A strategy for the selective imaging and inhibition of MT1-MMP to assess its functional roles in breast cancer invasion and metastasis

基质金属蛋白酶(MMPs)通过介导细胞外基质的降解促进肿瘤细胞的侵袭和血管生成，使其成为很有前途的抗癌药物靶点。MMPs家族由23个成员组成，结构同源性高，但在肿瘤进展中的功能不同。这种高度的结构同源性使得开发特定的抑制剂和其他化学工具来研究MMPs具有挑战性。此外，与丝氨酸或半胱氨酸蛋白酶不同，MMPs不使用蛋白质结合的亲核试剂来催化酰胺键的断裂。因此，它们缺乏一种可以被抑制剂靶向共价修饰酶的亲核剂。Matthew Bogyo教授的团队最近开发了一种新的方法，可以克服这些限制，并允许特定的靶向个别的基质金属蛋白酶。该策略包括将功能沉默的突变引入到基质金属蛋白酶中，以产生亲核试剂，用于通过小分子抑制剂和光学探针进行共价修饰，这些光学探针是针对新引入的残基而设计的。因此，可以产生对工程化的基质金属蛋白酶具有绝对选择性的探针，从而允许以高度的时间控制对所需的蛋白酶进行特定的成像和选择性的抑制。此外，这种酶工程/探针方法避免了以前针对MMP的敲除研究的局限性，如补偿和致死性问题。在这个提案中，我概述了将这项新开发的技术应用于乳腺癌小鼠模型的计划，以研究膜型1-基质金属蛋白酶1(MT1-MMP)在癌症进展中的作用。MT1-MMPs是MMPs中最有效的胶原酶，对癌细胞通过基质胶原基质的侵袭至关重要。胶原酶的这种突出作用很可能是基于它在细胞膜上的定位，使细胞外基质能够有重点的重塑。然而，需要更全面地了解MT1-MMPs的功能，特别是其特异性抑制的后果，才能剖析其在肿瘤发生中的特定作用，并验证其作为相关药物靶点的有效性。为了实现这一目标，我将使用基于CRISPR/Cas的基因组编辑的小鼠来表达与乳腺癌转移转基因模型杂交的工程MT1-MMPs。这一模型与基因工程抑制物相结合，将使我能够评估特定的MT1-基质金属蛋白酶抑制对肿瘤进展和转移种植的影响。此外，在原发肿瘤发展和转移部位通过选择性探针直接观察活性MT1-MMPs的定位将有助于深入了解其在疾病病理学中的主要作用。我相信，在这些高度新颖和强大的化学工具的帮助下，将有可能重新审视MT1-MMPs作为肿瘤治疗的药物靶点，并对MT1-MMPs的功能提供更好的理解，从而开发更有效的治疗策略。

Matrix metallo proteases (MMPs) facilitate tumor cell invasion and angiogenesis by mediating degradation of the extracellular matrix, making them promising anti-cancer drug targets. The family of MMPs consists of 23 members with high structural homology but diverse functions in tumor progression. This high degree of structural homology has made the development of specific inhibitors and other chemical tools to study MMPs challenging. Furthermore, in contrast to serine or cysteine proteases, MMPs do not use a protein bound nucleophile to catalyze cleavage of the amide bond. Thus, they lack a nucleophile that can be targeted by inhibitors to covalently modify the enzyme. The group of Prof. Matthew Bogyo has recently developed a new approach that overcomes these limitations and allows specific targeting of individual MMP proteases. The strategy involves the introduction of a functionally silent mutation into a MMP protease to generate a nucleophile for covalent modification by small molecule inhibitors and optical probes that have been designed to target the newly introduced residue. Thus, probes can be generated to be absolutely selective for the engineered MMP allowing specific imaging and selective inhibition of the desired protease with a high degree of temporal control. In addition this protease engineering/probe approach avoids limitations of previous knock out studies targeting MMPs like issues of compensation and lethality. In this proposal I outline plans to apply this newly developed technology to a mouse model of breast cancer to study the function of membrane type 1-matrix metalloproteinase 1 (MT1-MMP) in cancer progression. MT1-MMP is the most potent collagenase among the MMPs and is critical for cancer cell invasion through stromal collagen matrices. This outstanding role among the collagenases is most likely based on its localization at the cell membrane enabling focused ECM remodeling. However, a more complete understanding of MT1-MMP function and especially the consequences of its specific inhibition will be required to both dissect its specific role in tumorigenesis and to validate it as a relevant drug target. To accomplish this goal I will use mice that underwent CRISPR/Cas based genome editing to express the engineered MT1-MMP crossed with a transgenic model for metastasizing breast cancer. This model combined with an engineered inhibitor will allow me to evaluate the effect of specific MT1-MMP inhibition on tumor progression and metastatic seeding. Furthermore, direct visualization of active MT1-MMP localization by a selective probe during primary tumor development and at metastatic sites will provide insight into its primary function in disease pathology. I believe that with the help of these highly novel and powerful chemical tools, it will be possible to revisit MT1-MMP as a drug target in cancer treatment and provide a better understanding of MT1-MMP function so that more effective treatment strategies can be developed.