Principles for Designing Stimulus-Responsive Enzymes

总结： 将刺激反应设计到任何选择的酶中的能力将有助于我们的能力， 以极高的空间和时间精度询问和干预生物过程。通过 自下而上构建酶的刺激反应，洞察基本的生物物理学 自然系统中的变构效应的基本原理也将得到实现（通过建筑学习）。重点 这一建议是自下而上的刺激响应前药活化酶的发展， 协同结合蛋白质设计通过计算方法（Khare）与蛋白质工程 通过非天然氨基酸（UAA）诱变（Deiters）。非天然氨基酸的遗传掺入 允许以特定于位点的方式引入生物正交开关，并且计算建模使得 蛋白质活性位点和UAA的结构和构象景观的合理重新布线 原子分辨率的微环境。通过将这些最先进的技术与成熟的 详细的动力学，结构和生物物理特性的协议，我们假设，自下而上 框架引入蛋白质的刺激反应将被破译。作为我们的模型系统， 将使用羧肽酶G2/氮芥前药和胞嘧啶脱氨酶/5-氟尿嘧啶 酶/前药对，这两种药物都已在称为 定向酶前体药物疗法（DEPT）。它们在DEPT治疗环境中的效用将通过以下方式得到增强： 使它们可有条件地激活。在目标1中，我们将使用含偶氮苯的光响应UAA 用于使所设计的酶可光控制。在目标2中，我们将开发设计方法 可被组织特异性蛋白水解酶激活的酶的酶原化形式，如基质 金属蛋白酶在目标3中，我们将使用各种结构和生物物理技术来验证设计的 酶，并为进一步的设计迭代和建模方法的改进提供反馈。而 我们的重点是上述两种酶，然而，我们正在开发的方法将被转移到 控制各种其他酶的活性和递送，使其成为设计新的 刺激响应酶，并可能还允许前所未有的选择性和最佳的交付， 化疗

Summary: The ability to design stimulus-responsiveness into any enzyme of choice would aid in our ability to interrogate and intervene in biological processes with exquisitely high spatial and temporal precision. By constructing stimulus-responsiveness into enzymes from the bottom up, insights into fundamental biophysical principles underlying allosteric effects in natural systems will also be achieved (learning by building). The focus of this proposal is the bottom-up development of stimulus-responsive prodrug-activating enzymes by synergistically combining protein design through computational approaches (Khare) with protein engineering through unnatural amino acid (UAA) mutagenesis (Deiters). Genetic incorporation of unnatural amino acids allows introducing bio-orthogonal switches in a site-specific manner, and computational modeling enables rational rewiring of the structure and conformational landscape of the protein active site and UAA microenvironment with atomic resolution. By combining these state-of-the-art techniques with well-established protocols for detailed kinetic, structural, and biophysical characterization, we hypothesize that a bottom up framework for introducing stimulus-responsiveness in proteins will be deciphered. As our model systems, we will use the carboxypeptidase G2/nitrogen mustard prodrug and cytosine deaminase/5-fluorouracil enzyme/prodrug pairs, both of which have been extensively investigated in a chemotherapy application called directed enzyme prodrug therapy (DEPT). Their utility in a therapeutic setting for DEPT will be enhanced by rendering them conditionally activatable. In Aim 1, we will use azobenzene-containing photo-responsive UAAs for rendering the designed enzymes photocontrollable. In Aim 2, we will develop methodology for designing zymogenized versions of enzymes that can be activated by tissue-specific proteolytic enzymes, such as matrix metalloproteases. In Aim 3, we will use a variety of structural and biophysical techniques to validate designed enzymes and provide feedback for further design iterations and modeling methodology improvement. While our focus is on the two enzymes mentioned, the methods we are developing, however, will be transferable to the control of activity and delivery of a variety of other enzymes, making this a general approach to design new stimulus-responsive enzymes and potentially also allowing unprecedented selectivity and optimal delivery of chemotherapies.