喹啉类药物对多发性骨髓瘤的实验性治疗作用及其抑制组蛋白去乙酰化酶的分子机理

Repurposing old drugs for new indications is one of the most important and the most efficient strategies for drug discovery. Quinolines are a class of anti-infection or anti-malaria drugs characterized with minimal toxicity, low price and easy synthesis. In previous studies, we found that quinoline drugs including clioquinol (CLQ) and nitroxoline (NXQ) can inhibit proliferation of a panel of multiple myeloma (MM) cells at low concentrations. Histone deacetylases (HDACs) are frequently dysregulated in MM cells and associated with poor therapeutic outcomes and drug resistance. In the mechanistic study, we found that CLQ inhibited HDAC activity with evidence that it bound to HDAC, down-regulated HDAC expression at the protein level, and up-regulated the acetylated status of histone 3 (H3), an important indicator of inhibition of HDACs, but the underlying mechanisms are not clear. Since CLQ is a potent chelator of zinc ion which is vital for HDAC activity, we hypothesize that quinolines may inhibit HDAC activity by disrupting zinc in the active pocket of HDACs. In this proposal, we are trying to find out the best anti-myeloma quinoline drug by surveying all quinoline drugs that are clinical available by several assays involved in MM cell lines, primary MM samples and nude mice MM xenografts. Subsequently, we will dissect the mechanisms involved in HDAC inhibition. We will apply protocols such as enzymology, real-time PCR, immunoprecipitation, computer modeling, and mass spectrometry to work out how quinoline drugs interact with HDACs and inhibit their activities. In detail, we will (1) use molecular biology protocols to evaluate HDAC expression at both transcriptional and translational levels upon the treatment of quinoline drugs; (2) study the inhibition pattern and kinetic characteristics of HDACs by quinolines; (3) take advantage of computer modeling techniques to predict the interaction of quinoline drug with HDACs; (4) use immunoprecipitation-mass spectrometry (IP-MS) to analyze the quinoline drug-HDAC complexes. This study will provide a new line of evidence to support quinoline drugs in the treatment of MM and will also facilitate the development of anti-myeloma quinoline drugs.

"老药新用"是加快新药研究的主要途径之一。我们发现喹啉类药物氯碘羟喹、硝羟喹啉等可以诱导多发性骨髓瘤（MM）的凋亡，并具有毒性小、价格低等优点，将其开发成新的抗MM药物具有很好的临床意义。组蛋白去乙酰化酶（HDAC）的表达或功能失调与MM发生密切相关，我们发现氯碘羟喹可与HDAC结合，下调HDAC蛋白质水平，并提高组蛋白H3的乙酰化水平，提示其对HDAC有抑制作用，但具体机制不明。本项目将以MM细胞株、患者原代细胞和裸鼠模型为研究对象，筛选出具有抗MM作用的喹啉类药物，并用酶学、定量PCR、计算机模拟、免疫沉淀及质谱技术，研究喹啉类药物抑制HDAC的分子机理。主要包括：①喹啉类药物对HDAC转录、表达及功能的影响；②对HDAC酶活性的抑制作用及特征；③预测其与HDAC的结合位点和抑制模式；④免疫沉淀-质谱技术分析喹啉类药物与HDAC复合物的形成。本研究将为开发喹啉类抗MM药物提供理论基础。

目前常用的喹啉类药物包括氯碘羟喹（Clioquinol，CLQ）、硝羟喹啉（Nitroxoline，NXQ）、氯喹、伯氨喹、甲氟喹等。本项目中，我们以MM代表性细胞株RPMI-8226为研究对象完成了对喹啉类药物的筛选，发现NXQ诱导MM的凋亡程度最强， 2.5 μM的NXQ在12小时内即能诱导多种MM细胞的凋亡。NXQ对MM的影响在细胞和动物水平都得到了很好的验证。在机理研究方面，我们发现CLQ能够通过与HDAC的锌离子结合从而抑制HDAC的活性，且具有很强的选择性。以锌离子为活性中心的HDAC 共有三大类11个成员， HDAC的酶活性分析中发现CLQ特异性地抑制Class I 和IIa HDAC 的活性，对Class IIb（以及Class IV没有作用。当在细胞中加入锌离子时，CLQ对HDAC的抑制作用被削弱，进一步提示CLQ可以通过结合并干扰HDAC中的锌离子活性中心来抑制HDAC的功能。这一作用机制在我们的计算机模拟实验中得到了证实。一方面，CLQ通过其强大的金属阳离子螯合能力，与锌离子结合。另一方面，CLQ还可以与HDAC的活性口袋的多个氨基酸残基形成氢键和范德华力，进一步抑制HDAC的活性。. TRIM25在多发性骨髓瘤细胞中呈高水平表达，NXQ能够下调TRIM25的表达。我们进一步发现TRIM25通过与p53的结合诱导其降解。而加入硝基喹啉后，TRIM25的蛋白质水平降低，p53水平上升，提示硝基喹啉能够调节TRIM25/p53轴，诱导MM细胞的凋亡。. 除了诱导凋亡之外，我们发现喹啉类药物也能诱导MM细胞的自噬，并且发现这种自噬与凋亡关联，因为当自噬被抑制后，MM细胞对CLQ的敏感性下降。有意思的是，CLQ虽然不能改变自噬中重要蛋白质Beclin 1和PI3K的表达水平，但能抑制mTOR的酶活性，促进自噬激活复合物Beclin 1/VPS34 的形成。在Beclin 1 被敲低后，CLQ诱导的自噬被完全抑制。这些表明自噬参与了喹啉类药物的抗MM作用。. 此外，发现了多个小分子的抗多发性骨髓瘤的药物，阐明了多发性骨髓瘤细胞中重要转录因子c-Maf的降解机制。以上研究成果部分发表于JBC (2013)、 BJC (2014)、Blood (2016)、EJH (2014)、Anti-cancer Drugs (2016)等杂志。