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首页> 外文期刊>Journal of Biotechnology >In vivo selection of intrabodies specifically targeting protein-protein interactions: A general platform for an ''undruggable'' class of disease targets
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In vivo selection of intrabodies specifically targeting protein-protein interactions: A general platform for an ''undruggable'' class of disease targets

机译：体内针对蛋白质-蛋白质相互作用的体内抗体的选择：“非药物”类疾病靶标的通用平台

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Protein-protein interactions represent a major potential drug target for many human diseases, but these are unanimously considered undruggable with small chemical molecules. We have developed 3-SPLINT, a novel technology for the selection of antibodies that are intrinsically endowed with the ability to interfere with a given protein-protein interaction. The selection procedure exploits the recently described yeast SPLINT libraries of intrabodies, adapting them to a reverse-hybrid system, yielding the selection of recombinant antibodies that are able to disrupt a target protein-protein interaction in vivo. This class of antibodies should therefore perturb an individual protein-protein interaction, without perturbing the scaffolding function of the target protein in that complex, or other protein interactions of that same protein. We provide here a proof of concept of the technology, by the de novo selection of antibodies against two distinct interacting protein pairs: the GABARAP, which interact with the gamma2 subunit of GABA(A) receptor, and the p65 protein dimer, involved in the NF-KAPPAB-mediated signalling transduction pathway. Intrabodies selected against the latter were functionally validated in cells. Such antibodies, by interfering with the dimerization domain of p65, lead to an activation of the NF-KAPPAB-mediated transcriptional activity, which is normally inhibited by p65 knock-down RNAi. This provides a clear-cut demonstration that interfering with a protein interaction can be functionally very different from physically removing one of the interacting proteins. The 3-SPLINT approach provides a general and finer tool for the functional validation of selected protein interactions in protein networks, and is ideally applied to protein ''hubs'', displaying multiple distinct interactions. 3-SPLINT will therefore complement RNAi-based approaches, in the toolkit of target validation strategies, and is amenable to the systematic isolation of comprehensive sets of antibodies against most protein-protein interactions of a given protein network.
机译：蛋白质-蛋白质相互作用代表了许多人类疾病的主要潜在药物靶标，但是一致认为，这些相互作用不可与小化学分子一起使用。我们已经开发了3-SPLINT，这是一种用于选择具有固有干扰能力的蛋白，这种蛋白具有干扰给定蛋白质与蛋白质相互作用的能力。选择程序利用了最近描述的体内酵母SPLINT文库，使它们适应反向杂交系统，从而选择了能够破坏体内靶蛋白质与蛋白质相互作用的重组抗体。因此，这类抗体应扰动单个蛋白与蛋白的相互作用，而不会扰乱该复合物中靶蛋白的支架功能或同一蛋白的其他蛋白相互作用。我们通过从头开始选择针对两种不同的相互作用蛋白对的抗体来提供技术概念的证明：与GABA（A）受体的gamma2亚基相互作用的GABARAP，以及参与其中的p65蛋白二聚体NF-KAPPAB介导的信号转导途径。针对后者选择的体内抗体在细胞中进行了功能验证。此类抗体通过干扰p65的二聚化结构域，导致NF-KAPPAB介导的转录活性被激活，通常被p65敲低RNAi抑制。这提供了明确的证明，即干扰蛋白质相互作用的功能可能与物理上去除其中一种相互作用蛋白质的功能有很大不同。 3-SPLINT方法为蛋白质网络中所选蛋白质相互作用的功能验证提供了通用且更精细的工具，并且理想地应用于显示出多个不同相互作用的蛋白质“集线器”。因此，3-SPLINT将在靶标验证策略的工具包中补充基于RNAi的方法，并且适用于系统隔离针对给定蛋白质网络中大多数蛋白质-蛋白质相互作用的全面抗体组合。

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