Department of Chemistry and The Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, The State University of New York, 657 Natural Sciences Complex, Buffalo, New York 14260, USA.
J Am Chem Soc. 2009 Dec 2;131(47):17464-72. doi: 10.1021/ja906877y.
Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in noncoding regions of RNA. In DM1, the expansion is an rCUG triplet repeat, whereas the DM2 expansion is an rCCUG quadruplet repeat. Both RNAs fold into hairpin structures with periodically repeating internal loops separated by two 5'GC/3'CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 x 1 nucleotide UU loops while the DM2 repeat forms 2 x 2 nucleotide 5'CU/3'UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6'-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 x 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective for the DM2 RNA over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by a shorter distance. Indeed, here the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with K(d)'s of 20 nM (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective) and inhibit the RNA-protein interaction with IC(50)'s of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- x 3-fold). The trimer and tetramer also bind approximately 13- and approximately 63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and nontoxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and, (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.
肌强直性营养不良 1 型和 2 型（DM1 和 DM2，分别）是由非编码 RNA 中重复核苷酸的扩展引起的。在 DM1 中，扩展是 rCUG 三核苷酸重复，而 DM2 扩展是 rCCUG 四核苷酸重复。这两种 RNA 都折叠成发夹结构，内部环周期性重复，由两个 5'GC/3'CG 碱基对隔开。然而，环的大小不同：DM1 重复形成 1 x 1 核苷酸 UU 环，而 DM2 重复形成 2 x 2 核苷酸 5'CU/3'UC 环。当扩展的重复与 RNA 剪接调节剂 Muscleblind-like 1 蛋白（MBNL1）结合时，就会导致 DM，从而损害其功能。因此，这些疾病的一种潜在治疗策略是防止 MBNL1 与毒性 RNA 重复结合。以前，我们通过将 6'-N-5-己酰基卡那霉素 A（K）模块化组装到肽聚糖骨架上，设计了 DM2-MBNL1 相互作用的纳摩尔抑制剂。K 配体与 DM2 RNA 中富含嘧啶的内部 2 x 2 环具有高亲和力结合。从该研究中鉴定出的最佳化合物含有三个 K 模块，由四个丙基胺间隔模块隔开，对 DM2 RNA 的选择性是 DM1 RNA 的 20 倍。因为模块化组装的含 K 化合物也与 DM1 RNA 结合，尽管亲和力较低，而且由于环的大小不同，我们假设最佳的 DM1 RNA 结合剂可能显示出较短的 K 模块间隔。事实上，在这里，理想的 DM1 RNA 结合剂只有两个丙基胺间隔模块分隔 K 配体。在肽聚糖支架上显示三个和四个 K 模块的肽聚糖与 DM1 RNA 的结合 K(d)'为 20 nM（DM1 对 DM2 的选择性为 3 倍）和 4 nM（DM1 对 DM2 的选择性为 6 倍），并分别以 IC(50)'为 40 和 7 nM 抑制 RNA-蛋白相互作用。重要的是，通过将这两项研究结合起来，我们已经确定适当的间隔可以使结合选择性提高 60 倍（20- x 3 倍）。三聚体和四聚体与 DM1 RNA 的结合亲和力也分别提高了约 13 倍和 63 倍。模块化组装的化合物通过流式细胞术确定是细胞渗透和无毒的。结果表明，对于这两个系统：（i）可编程模块化组装方法可以为具有亲和力和特异性的 RNA 提供合成配体，这些配体超过天然蛋白质；并且，（ii）配体模块的间隔可以用于调整对一个 RNA 靶标的特异性。
