Light Activation of Enzyme Inhibition

描述（由申请人提供）：异常蛋白水解是许多疾病的标志，通常局限于某些器官和组织。不幸的是，了解人类疾病模型中异常蛋白水解的空间方面的基础研究很难开展，因为目前的抑制剂缺乏以特定位置和特定时间的方式激活的能力。此外，尽管小分子抑制剂可用于靶向异常蛋白水解，但在化疗干预期间，非靶组织中的抑制可能是副作用和毒性的重要原因。总的来说，为了研究异常蛋白酶活性的空间方面，我们不仅要能够测量，还要能够在人类疾病模型中以空间控制的方式抑制蛋白质水解。为了实现这一目标，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。我们的光激活方法针对分离酶和活细胞，并针对一类与炎症和癌症进展相关的重要半胱氨酸蛋白酶。我们开发了一种有效的笼化方法，通过金属结合来中和蛋白酶抑制剂的腈弹头，在可见光下快速释放和激活抑制剂，而不会产生有害的毒性。在此资助期间，我们建议朝着我们的长期目标迈出重要的一步，开发和应用光激活抑制剂的完整工具包，以研究半胱氨酸蛋白酶在活动物模型中的炎症和癌症中的作用。具体目标是：1)开发新型有效的笼型组织蛋白酶和半胱天冬酶抑制剂；2)了解笼型蛋白酶抑制剂的光化学性质并调节其释放波长；3)表征笼化配合物在活细胞中的行为；4)在小鼠骨肿瘤生长模型中实现组织蛋白酶和caspase的局部抑制。我们为这个项目组建了一个高度互补的跨学科研究团队，他们在工作和出版方面有着良好的记录。之所以选择骨肿瘤作为治疗靶点，是因为骨是前列腺癌的主要转移部位，而且大多数晚期前列腺癌患者会出现无法治愈的骨病变并发症。本研究旨在开发和验证光激活半胱氨酸蛋白酶抑制剂作为转移性骨病的基础研究工具和潜在治疗方法。文献资料证实了光动力疗法在活体动物模型中对骨肿瘤有效，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.