Diagnostic peptide-nanoparticle probes for profiling tumor protease activity

描述（申请人提供）：异常的蛋白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。蛋白水解事件负责揭示隐性ECM信号域，降解基底膜，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，但很少有技术可以检测和监测体内的活动。传统的离体技术，如肿瘤活组织检查和酶谱分析是侵入性的，并且提供的信息有限，因为蛋白酶活性在体内是高度相关和严格调控的。最近基于活性的荧光探针的工作受到可用的组织穿透报告器数量少的限制，这妨碍了对蛋白水解网络的研究和对组织深处肿瘤活性的检测。在这里，我们建议通过构建长循环肽纳米颗粒探针来规避这些挑战，这种探针可以通过尿液调查、感知和远程报告肿瘤活动。在这种策略中，氧化铁纳米颗粒被用作伴侣，将蛋白酶特异性肽库运送到肿瘤中，随后活性蛋白酶选择性切割释放肽片段，这些片段被肾脏系统清除到尿液中。这些肽片段预先设计了光不稳定触发器，以打开针对多路LC MS/MS定量优化的等压肽“报告器”。我们假设，在涉及荷瘤和无瘤小鼠的比较研究中，尿液的质谱分析将发现与体内蛋白酶活性相关的独特报告信号。量化的蛋白酶特征将被用作检测、监测和评估肿瘤对抗肿瘤治疗反应的指标。这种通过大量编码的远程报告器定量蛋白水解活性的策略将实现高度的多路复用，并允许在独立于解剖位置的部位检测肿瘤。我们希望这个平台具有广泛的实用性，并适用于许多蛋白酶依赖性疾病，如心血管疾病、凝血病和肝纤维化。

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.