De novo Designed Safranine Enzymes for Cancer Therapy (pilot)

蛋白质设计的新前沿是创造完全人工的酶。一个吸引人的设计 目标是构建用于生物还原性癌症治疗的改进的催化剂。目前这些 疗法利用天然存在的酶，所述酶又含有生物酶辅因子。一个主要 使用这些酶的缺点是它们激活的前药必须与天然的类似 出现代谢物。这导致这些前药在身体其他地方的显著活化， 化学毒性我建议设计完全人工的酶，可以激活新的前药， 与人体新陈代谢正交。这种从头设计方法也很好地利用了 非天然酶辅因子作为定制酶活性的基础。这反过来又扩大了 酶可能的化学反应性，允许前药的设计集中在低非特异性 活性，因此毒性低。具体来说，我计划通过计算机植入一个依赖黄素的 硝基还原酶活性位点的四个α螺旋束支架。然后，使用NMR定向迭代 我在过去成功地重新设计了酶，酶将被重新设计，以利用类黄素 番红0分子。这种分子的电子转移能量学更适合， 用于活化硝基芳香族前药而没有催化侧的生物黄素辅因子 反应.下一步需要一个同时的活性位点：前药重新设计过程，其中最佳的 可以产生活性无毒配对。这将使用人类细胞培养物进行测试，比较前药 在有和没有暴露于酶的情况下的毒性。 我是设计改进的酶用于生物还原性癌症治疗。设计和合成 与体内蛋白质完全不同的蛋白质， 它激活的药物这将使我们能够大大减少正常情况下遇到的毒性， 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.