Ligand discovery for delineating cholesterol homeostasis in the brain

阿尔茨海默病(AD)是一种长期的神经退行性疾病，在美国所有死亡原因中排名第六，其特征是淀粉样蛋白β沉积和神经纤维缠结。目前，还没有可以阻止或逆转疾病进展的药物，所有创造这种疗法的努力都失败了。最近的研究表明，脑内胆固醇稳态的异常与包括阿尔茨海默病在内的几种神经退行性疾病密切相关。CYP46A1酶促脑胆固醇转化为24S-羟基胆固醇是维持脑内胆固醇稳态的主要清除机制。细胞色素P46A1基因异常参与了AD的生理病理过程。因此，CYP46A1的药理调节代表了一种有吸引力的AD治疗方法。正电子发射断层扫描(PET)能够量化体内的生化过程，而合适的CYP46A1配体将极大地提高我们对AD生理病理条件下CYP46A1介导的胆固醇稳态的理解，否则无法通过体外(破坏性)分析获得。利用正电子发射计算机断层扫描技术对活体AD脑内的细胞色素P46A1进行定量研究，将有助于评估新的AD治疗药物的分布、靶点定位和剂量占有率。到目前为止，还没有成功的例子被证明成像CYP46A1用于临床，这表明我们在体内研究这一靶点的能力严重不足。因此，我们建议开发一种新型的PET配体来填补这一空白，作为AD的第一个翻译成像工具。 我们的配体[11C]CYP-507展示了第一个针对CYP46A1的成像原型，但由于边缘结合特异性和低脑渗透率而被终止。在我们的下一代中，我们成功地鉴定了一个具有高结合亲和力和高选择性的先导分子CYP-812。我们合成了CYP-812的11C-同位素，初步的PET研究证实，我们已经克服了CYP46A1配体开发的主要障碍，实现了：1)合理和区域特异性的脑摄取；2)适度的靶向性。虽然CYP-812是一个很有希望的先导，但为了提高大脑通透性、提高效力和结合特异性以及适当的脑动力学，正在寻求进一步的优化，用于跨物种(啮齿动物和非人类灵长类)的翻译成像研究，以实现AD研究的最佳CYP46A1定量。作为具体目标，我们将设计和准备一个可用11C或18F标记的CYP46A1调节剂的焦点文库，并评估它们在啮齿动物和非人灵长类动物中的活性和药物挑战期间的变化，以及死后人脑组织的放射自显影和生物学验证的能力。这项工作的影响不仅是开发第一个用于研究AD相关生物学过程的有效和选择性的CYP46A1 PET配体，而且最终通过在高等物种中的PET成像验证，促进该配体用于潜在的临床翻译和监测新型AD神经疗法的靶向反应。

Alzheimer’s disease (AD) is a long-term neurodegenerative disorder that ranks sixth in the leading cause of all deaths in the United States and features amyloid β protein deposition and neurofibrillary tangles. At present, there are no drugs available to halt or reverse disease progression, and all efforts to create such therapies have failed. Recent studies have demonstrated that abnormalities of cholesterol homeostasis in the brain are strongly associated with several neurodegenerative diseases, including AD. The CYP46A1 enzymatic conversion of brain cholesterol into 24S-hydroxycholesterol is the major elimination mechanism to maintain brain cholesterol homeostasis. Disturbances in CYP46A1 is implicated in the AD physiopathology. Therefore, pharmacological modulation of CYP46A1 represents an attractive AD therapeutic approach. Positron emission tomography (PET) is capable of quantifying biochemical processes in vivo, and a suitable CYP46A1 ligand would substantially improve our understanding of CYP46A1-mediated cholesterol homeostasis under AD physiopathological conditions otherwise inaccessible by ex vivo (destructive) analysis. Quantification of CYP46A1 in living AD brain by PET would provide the assessment of distribution, target engagement and dose occupancy of new AD therapeutics. To date, no successful examples have been demonstrated to image CYP46A1 for clinical use, representing a significant deficiency of our ability to study this target in vivo. Therefore, we propose to develop a novel PET ligand that can fill this void, as the first translational imaging tool for AD. Our ligand [11C]CYP-507 demonstrated the first prototype for imaging towards CYP46A1, but was discontinued due to marginal binding specificity and low brain penetration. In our next generation, we successfully identified a lead molecule, CYP-812, which showed high binding affinity and high selectivity. An 11C-isotopologue of CYP-812 was synthesized and preliminary PET studies confirmed that we have overcome the major obstacles for CYP46A1 ligand development by achieving: 1) reasonable and regional-specific brain uptake; 2) moderate target specificity. Though CYP-812 is a promising lead, further optimizations aimed at higher brain permeability, improved potency and binding specificity with proper brain kinetics are sought for translational cross-species (rodents and nonhuman primates) imaging studies to achieve optimal CYP46A1 quantification for AD research. As specific goals, we will design and prepare a focused library of CYP46A1 modulators amenable for labeling with 11C or 18F, and evaluate their ability to quantify CYP46A1 activity and changes during drug challenge in rodents and nonhuman primates, as well as autoradiography and biological validation in postmortem human brain tissues. The impact of this work is not only to develop the first potent and selective CYP46A1 PET ligand for the study of AD-related biological processes, but also ultimately, via PET imaging validation in higher species, to advance this ligand for potential clinical translation and monitor target response of novel AD neurotherapeutics.