Irreversible Inhibtion of Cerebellar Gli Transcription Factors by Cobalt (III) Complexes

Hedgehog(HH)信号通路的异常表达与Hedgehog(HH)信号通路的形成和 髓母细胞瘤(MB)瘤的一个亚群的增殖。MB主要是一种儿童脑癌， 70%的病例发生在10岁以下的儿童中。由HH信号驱动的SHH亚群是 在遗传原因和组织学上存在广泛的异质性，使得有效的治疗具有挑战性，并导致 惨淡的结局。目前的治疗策略包括肿瘤切除、颅骨脊髓照射和 化疗，但有短期和长期不良反应。在努力抑制HH途径的过程中， 研究的目标是Smoothens(Smo)蛋白，但由于下游突变而受阻 导致癌症复发的致死率要高得多。因此，这个项目试图以Gli蛋白为靶点，它 属于锌指转录因子家族，是HH途径的最终效应者。这个 Meade实验室开发了一系列钴(III)-席夫碱配合物(Co(III)-Sb)与转移因子共识偶联 特异性和不可逆地抑制目标锌指转录因子的序列。这项提议旨在彻底改变 Co(III)-Sb与Gli共有序列偶联生成Co(III)-Gli的方法 和不可逆的Gli蛋白抑制剂。该缓蚀剂将被偶联到Gd(III)标记的金纳米颗粒上 (AUNP)递送平台，以评估和成像体外和体内Gli抑制的有效性。 这项建议的第一个目标是重新设计将DNA偶联到Co(III)-Sb的合成路线。这个 Co(III)-Sb的赤道配体支架将带有炔基官能化，以允许点击 与叠氮功能化的Gli共有序列的接合，产生Co(III)-Gli。预计这将是 显著提高了当前共轭方法的产量和可扩展性。第二个和第三个 目的评价Co(III)-Gli对Gli抑制肿瘤生长的作用。CO(III)-Gli 将与DNA封顶的AuNP杂交，以产生优化的去杂交序列以释放 在生理温度下的试剂。Co(III)-Gli AuNPs将被Gd(III)磁共振成像标记 (MRI)造影剂提供了一个平台，用于在体外和体内绘制结合物的命运图。一种能力 Co(III)-Gli抑制外源性Gli的作用将在体外使用人胚胎肾脏细胞和在体进行评估 使用自然发展成SHH亚型MB肿瘤的小鼠模型。CO(III)-Gli有望抑制Gli 具有很高的特异性，导致肿瘤生长受到抑制。 该项目在短期和长期影响方面都完全符合美国国立卫生研究院的使命。它 将在短期内进一步发展关于小脑Gli转录因子抑制的知识，以及 为SHH亚型髓母细胞瘤的长期治疗提供一个平台。这个 该项目的成功将为提高存活率和质量提供一个独特的机会。 生命中最恶性的儿童脑癌。

Aberrant expression of the Hedgehog (Hh) signaling pathway is heavily implicated in the formation and proliferation of a subgroup of medulloblastoma (MB) tumors. MB is predominantly a pediatric brain cancer, with 70% of occurrences appearing in children under the age of 10. The SHH subgroup, driven by Hh signaling, is widely heterogeneous in genetic cause and histology, making effective treatment challenging and resulting in dismal outcomes. Current treatment strategies involve tumor resection, craniospinal irradiation, and chemotherapy, but suffer from short-term and long-term adverse effects. In efforts to inhibit the Hh pathway, research has targeted the protein Smoothened (Smo), but has been stunted by downstream mutations that lead to cancer recurrence with a much higher lethality. Thus, this project seeks to target Gli proteins, which belong to a family of zinc finger transcription factors (TFs) and are the final effectors of the Hh pathway. The Meade lab has developed a series of Cobalt (III)-Schiff base complexes (Co(III)-sb) coupled to TF consensus sequences that specifically and irreversibly inhibit zinc finger TFs of interest. This proposal seeks to reinvent the method for conjugating Co(III)-sb to the Gli consensus sequence to generate Co(III)-Gli, a highly specific and irreversible inhibitor of Gli proteins. This inhibitor will be conjugated to a Gd(III)-labeled gold nanoparticle (AuNP) delivery platform to evaluate and image the effectiveness of Gli inhibition both in vitro and in vivo. The first objective of this proposal is to redesign the synthetic route for coupling DNA to Co(III)-sb. The equatorial ligand scaffold of Co(III)-sb will be functionalized with an alkyne moiety to allow for clickable conjugation to an azide functionalized Gli consensus sequence, generating Co(III)-Gli. This is anticipated to significantly improve yields and scalability from the current conjugation method. The second and third objectives focus on evaluating the potency of Co(III)-Gli against Gli for the inhibition of tumor growth. Co(III)-Gli will be hybridized to a DNA capped AuNP to generate an optimized dehybridization sequence to release the agent at physiological temperature. Co(III)-Gli AuNPs will be labeled with Gd(III) magnetic resonance imaging (MRI) contrast agents to provide a platform for fate mapping the conjugates both in vitro and in vivo. The ability of Co(III)-Gli to inhibit exogenous Gli will be evaluated in vitro using human embryonic kidney cells and in vivo using a murine model that natively develops SHH subtype MB tumors. Co(III)-Gli is anticipated to inhibit Gli with high specificity, resulting in suppressed tumor growth. This project fully aligns with the mission of the NIH both in its short-term and long-term implications. It will further develop knowledge about inhibition of cerebellar Gli transcription factors in the short-term, and generate a platform for enhancing current treatment options for SHH subtype medulloblastoma long-term. The success of this project would present a unique opportunity for improving the survival rate and resulting quality of life for the most malignant childhood brain cancer.