Diagnostic peptide-nanoparticle probes for profiling tumor protease activity

描述(申请人提供)：异常的蛋白水解酶活性在许多复杂的肿瘤过程中是必不可少的，包括生长、侵袭和转移。蛋白分解事件负责揭示隐秘的ECM信号域，降解基底膜，并激活包括受体和生长因子在内的一系列分子。尽管它们很重要，但几乎没有检测和监测体内活动的技术。传统的体外技术，如肿瘤活检和酶谱分析，是侵入性的，提供的洞察力有限，因为蛋白酶活性在体内具有高度的背景和严格的调控。最近基于活性的荧光探针的工作受到少数可穿透组织的记者的限制，排除了对蛋白质分解网络的研究和对组织深处部位肿瘤活性的检测。在这里，我们建议通过构建可以通过尿液测量、感知和远程报告肿瘤活动的长循环多肽-纳米颗粒探针来绕过这些挑战。在这一策略中，氧化铁纳米颗粒被用作伴侣，将蛋白水解酶特异性的多肽库传递给肿瘤，然后被活性蛋白水解酶选择性切割，释放出被肾脏系统清除到尿液中的多肽片段。这些多肽片段预先设计了光不稳定的触发剂，以传递为多路LC MS/MS定量而优化的同压多肽“Reports”。我们假设，在涉及荷瘤和非肿瘤小鼠的比较研究中，对尿液的质谱分析将发现独特的报告信号，可以与体内的蛋白酶活性相关。量化的蛋白酶信号将被用作检测、监测和评估肿瘤对抗肿瘤治疗的反应的指标。这种通过大量编码的远程报告来量化蛋白质分解活性的策略将实现高度的多元化，并将允许在与解剖位置无关的位置检测肿瘤。我们希望这个平台具有广泛的实用性，并适用于一些依赖蛋白水解酶的疾病，如心血管疾病，凝固性疾病，和肝纤维化。

DESCRIPTION (provided by applicant): Aberrant protease activity is essential in many complex tumor processes including growth, invasion and metastasis. Proteolytic events are responsible for unveiling cryptic ECM signaling domains, degrading basement membranes, and activating a suite of molecules including receptors and growth factors. Despite their importance, few technologies exist for detecting and monitoring activities in vivo. Traditional ex vivo techniques like tumor biopsy followed by zymography assays are invasive and provide limited insight since protease activity is highly contextual and tightly regulated in vivo. Recent work with activity-based fluorescent probes are limited by the small number of tissue-penetrating reporters available, precluding studies of proteolytic networks and detection of tumor activity at sites deep within tissue. Here, we propose to circumvent these challenges by constructing long circulating peptide-nanoparticle probes that can survey, sense and remotely report on tumor activity through the urine. In this strategy, iron oxide nanoparticles are utilized as chaperones to deliver protease-specific peptide libraries to tumors whereupon selective cleavage by active proteases releases peptide fragments that are cleared by the renal system into urine. These peptide fragments are predesigned with photo-labile triggers to uncage isobaric peptide "reporters" optimized for multiplexed LC MS/MS quantification. We hypothesize that mass spectrometric analysis of urine in comparative studies involving tumor-bearing and tumor-free mice will uncover unique reporter signatures that can be correlated with protease activity in vivo. Quantified protease signatures will be utilized as a metric for detecting, monitoring and evaluating tumor responses to anti-tumor therapies. This strategy of quantifying proteolytic activity through mass-encoded remote reporters will enable a high degree of multiplexing and will permit the detection of tumors at sites independent of anatomical position. We expect this platform to have broad utility and amenable to a number of protease-dependent diseases such as cardiovascular diseases, coagulopathies, and liver fibrosis.