A Highly Specific, Tissue-Permeable Inhibitor of Gli Transcription Factors

描述（由申请人提供）：Hedgehog（Hh）通路的异常信号传导与基底细胞癌（BCC）的形成和进展密切相关，基底细胞癌是一种影响3/10白人的皮肤癌。BCC的标准治疗包括手术切除整个肿瘤，但这种手术可能会留下严重的疤痕。研究人员一直在研究通过化学抑制Hh通路发挥作用的替代疗法。到目前为止，大多数疗法都抑制Smoothened（Smo），这是Hh通路的上游成员，但该通路后来产生了耐药性。因此，拟议的项目旨在靶向Gli蛋白，这是一组锌指转录因子（TF），作为Hh途径的末端步骤。米德教授的实验室开发了一系列钴（III）-席夫碱（Co（III）-sb）配合物，它们是锌指TF的高度特异性和不可逆抑制剂。通过将Co（III）-sb与Gli靶结合序列（5 '-GACCACCCA-3'）缀合以制备Co（III）-Gli，预期对Gli蛋白的高度特异性和有效抑制。该提案还寻求将荧光改性的Co（III）-Gli抑制剂连接到金纳米颗粒（AuNP）上，使得“Au-Gli-Co.“这将允许：1）Gli抑制剂的局部递送，2）局部递送，因为Co（III）-Gli可以通过光诱导从AuNP释放，和3）荧光活性物质的容易的可视化和跟踪 在体外和体内的代理。 该提案的第一个目标是合成和表征独特的Au-Gli-Co颗粒。Co（III）-Gli将使用DNA杂交方法连接到AuNP上。它可以使用近红外光从AuNP中去杂交（并因此释放），这会导致AuNP等离子体共振并产生热量。第二个和第三个目标是验证Hh通路抑制在体外和体内。将在两种细胞系上进行细胞实验，以确定抑制剂靶向Gli的功效和特异性。将使用3D筏培养模型评估组织渗透和细胞输送。体内实验将确定局部应用和激光活化后该试剂治疗BCC肿瘤的能力。将使用在用他莫昔芬诱导后表达Hh介导的BCC肿瘤的小鼠模型。 该提案符合NIAMS的长期研究和资助计划。该项目涉及使用小分子开发一种新的皮肤癌治疗方法，此外还提出了一种有效和可控的经皮药物输送系统。它可能用于治疗早期或晚期BCC。与许多皮肤病一样，BCC往往比更具侵袭性的癌症受到较少的关注，但该项目的成功实施将对改善患者的生活质量产生重大影响。

DESCRIPTION (provided by applicant): Aberrant signaling of the Hedgehog (Hh) pathway is heavily linked to the formation and progression of basal cell carcinomas (BCC), a skin cancer that affects 3 in 10 Caucasians. The standard treatment of BCC involves surgical excision of the entire tumor, but this procedure can leave heavy scarring. Researchers have been studying alternate therapy options that act by chemically inhibiting the Hh pathway. Most therapies so far inhibit smoothened (Smo), an upstream member of the Hh pathway, but the pathway later develops resistance. Thus, the proposed project aims to target Gli proteins, a set of zinc finger transcription factors (TFs) that act as the terminal step in the Hh pathway. Professor Meade's lab has developed a series of cobalt(III)-Schiff base (Co(III)-sb) complexes that are highly specific and irreversible inhibitors of zinc finger TFs. By conjugating Co(III)-sb to the Gli targe binding sequence (5'-GACCACCCA-3') to make Co(III)-Gli, highly specific and potent inhibition of Gli proteins is anticipated. This proposal additionally seeks to attach fluorescently modified Co(III)-Gli inhibitor to a gold nanoparticle (AuNP), making "Au-Gli-Co." This will allow for: 1) topical delivery of the Gli inhibitor, 2) localized delivery, since Co(III)-Gli can be released fro the AuNP by inducing with light, and 3) facile visualization and tracking of the fluorescent active agent in vitro and in vivo. The first objective of the proposal is to synthesize and characterize the unique Au-Gli-Co particle. Co(III)-Gli will be attached to the AuNP using DNA hybridization methods. It can become dehybridized (and therefore released) from the AuNP using near-infrared light, which causes AuNP plasmon resonance and generates heat. The second and third objectives are to validate Hh pathway inhibition in vitro and in vivo. Cellular experiments will be performed on two cells lines to determine the inhibitor's efficacy and specificity for targeting Gli. A 3D raft cultre model will be used to assess tissue penetration and cellular delivery. In vivo experiments will determine the ability of the agent to treat BCC tumors after topical application and laser activation. A mouse model that expresses Hh-mediated BCC tumors upon induction with tamoxifen will be used. This proposal meets the long-range research and funding plans of the NIAMS. The project involves the development of a new skin cancer treatment with the use of small molecules, in addition to proposing an efficient and controlled system of transcutaneous drug delivery. It could have application in treating early or later stage BCC. Like many skin diseases, BCC tends to garner less attention than more aggressive cancers, but the successful implementation of this project would have significant implications in improving patients' quality of life.