Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures.
Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures.
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DOI：
10.1016/j.jbiotec.2021.10.001
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发表时间：
2021-12-10
影响因子：
4.1
通讯作者：
Littleton J
中科院分区：
文献类型：
--
作者：
Rogers DT;Pomerleau F;Kelley Z;Brown D;Lynn B;Gerhardt GA;Littleton J
多巴胺转运体（DAT）是药物使用障碍（sud）的靶标，而具有低滥用潜力的“非经典”DAT抑制剂是治疗的候选药物。来自红半边莲的洛比林是一种非典型的DAT抑制剂铅。碱的化学合成具有挑战性；因此，“目标导向进化”被用于先导优化。靶蛋白在植物细胞中表达，在靶蛋白功能抑制赋予生存优势的条件下选择突变细胞群。幸存的突变体被“埋设”用于目标活动。应用于表达人类DAT的突变L. cardinalis细胞群，我们鉴定了20个过量产生DAT抑制剂的突变体。微量分析优先考虑新的洛比林衍生物，我们首先研究了水溶性更高的洛比林n -氧化物。它抑制大鼠突触体[3H]DA摄取，IC50与洛比林相似。反复将DA显微注射到大鼠纹状体中，洛比林产生短暂的DA清除率降低。相反，脑碱n -氧化物延长了DA峰振幅，特别是在腹侧纹状体。洛比林n -氧化物也产生了与简单的DAT抑制不一致的峰后DA清除的复杂变化。这种不寻常的DAT相互作用可能被证明对治疗sud有用。该研究证明了植物细胞靶向进化对优化化学合成困难的先导化合物的价值。
The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and “non-classical”“ DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, “target-directed evolution” was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are “mined” for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.
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影响因子：
4.1
作者：
Mfuh AM;Larionov OV
通讯作者：
Larionov OV
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3
作者：
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