Wang Huabo, Ramakrishnan Anand, Fletcher Steven, Prochownik Edward V
Section of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA 15224, USA.
GE Healthcare, Piscataway, NJ 08854, USA.
J Biol Methods. 2015;2(2). doi: 10.14440/jbm.2015.54.
The use of small molecules to interfere with protein-protein interactions has tremendous therapeutic appeal and is an area of intense interest. Numerous techniques exist to assess these interactions and their disruption. Many, however, require large amounts of protein, do not allow interactions to be followed in real time, are technically demanding or require large capital expenditures and high levels of expertise. Surface plasmon resonance (SPR) represents a convenient alternative to these techniques with virtually none of their disadvantages. We have devised an SPR-based method that allows the heterodimeric association between the c-Myc (Myc) oncoprotein and its obligate partner Max to be quantified in a manner that agrees well with values obtained by other methods. We have adapted it to examine the ability of previously validated small molecules to interfere with Myc-Max heterodimerization and DNA binding. These inhibitors comprised two distinct classes of molecules that inhibit DNA binding by preventing Myc-Max interaction or distorting pre-formed heterodimers and rendering them incapable of DNA binding. Our studies also point out several potential artifacts and pitfalls to be considered when attempting to employ similar SPR-based methods. This technique should be readily adaptable to the study of other protein-protein interactions and their disruption by small molecules.
使用小分子干扰蛋白质-蛋白质相互作用具有巨大的治疗吸引力，是一个备受关注的领域。存在多种技术来评估这些相互作用及其破坏情况。然而，许多技术需要大量蛋白质，不允许实时跟踪相互作用，技术要求高，或者需要大量资金投入和高水平专业知识。表面等离子体共振（SPR）是这些技术的一种便捷替代方法，几乎没有它们的缺点。我们设计了一种基于SPR的方法，该方法能够以与其他方法获得的值高度一致的方式对c-Myc（Myc）癌蛋白与其必需伴侣Max之间的异二聚体结合进行定量。我们对其进行了改进，以检测先前经验证的小分子干扰Myc-Max异二聚化和DNA结合的能力。这些抑制剂包括两类不同的分子，它们通过阻止Myc-Max相互作用或扭曲预先形成的异二聚体并使其无法结合DNA来抑制DNA结合。我们的研究还指出了在尝试采用类似的基于SPR的方法时需要考虑的几个潜在假象和陷阱。该技术应该很容易适用于研究其他蛋白质-蛋白质相互作用及其被小分子破坏的情况。
