De novo Designed Safranine Enzymes for Cancer Therapy (pilot)

蛋白质设计的新前沿是创造完全人造的酶。一个吸引人的设计 目标是构建用于生物还原癌症治疗的改进催化剂。目前，这些 治疗方法利用自然产生的酶，而这些酶又含有生物酶辅因子。一位少校 使用这些酶的缺点是，它们激活的前药必然与天然药物相似。 产生代谢物的。这会导致这些前体药物在身体其他部位显著激活，从而导致 化学毒性。我建议设计完全人造的酶，可以激活新的前体药物，这些药物 与人类的新陈代谢是正交的。这种从头开始的设计方法也很好地利用了 非天然酶辅因子作为量身定制酶活性的基础。这反过来又扩大了 酶的化学反应活性是可能的，这使得前药的设计可以专注于低非特异性 活性高，因此毒性低。具体地说，我计划通过计算植入一个黄素依赖的 硝基还原酶在四个α螺旋束支架上的活性部位。然后，使用核磁共振定向迭代 我在过去成功地进行了重新设计过程，酶将被重新设计以利用类黄素 藏红花碱0分子。这种分子的电子转移能量学更适合和更 比生物黄素辅因子更强的激活硝基芳香族前体药物而不催化侧 反应。下一步需要同时进行活性部位：前药重新设计过程，其中最优 可以创建主动的无毒配对。这将用人类细胞培养进行测试，比较前药 有或没有暴露在酶中的毒性。 我的任务是设计用于生物还原癌症治疗的改良酶。设计与综合 与人体中存在的蛋白质完全不同的蛋白质也允许我们极大地重新设计 它激活的药物。这将使我们能够极大地减少正常情况下 化疗。

The new frontier in protein design is the creation of completely artificial enzymes. One attractive design target is the construction of improved catalytic agents for bioreductive cancer therapy. Currently, these therapies utilize naturally occurring enzymes, which in turn contain biological enzyme cofactors. A major drawback of using these enzymes is that the prodrugs which they activate are necessarily similar to naturally occuring metabolites. This results in significant activation of these prodrugs elsewhere in the body, causing chemotoxicity. I propose to design completely artificial enzymes that can activate new prodrugs which are orthogonal to human metabolism. This de novo design approach also lends itself well to the utilization of non-natural enzyme cofactors as the basis for tailored enzyme activities. This in turn widens the scope of chemical reactivity possible for the enzyme, permitting the design of the prodrug to focus on low nonspecific activity and consequently, low toxicity. In specific, I plan to computationally implant a flavin-dependent nitroreductase active site on a four alpha helix bundle scaffold. Then, using the NMR-directed iterative redesign process that I have had success with in the past, the enzyme will be redesigned to utilize the flavinlike safranine 0 molecule. This molecule has electron transfer energetics that are better suited and more robust than the biological flavin cofactors for activating nitraromatic prodrugs without catalyzing side reactions. The next step entails a simulataneous active site:prodrug redesign process wherein an optimally active non-toxic pairing can be created. This will be tested using human cell cultures, comparing prodrug toxicity with and without exposure to the enzyme. I am proposign to design improved enzymes for bioreductive cancer therapy. Designing and synthesizing proteins which are completely different from those that exist in the body allows us to also greatly redesign the drugs it activates. This should allow us to greatly reduce the toxicity normal encountered in chemotherapy.