Melillo Giovanni
Developmental Therapeutics Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, USA.
Methods Enzymol. 2007;435:385-402. doi: 10.1016/S0076-6879(07)35020-9.
The tremendous progress in our understanding of the molecular mechanisms underlying the presence and consequences of hypoxia in human cancers has been accompanied by renewed enthusiasm for the development of therapeutic strategies targeting hypoxic cells signaling pathways. Hypoxia-inducible factor 1 (HIF-1), a key transcriptional activator that mediates hypoxic responses, has been the focus of intense investigation and efforts to identify small molecule inhibitors or novel strategies for HIF-1 inhibition have multiplied over the last few years. Despite challenges associated with targeting transcription factors, which hamper these efforts, several strategies have been pursued. In this chapter, protocols related to screening assays, both cell-based and cell-free, are described and discussed in the context of their application for the identification of HIF-1 inhibitors. While cell-based assays offer the opportunity to reveal unidentified components of the hypoxic cell signaling pathway, cell-free targeted approaches may lead to the identification of more selective HIF-1 inhibitors. Validation of "hits" and characterization of their mechanism of action are essential for a rational development of putative HIF-1 inhibitors in preclinical models and early clinical trials.
我们对人类癌症中缺氧的存在及其后果所涉及分子机制的理解取得了巨大进展，与此同时，人们对开发针对缺氧细胞信号通路的治疗策略再度充满热情。缺氧诱导因子1（HIF-1）是介导缺氧反应的关键转录激活因子，一直是深入研究的焦点，在过去几年里，识别小分子抑制剂或采用新策略抑制HIF-1的努力成倍增加。尽管针对转录因子存在阻碍这些努力的挑战，但人们还是采用了多种策略。在本章中，将在用于识别HIF-1抑制剂的应用背景下，描述和讨论与基于细胞和无细胞的筛选测定相关的方案。虽然基于细胞的测定提供了揭示缺氧细胞信号通路中未识别成分的机会，但无细胞靶向方法可能会导致识别出更具选择性的HIF-1抑制剂。在临床前模型和早期临床试验中合理开发假定的HIF-1抑制剂，对“命中”结果进行验证及其作用机制的表征至关重要。
