Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors
Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors
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DOI：
10.1021/ja802977h
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发表时间：
2008-07-23
影响因子：
15
通讯作者：
Shoichet, Brian K.
中科院分区：
文献类型：
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作者：
Coan, Kristin E. D.;Shoichet, Brian K.
早期药物发现中的许多假阳性归因于有机分子胶体状聚集体的非特异性抑制。尽管它们很普遍，但人们对聚集体浓度、结构或动态平衡知之甚少。酶的结合机制、化学计量和亲和力仍不确定。为了研究浓度的基本问题，我们使用流式细胞术对聚集颗粒进行了计数。对于七个聚集体形成分子，直到单体浓度超过阈值才观察到聚集体，表明“临界聚集浓度”（CAC）。在 CAC 之上，聚集体计数随着有机材料的添加而线性增加，而在 CAC 之下稀释时颗粒会分散。单体有机分子的浓度在 CAC 之上恒定，聚集体颗粒的尺寸也是如此。对于形成大聚集体的两种化合物，尼卡地平和咪康唑，我们通过流式细胞术直接测量颗粒数，确定刚好高于 CAC 的聚集体浓度范围为 5 至 30 fM。通过将酶的抑制与这两种药物的聚集体计数相关联，我们确定结合的化学计量为每个聚集体颗粒约 10 000 个酶分子。使用尼卡地平和咪康唑聚集颗粒的测量体积（分别为 2.1 x 10(11) 和 4.7 x 10(10) 埃(3)）、计算单体体积，以及通过 CAC 所有其他单体形成聚集颗粒的观察结果，我们发现聚集体是致密堆积的颗粒。最后，考虑到它们的尺寸和酶化学计量，所有螯合酶都可以舒适地容纳在聚集体的表面上。
Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a "critical aggregation concentration" (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10 000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 x 10(11) and 4.7 x 10(10) angstrom(3), respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate.