Dexamethasone-methotrexate:: An efficient chemical inducer of protein dimerization in vivo
Dexamethasone-methotrexate:: An efficient chemical inducer of protein dimerization in vivo
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DOI：
10.1021/ja9941532
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发表时间：
2000-05-03
影响因子：
15
通讯作者：
Cornish, VW
中科院分区：
文献类型：
--
作者：
Lin, HN;Abida, WM;Cornish, VW
可以在 ViVo 中控制细胞过程的细胞渗透性小分子是生物化学和细胞生物学基础研究不可或缺的一部分。例如，异丙基-β-D-硫代半乳糖苷 (IPTG) 几十年来一直用于通过稳定 lac 阻遏物的构象来激活基因转录，同时降低操纵子亲和力。这种方法的巧妙延伸涉及使用二聚配体或“二聚化化学诱导剂”(CID) 来操纵 ViVo 中的蛋白质-蛋白质相互作用。 1-6 CID 的灵感来自于对天然免疫抑制剂 FK506 和雷帕霉素作用机制的研究。 7, 8 研究发现，这两种化合物的免疫抑制活性是由于它们使两种原本不相互作用的蛋白质二聚化。为了概括这种分子机制，Spencer 等人。 1993 年表明，两个通过 C21 烯丙基连接的 FK506 分子可以寡聚与 FKBP12 融合的蛋白质。 CID 特别强大，因为只需将感兴趣的蛋白质与 CID 受体融合即可反复使用相同的二聚配体。迄今为止描述的大多数 CID 是 FK5061、3、4 或 FK506 类似物、5、6 的二聚体，尽管也报道了其他配体。 2 鉴于 CID 的广泛用途，我们试图设计一种优化的 CID，它可以很容易地由市售材料制备，并且基于不同的配体-受体对。在这里，我们报道了这样一种化合物：异二聚体地塞米松-甲氨蝶呤分子，可以在 ViVo 中有效地二聚化蛋白质（图 1）。我们选择基于充分表征的配体-受体对地塞米松 (Dex)-糖皮质激素受体 (GR) 和甲氨蝶呤 (Mtx)-二氢叶酸还原酶 (DHFR) 构建异二聚体 CID。 Dex 和 Mtx 都具有可以轻松修改而不破坏受体结合的化学功能。 12-15 由于 Dex 和 Mtx 与其各自受体的相互作用已得到充分表征，因此 9、10、14、15 未来 Dex-Mtx 系统的表征和优化也应该得到促进。大鼠糖皮质激素受体 (rGR) 与 Dex 的结合 KD 为 5 nM，并且已分离出对 Dex 亲和力增加的 rGR 突变体。 9 类固醇地塞米松已被广泛用作细胞渗透性小分子，以调节体内 GR 融合蛋白的活性和核定位。 10 最近，报道了一种酵母“三杂交”系统，其中 GR 融合体和 FKBP12 融合体可以通过小分子 Dex-FK506 二聚化。 11 同样，由于 Mtx 对 DHFR 具有皮摩尔亲和力，DHFR 融合蛋白已用于各种生化应用 16、17。 18 Dex 和 Mtx 均可市售，并且 Mtx 可以很容易地从简单的起始材料合成。 Dex-Mtx异二聚体的逆向合成分析如方案1所示。该合成基于之前Dex和Mtx衍生物的合成。 12, 19-21 合成的目的是使两个配体之间的化学连接体能够轻松改变。两种配体均作为硫醇衍生物引入二卤连接基中。用高碘酸盐氧化裂解后，使用标准肽偶联试剂用胱胺对 Dex 进行衍生化。只需用同型半胱氨酸取代谷氨酸，Mtx 中的 γ-羧酸盐就被硫醇取代。以叔丁基酯和二硫化物形式保护的同型半胱氨酸与 4-甲基氨基苯甲酸偶联。使用三正丁基膦将所得的 Dex 和 Mtx 二硫化物衍生物还原为其相应的硫醇，并在一锅反应中将两种硫醇偶联到二溴连接基上......
Cell-permeable small molecules that can control cellular processes in ViVo are integral to basic research in biochemistry and cell biology. Isopropyl-β-D-thiogalactoside (IPTG), for example, has been used for decades to activate gene transcription by stabilizing a conformation of lac repressor with reduced operator affinity. A clever extension of this approach involves the use of dimeric ligands or “chemical inducers of dimerization”(CIDs) to manipulate protein-protein interactions in ViVo. 1-6 The inspiration for CIDs were studies of the mechanism of action of the natural immunosuppressants FK506 and rapamycin. 7, 8 It was found that the immunosuppressant activity of both compounds results from the fact that they dimerize two proteins that otherwise do not interact. To generalize this molecular mechanism, Spencer et al. showed in 1993 that two FK506 molecules tethered via their C21 allyl groups could oligomerize proteins fused to FKBP12. CIDs are particularly powerful because the same dimeric ligand can be used over and over again simply by fusing the proteins of interest to the receptors for the CID. The majority of CIDs described to date are dimers of FK5061, 3, 4 or FK506-analogues, 5, 6 although other ligands have been reported also. 2 Given the broad utility of CIDs, we sought to design an optimized CID that could be prepared readily from commercially available materials and that was based on different ligand-receptor pairs. Here, we report such a compound: a heterodimeric dexamethasone-methotrexate molecule that can dimerize proteins efficiently in ViVo (Figure 1).We chose to build a heterodimeric CID based on the wellcharacterized ligand-receptor pairs dexamethasone (Dex)-glucocorticoid receptor (GR) and methotrexate (Mtx)-dihydrofolate reductase (DHFR). Both Dex and Mtx present chemical functionality that can be modified readily without disrupting receptor binding. 12-15 Because the interactions of both Dex and Mtx with their respective receptors are well characterized, 9, 10, 14, 15 future characterization and optimization of the Dex-Mtx system should also be facilitated. The rat glucocorticoid receptor (rGR) binds Dex with a KD of 5 nM, and mutants of rGR with increased affinity for Dex have been isolated. 9 The steroid dexamethasone has been used extensively as a cell-permeable small molecule to regulate the activity and nuclear localization of GR fusion proteins in vivo. 10 Recently, a yeast “three-hybrid” system has been reported in which a GR-fusion and an FKBP12-fusion could be dimerized by the small molecule Dex-FK506. 11 Likewise, DHFR fusion proteins have been used for a variety of biochemical applications16, 17 due to Mtx’s picomolar affinity for DHFR. 18 Both Dex and Mtx are commercially available, and Mtx can be synthesized readily from simple starting materials. The retro-synthetic analysis of the Dex-Mtx heterodimer is shown in Scheme 1. The synthesis is based on previous syntheses of Dex and Mtx derivatives. 12, 19-21 The synthesis is designed to allow the chemical linker between the two ligands to be varied readily. Both ligands were introduced as thiol derivatives to a di-halo linker. Following oxidative cleavage with periodate, Dex was derivatized with cystamine using standard peptide coupling reagents. The γ-carboxylate in Mtx was replaced with a thiol simply by replacing glutamate with homocysteine. Homocysteine, protected as the tert-butyl ester and disulfide, was coupled to 4-methylaminobenzoic acid. The resulting Dex and Mtx disulfide derivatives were reduced to their corresponding thiols using tri-n-butylphosphine, and the two thiols were coupled to a di-bromo linker in a one-pot reaction …