛋白自组装的化学计量和亚化学计量抑制。
DOI：
10.1021/bi950523x
复制
发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
Timasheff,SN
中科院分区：
文献类型：
--
作者：
Perez-Ramirez,B;Andreu,JM;Gorbunoff,MJ;Timasheff,SN
研究了几种秋水仙碱（COL）结构类似物对微管蛋白自组装的化学计量和亚化学计量抑制作用的机制。根据一个简单的模型分析抑制数据，该模型考虑了正常微管生长常数Kg，等于Cr-1（Cr是微管形成的临界浓度），以及药物与微管蛋白的结合常数Kb。以这种方式，确定微管抑制常数（Ki）的值，其是微管蛋白-药物复合物与生长微管末端的结合常数（其停止微管生长）。各种秋水仙碱类似物对微管抑制的分析结果表明，所有的抑制作用都可以用这个模型合理地表达。最强的抑制剂是秋水仙碱（COL）、别秋水仙碱（ALLO）和联苯酮类似物2，3，4-三甲氧基-4 '-乙酰基-1，1'-联苯（TKB），它们的Ki值基本相同，为（2.1 ± 0.3）× 106 M-1。MTC是秋水仙碱的二环类似物，Ki= 5.6 × 105 M ~（-1）。最引人注目的结果是环酚酮甲基醚（TME），这是COL的C环，它与微管蛋白的结合非常弱（Kb= 3.5 × 102 M-1），是一种亚化学计量的抑制剂。其Ki值为8.7 × 105 M ~（-1），与MTC的强度相同，表明A环对诱导组装抑制作用的贡献很小或没有贡献。这三种联苯以相似的亲和力与微管蛋白结合，跨越了从强亚化学计量抑制（TKB）到2，3，4-三甲氧基-4 '-甲氧羰基-1，1'-联苯（TCB）的化学计量抑制和甲氧基衍生物2，3，4，4 '-四甲氧基-1，1'-联苯（TMB）的中间模式的光谱。在50%抑制率下，微管蛋白与药物结合的程度（r）约为100%。对于TKB、ALLO和COL，2%，即每40 - 50个游离蛋白分子有一个配体微管蛋白（亚化学计量）。该比例对于TCB（化学计量）为1：1.5，对于TMB（中间体）为1：6。对于单环化合物TME，其为1：25。化学计量比的级数与Ki成正比，与Kb值无关，表明化学计量比受Ki的控制，且与Ki之间存在密切的热力学联系。各种药物的抑制能力的比较确定了环C或C '上的羰基的强亚化学计量抑制的需要。此外，该基团必须通过与蛋白质的相互作用或通过环B赋予的结构刚性来正确定向，如在ALLO中。简单的链接平衡模型，在本文中开发允许对齐的药物沿着一个连续的范围从化学计量到强亚化学计量模式的微管抑制。此外，它表明，以前确定的两个类是由一个单一的作用机制描述的单调进展的频谱的两端。
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 considerationKg, the normal microtubule growth constant, equal to Cr-1(Cr is the critical concentration for microtubule formation), andKb, the binding constant of the drug to tubulin. In this manner, the value of the microtubule inhibition constant (Ki), which is the binding constant of the tubulin−drug complex to the end of a growing microtubule (which stops the microtubule growth), was determined. The results of the analysis of microtubule inhibition by the various colchicine analogues show that all the inhibitions can be expressed reasonably by this model. The strongest inhibitors found were colchicine (COL), allocolchicine (ALLO), and the biphenyl keto analogue 2,3,4-trimethoxy-4‘-acetyl-1,1‘-biphenyl (TKB), which had essentially identical values ofKi= (2.1 ± 0.3) × 106M-1. MTC, the two-ring analogue of colchicine, was weaker (Ki= 5.6 × 105M-1). A most striking result was that tropolone methyl ether (TME), which is ring C of COL, and which binds very weakly to tubulin (Kb= 3.5 × 102M-1), is a substoichiometric inhibitor. ItsKivalue of 8.7 × 105M-1makes it identical in strength to MTC, suggesting that ring A makes little or no contribution to the induction of assembly inhibition. The three biphenyls, which bind to tubulin with similar affinity, spanned the spectrum from strong substoichiometric inhibition (TKB) to stoichiometric inhibition for 2,3,4-trimethoxy-4‘-carbomethoxy-1,1‘-biphenyl (TCB) and an intermediate mode for the methoxy derivative 2,3,4,4‘-tetramethoxy-1,1‘-biphenyl (TMB). The extent of tubulin bound 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 withKiand was totally unrelated to the values ofKb, which indicated the control of the stoichiometry byKiand the close thermodynamic linkage betweenrandKi. Comparison of the inhibitory capabilities of the various drugs identified the need for strong substoichiometric inhibition of a carbonyl group on ring C or C‘. Furthermore, this group must be properly oriented by interaction with the protein or by the structural rigidity imparted by ring B, as in ALLO. The simple linked equilibrium model developed in this paper permits the alignment of drugs along a continuum that ranges from stoichiometric to strong substoichiometric modes of microtubule inhibition. Furthermore, it shows that the previously identified two classes are the two ends of a monotonously progressing spectrum described by a single mechanism of action.