Amrita Center for Nanosciences and Molecular Medicine, Amrita Vishwavidyapeetham University, Cochin, India.
Mol Pharm. 2012 Nov 5;9(11):3062-78. doi: 10.1021/mp300172e. Epub 2012 Oct 5.
Deregulated protein kinases play a very critical role in tumorigenesis, metastasis, and drug resistance of cancer. Although molecularly targeted small molecule kinase inhibitors (SMI) are effective against many types of cancer, point mutations in the kinase domain impart drug resistance, a major challenge in the clinic. A classic example is chronic myeloid leukemia (CML) caused by BCR-ABL fusion protein, wherein a BCR-ABL kinase inhibitor, imatinib (IM), was highly successful in the early chronic phase of the disease, but failed in the advanced stages due to amplification of oncogene or point mutations in the drug-binding site of kinase domain. Here, by identifying critical molecular pathways responsible for the drug-resistance in refractory CML patient samples and a model cell line, we have rationally designed an endogenous protein nanomedicine targeted to both cell surface receptors and aberrantly activated secondary kinase in the oncogenic network. Molecular diagnosis revealed that, in addition to point mutations and amplification of oncogenic BCR-ABL kinase, relapsed/refractory patients exhibited significant activation of STAT5 signaling with correlative overexpression of transferrin receptors (TfR) on the cell membrane. Accordingly, we have developed a human serum albumin (HSA) based nanomedicine, loaded with STAT5 inhibitor (sorafenib), and surface conjugated the same with holo-transferrin (Tf) ligands for TfR specific delivery. This dual-targeted "transferrin conjugated albumin bound sorafenib" nanomedicine (Tf-nAlb-Soraf), prepared using aqueous nanoprecipitation method, displayed uniform spherical morphology with average size of ∼150 nm and drug encapsulation efficiency of ∼74%. TfR specific uptake and enhanced antileukemic activity of the nanomedicine was found maximum in the most drug resistant patient sample having the highest level of STAT5 and TfR expression, thereby confirming the accuracy of our rational design and potential of dual-targeting approach. The nanomedicine induced downregulation of key survival pathways such as pSTAT5 and antiapoptotic protein MCL-1 was demonstrated using immunoblotting. This study reveals that, by implementing molecular diagnosis, personalized nanomedicines can be rationally designed and nanoengineered by imparting therapeutic functionality to endogenous proteins to overcome clinically important challenges like molecular drug resistance.
失调的蛋白激酶在肿瘤的发生、转移和耐药性方面起着非常关键的作用。尽管针对许多类型癌症的分子靶向小分子激酶抑制剂（SMI）是有效的，但激酶结构域的点突变赋予了耐药性，这是临床上面临的主要挑战。一个典型的例子是由 BCR-ABL 融合蛋白引起的慢性髓性白血病（CML），其中 BCR-ABL 激酶抑制剂伊马替尼（IM）在疾病的早期慢性期非常成功，但由于癌基因的扩增或激酶结构域药物结合位点的点突变而在晚期阶段失败。在这里，通过鉴定负责耐药性的关键分子途径在难治性 CML 患者样本和模型细胞系中，我们合理设计了一种针对细胞表面受体和致癌网络中异常激活的二级激酶的内源性蛋白纳米药物。分子诊断显示，除了点突变和致癌 BCR-ABL 激酶的扩增外，复发/难治性患者表现出 STAT5 信号的显著激活，同时细胞膜上转铁蛋白受体（TfR）的表达也有相关性。因此，我们开发了一种基于人血清白蛋白（HSA）的纳米药物，负载有 STAT5 抑制剂（索拉非尼），并在表面用全铁蛋白（Tf）配体偶联，用于 TfR 特异性递药。这种双重靶向的“转铁蛋白结合白蛋白结合索拉非尼”纳米药物（Tf-nAlb-Soraf），采用水性纳米沉淀法制备，具有均匀的球形形态，平均粒径约为 150nm，药物包封效率约为 74%。发现纳米药物对 TfR 的摄取增加，并且在具有最高 STAT5 和 TfR 表达水平的最耐药患者样本中表现出最强的抗白血病活性，从而证实了我们合理设计的准确性和双重靶向方法的潜力。使用免疫印迹法证明了纳米药物诱导的关键存活途径如 pSTAT5 和抗凋亡蛋白 MCL-1 的下调。这项研究表明，通过实施分子诊断，可以合理设计个性化纳米药物，并通过赋予内源性蛋白质治疗功能来对其进行纳米工程设计，从而克服临床上重要的挑战，如分子耐药性。
