Discovering hybrid inhibitors for tumor microenvironment disruption

尽管肿瘤中的胞外蛋白水解酶和多肽酶具有促癌活性 微环境是众所周知的，在基本和翻译环境中单一酶的特定破坏 这仍然是一个根本性的挑战。例如，使用泛特定的化学抑制剂的基质 金属蛋白酶(MMPs)在癌症动物模型中的作用已证明干扰了MMPs的活性 可以延缓癌症的进展，但这些抑制物的临床翻译已经失败，主要是由于缺乏抑制物 专一性。努力发现特定的抑制剂或快速实施基因方法来干扰 在肿瘤微环境中活跃的MMPs和其他酶仍然具有挑战性。正在出现的发现 提示MMPs在肿瘤微环境中调节信号和免疫功能，使高度 对阐明MMPs在肿瘤微环境中的广泛作用至关重要的特殊试剂 并建立具有潜在临床应用前景的新一代抑制剂。 在这项申请中，我们建议建立一个发现高特异性酶抑制剂的平台 在高吞吐量方面。我们的基础技术结合了酵母展示和非标准氨基的使用 用于构建和评估缓蚀剂的酸，其结构不能用常规方法获得 接近了。我们假设，在抗体框架内精确定位小分子将产生 二价“杂交抑制剂”，保持抗原特异性，同时获得强大的抑制能力。我们会 由于提供了基于酵母的发现平台，因此我们将目标对准了MMP-2、-7、-9和-14 这些工具将使我们能够探索我们平台的功能。我们将建立 我们的平台使用以下两个具体目标：1)在抗体中识别有效的小分子定位 以实现高效的混合发现。在这个目标中，我们将定量地探索小分子的影响 抗体可变区内的连接位点、官能团和连接子，以确定最有希望的 将这些因素结合起来进行大规模的杂交发现。2)建立数量关系 抑制物特性和细胞侵袭。在这个目标中，我们将比较我们从酵母中获得的数据 使用标准生化和基于细胞的分析的数据进行测量，以设定数字目标 在杂交发现过程中，研究MMPs在细胞侵袭中的作用。 这里提出的平台将产生具有结构和特异性的分子试剂 使用现有的基于小分子或蛋白质的方法获取。由此产生的基质金属蛋白酶抑制剂可能会起到 作为治疗的线索，因为它们的高度特异性将克服以前治疗的关键缺点 候选人。在肿瘤微环境中进行精确的酶破坏也将带来更好的理解 在分子和系统水平上的肿瘤生物学。最后，我们预计，混合方法将 酶抑制将适用于任何酶家族的成员特异性靶向。

Although cancer-promoting activities of extracellular proteases and peptidases in the tumor microenvironment are well known, the specific disruption of a single enzyme in basic and translational settings remains a fundamental challenge. For example, the use of pan-specific chemical inhibitors of matrix metalloproteinases (MMPs) in animal models of cancer has demonstrated that interfering with MMP activity can slow cancer progression, but clinical translation of these inhibitors has failed, largely due to lack of inhibitor specificity. Efforts to discover specific inhibitors or rapidly implement genetic approaches to interfere with MMPs and other enzymes active in the tumor microenvironment remain challenging. Emerging findings indicate that MMPs modulate signaling and immune function in the tumor microenvironment, making highly specific reagents critical to elucidate the wide range of roles MMPs play within the tumor microenvironment and to establish a new generation of inhibitors for potential clinical applications. In this application, we propose to establish a platform for the discovery of highly specific enzyme inhibitors in high throughput. Our underlying technology combines the use of yeast display and noncanonical amino acids to construct and evaluate inhibitors with structures that are not accessible using conventional approaches. We hypothesize that precisely positioning small molecules within an antibody framework will yield bivalent “hybrid inhibitors” that retain antigen specificity while gaining potent inhibitory capabilities. We will implement our yeast-based discovery platform by targeting MMP-2, -7, -9, and -14 because of the availability of numerous MMP-related tools that will allow us to explore the capabilities of our platform. We will establish our platform using the following two specific aims: 1) Identify effective small molecule positioning in antibodies to enable efficient hybrid discovery. In this Aim, we will quantitatively explore the effects of small molecule attachment sites, functional groups, and linkers within antibody variable regions to identify the most promising combinations of these factors for large-scale hybrid discovery. 2) Establish quantitative relationships between inhibitor properties and cell invasion. In this Aim, we will compare data we derive from yeast-based measurements with data from standard biochemical and cell-based assays in order to set numerical targets during hybrid discovery efforts and to examine the roles of MMPs in cell invasion. The platform proposed here will yield molecular reagents with structures and specificities that cannot be accessed using existing small molecule- or protein-based approaches. The resulting MMP inhibitors may serve as therapeutic leads, as their high specificities will overcome key shortcomings of previous therapeutic candidates. Precise enzyme disruption in the tumor microenvironment will also lead to a better understanding of tumor biology at both molecular and systems levels. Finally, we anticipate that the hybrid approach to enzyme inhibition will be applicable to member-specific targeting of any family of enzymes.