The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters
The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters
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DOI：
10.1073/pnas.97.24.13003
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发表时间：
2000-11-21
影响因子：
11.1
通讯作者：
Rothbard, JB
中科院分区：
文献类型：
--
作者：
Wender, PA;Mitchell, DJ;Rothbard, JB
某些蛋白质含有亚基，使其能够主动转运穿过质膜进入细胞。在HIV-1的特定情况下，该亚基是基本结构域达特（49-57）（RKKRRQRRR）。为了建立这种易位过程的最佳结构要求，从而开发可以将药剂递送到细胞中的改进的分子转运蛋白，制备了Tat 49 -57的一系列类似物，并通过流式细胞术测定它们在Jurkat细胞中的细胞摄取。所有截短的和丙氨酸取代的类似物都表现出减少的细胞摄取，表明达特（49-57）的阳离子残基在其摄取中起主要作用。然而，单独的电荷不足以转运，因为几种阳离子氨基酸（组氨酸、赖氨酸和鸟氨酸）的寡聚体在细胞摄取中不如Taf（49-57）有效。相比之下，如通过Michaelis-Menton动力学分析测定的，9-mer L-精氨酸（R9）在细胞摄取方面比达特（49-57）有效20倍。D-精氨酸寡聚体（r9）表现出甚至更大的摄取速率增强（>100倍）。总的来说，这些研究表明，达特（49-57）的胍基在促进细胞摄取方面比电荷或骨架结构发挥更大的作用。在此基础上，我们设计合成了一类多胍类肽衍生物。值得注意的是，与达特（49-57）甚至与r9相比，在胍头基和骨架之间含有六亚甲基间隔基（N-hxg）的类肽类似物的子集表现出显著增强的细胞摄取。总的来说，已经开发了一种转运蛋白，其上级于达特（49-57），具有蛋白酶抗性，并且更容易和经济地制备。
Certain proteins contain subunits that enable their active translocation across the plasma membrane into cells. In the specific case of HIV-1, this subunit is the basic domain Tat(49-57) (RKKRRQRRR). To establish the optimal structural requirements for this translocation process, and thereby to develop improved molecular transporters that could deliver agents into cells, a series of analogues of Tat49-57 were prepared and their cellular uptake into Jurkat cells was determined by flow cytometry. All truncated and alanine-substituted analogues exhibited diminished cellular uptake, suggesting that the cationic residues of Tat(49-57) play a principal role in its uptake. Charge alone, however, is insufficient for transport as oligomers of several cationic amino acids (histidine, lysine, and ornithine) are less effective than Taf(49-57) in cellular uptake. In contrast, a 9-mer of L-arginine (R9) was 20-fold more efficient than Tat(49-57) at cellular uptake as determined by Michaelis-Menton kinetic analysis. The D-arginine oligomer (r9) exhibited an even greater uptake rate enhancement (>100-fold). Collectively, these studies suggest that the guanidinium groups of Tat(49-57) play a greater role in facilitating cellular uptake than either charge or backbone structure. Based on this analysis, we designed and synthesized a class of polyguanidine peptoid derivatives. Remarkably, the subset of peptoid analogues containing a six-methylene spacer between the guanidine head group and backbone (N-hxg), exhibited significantly enhanced cellular uptake compared to Tat(49-57) and even to r9. Overall, a transporter has been developed that is superior to Tat(49-57), protease resistent, and more readily and economically prepared.