Light Activation of Enzyme Inhibition

描述(申请人提供)：异常蛋白分解是许多病理学的特征，通常局限于某些器官和组织。不幸的是，理解人类疾病模型中异常蛋白分解的空间方面的基础研究很难进行，因为目前的抑制物缺乏以特定地点和时间的方式被激活的能力。此外，尽管小分子抑制剂可用于靶向异常的蛋白分解，但在非靶向组织中的抑制可能是化疗干预期间副作用和毒性的重要原因。一般来说，我们必须不仅能够测量，而且能够以空间控制的方式抑制人类疾病模型中的蛋白分解，以便研究异常蛋白水解酶活性的空间方面。为了这个目标，我们开发了一种令人兴奋的新方法，利用光对酶抑制进行空间和时间控制。我们的光活化方法针对分离的酶和活细胞，并针对与炎症和癌症进展相关的一类重要的半胱氨酸蛋白酶。我们开发了一种有效的笼化方法，通过金属结合中和蛋白酶抑制剂的丁腈弹头，在可见光下发生快速释放和激活，而不会造成有害毒性。在这一资助期间，我们建议朝着我们的长期目标迈出重要的一步，开发和应用一套完整的光激活抑制剂工具箱，以在活体动物模型中研究半胱氨酸蛋白酶在炎症和癌症中的作用。具体目标是1)开发新型有效的笼状组织蛋白酶和半胱氨酸天冬氨酸蛋白酶抑制剂，2)了解笼状蛋白酶抑制剂的光化学并调节释放波长，3)表征笼状复合体在活细胞中的行为，4)在小鼠骨肿瘤生长模型中实现对组织蛋白酶和半胱氨酸酶的局部抑制。我们已经为这个项目组建了一个高度互补和跨学科的研究团队，他们在合作和出版方面有着良好的记录。之所以选择以骨肿瘤为靶点，是因为骨是前列腺癌的主要转移部位，而大多数晚期疾病患者都经历了无法治愈的骨损伤并发症。这项建议旨在开发和验证光活化半胱氨酸蛋白酶抑制剂作为基础研究工具，以及转移性骨疾病的潜在治疗方法。文献资料证实，光动力疗法对活体动物模型中的骨肿瘤是有效的，这有力地支持了我们的研究。

DESCRIPTION (provided by applicant): Aberrant proteolysis, a hallmark of many pathologies, is often localized to certain organs and tissues. Unfortunately, basic studies to understand spatial aspects of aberrant proteolysis in human disease models are difficult to carry out, because current inhibitors lack the ability to be activated in a location- and time-specific fashio. Furthermore, even though small molecule inhibitors are available to target aberrant proteolysis, inhibition in non-target tissues can be a significant cause of side effects and toxicity during chemotherapeutic interventions. In general, we must be able to not only measure, but also inhibit proteolysis in a spatially controlled fashion in human disease models, in order to study spatial aspects of aberrant protease activity. Towards this goal we have developed an exciting new method that gives spatial and temporal control over enzyme inhibition using light. Our photoactivated approach works against isolated enzymes and in live cells, and targets an important class of cysteine proteases associated with inflammation and cancer progression. We developed an effective caging method based on neutralizing nitrile warheads of protease inhibitors through metal binding, where fast inhibitor release and activation occur with visible light, without causing deleterious toxicity. During this grant period, we propose to make a significant step towards our long-term goal, to develop and apply a complete toolkit of light-activated inhibitors to investigate the role of cysteine proteases in inflammation and cancer in live animal models. Specific Aims are 1) to develop new, potent versions of caged cathepsin and caspase inhibitors, 2) to understand the photochemistry of caged protease inhibitors and tune the wavelength of release; 3) to characterize the behavior of caged complexes in living cells; 4) to achieve local inhibition of cathepsin and caspase enzymes in a murine model of bone tumor growth. We have assembled a highly complementary and interdisciplinary research team for this project that has a proven track record of working and publishing together. Targeting of bone tumors was chosen, because bone is the primary site of metastasis for prostate cancer and most patients with advanced disease experience complications from bone lesions that are incurable. This proposal aims to develop and validate light-activated cysteine protease inhibitors as basic research tools, and potential therapeutics in metastatic bone disease. Literature data confirm that photodynamic therapy is effective against bone tumors in live animal models, which strongly supports our studies.