Massively parallel, computationally guided design of a proenzyme.
Massively parallel, computationally guided design of a proenzyme.
复制标题
DOI：
10.1073/pnas.2116097119
复制
发表时间：
2022-04-12
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质已经显示出作为治疗和诊断的前景，但它们的有效性受到我们无法在空间上靶向其活性的限制。为了克服这一限制，我们开发了一种计算指导的方法来设计无活性的酶原或酶原，它们通过蛋白酶的切割而被激活。由于蛋白酶在各种组织和疾病状态（包括癌症）中差异表达，因此这些酶原可以靶向所需的微环境。我们在治疗相关蛋白羧肽酶G2（CPG 2）上测试了我们的方法。我们设计了Pro-CPG 2，其被抑制80%至98%，并且在蛋白酶处理后部分至完全可再活化。所开发的方法学经过进一步的改进，可以为常规设计以空间控制方式起作用的基于蛋白酶激活蛋白质的治疗和诊断铺平道路。将设计的蛋白质的活性限制在特定的微环境中将具有广泛的应用，例如通过酶实现细胞类型特异性治疗作用，同时避免脱靶效应。虽然许多天然酶被合成为可以通过蛋白水解激活的无活性酶原，但重新设计任何选择的酶以类似地刺激响应是具有挑战性的。在这里，我们开发了一个大规模并行计算设计，筛选和下一代测序为基础的方法，酶原设计。对于模型系统，我们采用羧肽酶G2（CPG 2），一种临床批准的酶，可用于治疗癌症和控制药物毒性。最有效设计的变体的详细动力学表征表明，与未修饰的蛋白质相比，它们被抑制了约80%，并且它们的活性在与位点特异性蛋白酶孵育后完全恢复。在基于设计模型的亲和催化结构域之间引入二硫键将抑制程度增加到98%，但降低了通过蛋白水解恢复活性的程度。当在细胞培养物中评价时，所选的含二硫化物的酶原相对于完全活化的酶表现出显著较低的活性。结构和热力学特性提供了详细的见解前结构域结合和抑制机制。所描述的方法是通用的，可以使各种前蛋白的设计与精确的空间调控。
Proteins have shown promise as therapeutics and diagnostics, but their effectiveness is limited by our inability to spatially target their activity. To overcome this limitation, we developed a computationally guided method to design inactive proenzymes or zymogens, which are activated through cleavage by a protease. Since proteases are differentially expressed in various tissues and disease states, including cancer, these proenzymes could be targeted to the desired microenvironment. We tested our method on the therapeutically relevant protein carboxypeptidase G2 (CPG2). We designed Pro-CPG2s that are inhibited by 80 to 98% and are partially to fully reactivatable following protease treatment. The developed methodology, with further refinements, could pave the way for routinely designing protease-activated protein-based therapeutics and diagnostics that act in a spatially controlled manner. Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
登录
查看更多内容
影响因子：
5.4
作者：
Johnson, R. J.;Lin, Shawn R.;Raines, Ronald T.
通讯作者：
Raines, Ronald T.
影响因子：
5.7
作者：
Jozic, D;Bourenkow, G;Maskos, K
通讯作者：
Maskos, K
影响因子：
4.8
作者：
Lindner, HA;Lunin, VV;Ménard, R
通讯作者：
Ménard, R
影响因子：
64.8
作者：
Bhardwaj, Gaurav;Mulligan, Vikram Khipple;Bahl, Christopher D.;Gilmore, Jason M.;Harvey, Peta J.;Cheneval, Olivier;Buchko, Garry W.;Pulavarti, Surya V. S. R. K.;Kaas, Quentin;Eletsky, Alexander;Huang, Po-Ssu;Johnsen, William A.;Greisen, Per Jr;Rocklin, Gabriel J.;Song, Yifan;Linsky, Thomas W.;Watkins, Andrew;Rettie, Stephen A.;Xu, Xianzhong;Carter, Lauren P.;Bonneau, Richard;Olson, James M.;Coutsias, Evangelos;Correnti, Colin E.;Szyperski, Thomas;Craik, David J.;Baker, David
通讯作者：
Baker, David
影响因子：
12.4
作者：
Choi KY;Swierczewska M;Lee S;Chen X
通讯作者：
Chen X