Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments
Rigorous analysis of the interaction between proteins and low water-solubility drugs by qNMR-aided NMR titration experiments
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DOI：
10.1039/d1cp03175a
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发表时间：
2021-09-17
影响因子：
3.3
通讯作者：
Sugase，Kenji
中科院分区：
文献类型：
--
作者：
Hirakawa，Takuya;Walinda，Erik;Sugase，Kenji
药物的设计和验证是基于它们与靶蛋白相互作用的物理化学数据。然而，对于低水溶性药物，如果没有准确的沉淀估计，定量分析实际上是不可能的。本文将定量核磁共振与核磁共振滴定实验相结合，严格量化了低水溶性药物吡美莫司与其靶蛋白FKBP12的相互作用。值得注意的是，在考虑沉淀和不考虑沉淀的情况下估计的解离常数相差十倍以上。此外，该方法使我们能够定量FKBP12-pimecrolimus相互作用，即使在使用蛋白拥挤剂BSA建立的拥挤条件下。值得注意的是，在拥挤的环境下，fkbp12 -吡美莫司的相互作用受到轻微阻碍，这可以解释为BSA与药物分子的短暂结合。总的来说，所描述的方法将有助于量化低水溶性药物的结合特性，并阐明药物在包括活细胞在内的复杂拥挤溶液中的行为。
Drugs are designed and validated based on physicochemical data on their interactions with target proteins. For low water-solubility drugs, however, quantitative analysis is practically impossible without accurate estimation of precipitation. Here we combined quantitative NMR with NMR titration experiments to rigorously quantify the interaction of the low water-solubility drug pimecrolimus with its target protein FKBP12. Notably, the dissociation constants estimated with and without consideration of precipitation differed by more than tenfold. Moreover, the method enabled us to quantitate the FKBP12–pimecrolimus interaction even under a crowded condition established using the protein crowder BSA. Notably, the FKBP12–pimecrolimus interaction was slightly hampered under the crowded environment, which is explained by transient association of BSA with the drug molecules. Collectively, the described method will contribute to both quantifying the binding properties of low water-solubility drugs and to elucidating the drug behavior in complex crowded solutions including living cells.