Development of a Novel Inhibitor for Hepatitis B

项目总结/摘要 CADA类似物TL 020已被确定为NIAID、DMID中新的抗B型肝炎先导化合物 体外抗病毒筛选程序。其抑制HBV（病毒体）DNA分泌的EC_（50）为0.5-0.6 在二次筛选中发现其抑制细胞内DNA的EC 50为1.2 µM。TL 020具有 相对低的细胞毒性，在这些测定中SI 50值范围为40-80。CADA化合物具有 发现了一种选择性抑制某些蛋白质表达的新机制。先导化合物， CADA通过下调免疫细胞表面上的CD 4来作为抗HIV剂。它直接绑定了 在翻译过程中新生CD 4的信号肽，并抑制其跨膜转运的细胞， 内质网（ER）。CADA选择性结合人CD 4的25-残基信号肽，但不结合非- 灵长类（例如小鼠）CD 4，明显结合特定氨基酸残基。它是一种独特的小分子制剂 已经显示其通过以下方式抑制选择蛋白质跨ER膜的共翻译易位： 结合其信号肽 还发现CADA降低人蛋白分拣蛋白的细胞表面表达，分拣蛋白是 与许多疾病有关，包括额颞叶变性，自闭症，阿尔茨海默病， 动脉粥样硬化和乳腺癌。CADA化合物明显结合分拣蛋白信号肽，其具有类似的结合活性。 亲和力，但最大功效略低于CD 4。在分析了信号的结构后， 促甲状腺激素受体（TSHR）的肽，一项合作开始测试CADA 用于降低TSHR表达以治疗Graves病的化合物。据推测，化合物 被鉴定为下调TSHR，但不是CD 4或分拣蛋白！这些结果显示了裁剪的潜力 用于选择性地降低具有治疗意义的特定蛋白质的表达的化合物的结构。 CADA化合物不抑制任何DNA聚合酶，这是抗HBV最常见的作用机制 毒品我们的主要假设是，由于其新的作用机制，TL 020减少了一种新的细胞因子的表达。 以前未鉴定的HBV复制所需的宿主细胞蛋白。 本拟议项目的具体目标涉及以下三个总体目标： 1.确定TL 020的作用机制，包括其分子靶点。 2.通过合成TL 020类似物和体外筛选抗HBV效力进行先导优化。 3.后续体内研究有效候选药物的初步临床前药代动力学评价。 本项目中的拟议活动是使用标准方法调查该机制， 其中TL 020抑制HBV复制，并且还检测TL 020对宿主蛋白表达的影响。 将合成TL 020的新类似物以检查结构-活性关系。这些和以前 合成的CADA化合物将被筛选的效力，毒性和溶解性的目的，铅 优化.具有最佳物理性质的最具选择性的化合物将经历药代动力学 评价细胞渗透性和代谢稳定性，以确定用于临床前评价的候选物， vivo. 该项目完全符合R21计划的高风险高回报模型。的 潜在的风险是，可能无法确定TL 020的作用机制，或者具有适当 这些都是任何药物开发项目的风险。的 潜在的回报是，设计抗HBV药物的新靶点将被确定，一种实用的药物， 慢性HBV感染的治疗将得到发展，并对如何靶向信号肽有了新的认识 这些小分子将能够设计出治疗多种疾病和病症的新药。

PROJECT SUMMARY/ABSTRACT CADA analog TL020 has been identified as a new anti-hepatitis B lead compound in the NIAID, DMID in-vitro antiviral screening program. Its EC50 for inhibition of secreted HBV (virion) DNA was found to be 0.5-0.6 µM and its EC50 for inhibition of intracellular DNA was found to be 1.2 µM in the secondary screen. TL020 has relatively low cytotoxicity, with SI50 values ranging from 40-80 in these assays. CADA compounds have uncovered a novel mechanism for selectively inhibiting expression of certain proteins. The lead compound, CADA, acts as an anti-HIV agent by down-modulating CD4 on the surface of immune cells. It directly binds the signal peptide of nascent CD4 during translation and inhibits its translocation across the membrane of the endoplasmic reticulum (ER). CADA selectively binds the 25-residue signal peptide of human CD4, but not non- primate (e.g. mouse) CD4, apparently binding specific amino acid residues. It is a unique small-molecule agent that has been shown to inhibit co-translational translocation of select proteins across the ER membrane by binding its signal peptide. CADA has also been found to decrease cell-surface expression of the human protein sortilin, which is implicated in numerous diseases, including frontotemporal lobar degeneration, autism, Alzheimer's disease, atherosclerosis, and breast cancer. CADA compounds apparently bind the sortilin signal peptide with similar affinity, but the maximum efficacy is somewhat less than for CD4. After analyzing the stucture of the signal peptide of the thyroid stimulating hormone receptor (TSHR), a collaboration was initiated to test CADA compounds for decreasing expression of TSHR for treatment Graves disease. As hypothesized, compounds were identified that down-modulate TSHR, but not CD4 or sortilin! These results show the potential for tailoring the structures of compounds for selectively decreasing expression of specific proteins of therapeutic interest. CADA compounds do not inhibit any DNA polymerase, the most common mechanism of action of anti-HBV drugs. Our main hypothesis is that due to its novel mechanism of action, TL020 decreases expression of a previously unidentified host cell protein that is required for HBV replication. The Specific Aims of this proposed project address the following three overall goals: 1. Identification of the mechanism of action of TL020, including its molecular target. 2. Lead optimization by synthesis of TL020 analogs and screening anti-HBV potency in vitro. 3. Initial preclinical pharmacokinetic evaluation of potent candidates for follow-on studies in vivo. The proposed activities in this project are to use standard methods to investigate the mechanism by which TL020 inhibits HBV replication and to also examine the effects of TL020 on host protein expression. New analogs of TL020 will be synthesized to examine structure-activity relationships. These and previously synthesized CADA compounds will be screened for potency, toxicity and solubility for the purpose of lead optimization. The most selective compounds with the best physical properties will undergo pharmacokinetic evaluation for cell permeability and metabolic stability in order to identify candidates for preclinical evaluation in vivo. The proposed project fits perfectly into the high-risk high-reward model of the R21 program. The potential risks are that the mechanism of action of TL020 might not be identified or that a drug with suitable properties for in vivo studies might not be found. These are the risks of any drug development program. The potential rewards are that a new target for designing anti-HBV drugs will be identified, a practical drug for therapy of chronic HBV infection will be developed, and a new understanding of how to target signal peptides with small molecules that will enable the design of novel drugs for numerous diseases and conditions.