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白种人的皮肤癌。BCC的标准治疗包括手术切除整个肿瘤，但这个过程会留下严重的疤痕。研究人员一直在研究通过化学方式抑制Hh通路的替代疗法。迄今为止，大多数疗法抑制平滑（Smo），这是Hh通路的上游成员，但该通路后来产生耐药性。因此，该项目的目标是Gli蛋白，这是一组锌指转录因子（TFs），在Hh通路中起着终端作用。Meade教授的实验室开发了一系列钴(III)-希夫碱（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偶联到Gli靶蛋白结合序列（5'-GACCACCCA-3'）上生成Co(III)-Gli，预计将具有高度特异性和有效的Gli蛋白抑制作用。该方案还寻求将荧光修饰的Co(III)-Gli抑制剂附着在金纳米颗粒（AuNP）上，制成“Au-Gli-Co”。这将允许：1)局部递送Gli抑制剂，2)局部递送，因为Co(III)-Gli可以通过光诱导从AuNP中释放，3)易于可视化和跟踪荧光活性

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.