Engineering mono- and multi-valent inhibitors on a modular scaffold.
Engineering mono- and multi-valent inhibitors on a modular scaffold.
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DOI：
10.1039/d0sc03175e
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发表时间：
2021-01-21
期刊：
影响因子：
8.4
通讯作者：
Itzhaki LS
中科院分区：
文献类型：
--
作者：
Diamante A;Chaturbedy PK;Rowling PJE;Kumita JR;Eapen RS;McLaughlin SH;de la Roche M;Perez-Riba A;Itzhaki LS
我们利用重复蛋白的简单模块化结构，以不同的可编程几何形状创建单功能和多功能展示平台，并通过构建关键信号通路的有效抑制剂来证明概念。在这里，我们利用简单的，超稳定的，模块化的结构的共识设计的tetratricopeptide重复蛋白（CTPR）创建一个平台，能够显示单一以及多个功能和不同的可编程的几何排列，通过嫁接非螺旋短线性结合基序（SLiM）到相邻的重复之间的环。作为概念证明，我们构建了合成的CTPR来结合和抑制人端锚聚合酶蛋白（hTNKS），该蛋白在Wnt信号传导中起关键作用，并在癌症中上调。组装一系列单价和多价hTNKS粘合剂。为了充分利用模块化支架并进一步使多价几何结构多样化，我们设计了具有两种不同形式的结合模块，一种是单体，另一种是三聚体。我们表明，设计的蛋白质是稳定的，正确折叠，并能够结合和抑制hTNKS的细胞活性，导致Wnt途径的下调。CTPR蛋白阵列和hTNKS靶标中的多价性导致大分子组装体的形成，其可以在体外和细胞中可视化。当通过纳米颗粒包封递送到细胞中时，多价CTPR蛋白显示出异常的活性。它们能够抑制Wnt信号传导，其中小分子抑制剂迄今为止未能成功。我们的研究结果表明，CTPR平台具有巨大的潜力，可以利用一系列SLiM并组装具有内置多价能力和精确预编程几何形状的合成结合分子。
We exploit the simple modular architecture of repeat proteins to create a platform for single- and multi-functional display in diverse programmable geometries and demonstrate proof of concept by building potent inhibitors of a key signalling pathway. Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.