Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.
Bioconjug Chem. 2010 Sep 15;21(9):1691-702. doi: 10.1021/bc100292x.
Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both the site of conjugation and, to a lesser extent, the propinquity of the monomers within the conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth at a lower dose and with less frequent administration than does the monomer. This effect is attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the trimer is ∼300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for the treatment of lung cancer. These data encourage the development of mammalian ribonucleases for the treatment of human cancers.
定点交联可以生成具有特定价态的均一的多聚体蛋白质。胰型核糖核酸酶是一个特别有吸引力的靶标，因为它们的天然二聚体可以进入哺乳动物细胞，逃避细胞质核糖核酸酶抑制剂（RI），并发挥其毒性的核糖核酸酶活性。在这里，我们报告了使用八种不同的巯基反应性交联试剂来制备四种胰型核糖核酸酶的二聚体和三聚体缀合物。连接的部位，以及在较小程度上，缀合物中单体的邻近性，调节与 RI 的亲和力，从而调节细胞毒性。然而，多聚体的细胞毒性在体外受到其增加的流体力学半径的影响，这会减弱细胞质内的进入。牛胰核糖核酸酶（RNase A）的一种 RI 逃避单体变体抑制小鼠前列腺和肺癌肿瘤的生长。RI 逃避的三聚体缀合物以比单体更低的剂量和更频繁的给药抑制肿瘤生长。这种效果归因于三聚体在循环中的增强持久性。从分子基础上讲，三聚体的效力比厄洛替尼高约 300 倍，厄洛替尼已用于治疗肺癌。这些数据鼓励开发用于治疗人类癌症的哺乳动物核糖核酸酶。
