Molecular design modelling and rationalisation of mode of action of synthetic retinoids in cellular development processes.

干细胞生物学具有提供特定类型的细胞和组织用于研究、药物开发和细胞替代疗法的潜力。从干细胞工程化特定组织类型依赖于能够以可重复的方式指导细胞分化并且是稳定的。全反式视黄酸（ATRA）用于体外诱导细胞分化，然而，它会降解，从而产生可以以非预期方式影响分化的产物。为了提高细胞分化控制的效率，我们已经生产并测试了许多合成的类维生素A，它们具有显著提高的化学和物理稳定性。这些化合物影响人类多能干细胞和神经母细胞瘤细胞的发育变化，并且像它们的天然对应物一样，它们诱导许多效应，包括成熟细胞类型（包括神经元）的形成和细胞凋亡。我们已经开始建立一个结果的主体，第一个例子正在印刷中，我们现在需要能够理解结构与生物效应，以便设计下一代更具体的小分子。特别重要的是设计化合物的能力，所述化合物不仅充当ATRA或其他RA异构体的类似物，而且就所使用的结构单元而言是良性的，并且如果用于未来的组织再生应用，则不会引起毒性作用。我们建议进行分子模拟研究，以检查合成类维生素A与天然系统相比的结构-活性关系。基于30多种合成结构和3种天然RA化合物，可获得足够的生物学数据。这些化合物中的许多与核受体的竞争性结合特性是已知的，并且定期评估其他化合物。这为CASE项目提供了所需的数据，该项目将对每种合成化合物的作用模式进行分子水平的理解，并从中设计出新的化合物系列。全反式维甲酸是一种相对灵活的配体，以高亲和力结合所有RAR。类似地，9-顺式-RA的相对柔性允许其结合RXR以及RAR，这导致在治疗应用中靶向受体的低特异性，产生限制现有类维生素A的治疗潜力的副作用。合成配体可以被设计为构象刚性的，从而允许以更高的特异性靶向特定RAR的相当大的范围。RAR的结构，特别是RAR γ的配体结合口袋及其与一系列天然和合成配体的相互作用，已在高分辨率下阐明。这些数据允许RAR α和RAR β的配体结合结构域及其与RAR α和RAR β特异性激动剂的相互作用被建模，促进具有亚型特异性的新型化合物的设计。这将通过模拟配体和不同RAR的结合结构域之间的相互作用来实现，特别是关于羧基末端螺旋12的构象，其覆盖受体上的结合口袋，并且其构象对于共激活子/共阻遏子募集和解离是必需的。该项目将涉及：a）使用分子力学、分子动力学、DFT和/或MP2计算对所有化合物进行建模，并相互比较，以及通过单晶X射线衍射确定的实验结构; B）对每个结构进行RAR和RXR系统的虚拟结合研究，以获得估计结合能力的近似等级顺序; c）将这些数据与实际SAR筛选数据进行比较，以寻找虚拟和实际结合之间的相关性。第二代系统将设计用于进一步的合成和筛选。

Stem cell biology has potential to provide cells and tissues of specific types for use in research, the development of pharmaceuticals and cell replacement therapies. Engineering specific tissue types from stem cells is dependent on being able to direct cell differentiation in a repeatable manner and are stable. All trans-retinoic acid (ATRA) is used to induce cell differentiation in vitro, however, it degrades resulting in products that can effect differentiation in ways not intended. To improve the efficiency of cell differentiation control, we have produced and tested a number of synthetic retinoids which have significantly improved chemical and physical stability. These compounds effect developmental change in human pluripotent stem cells and neuroblastoma cells, and like their natural counterparts, they induce a number of effects, including the formation of mature cells types, including neurons, and cell apoptosis. We have begun to build up a body of results, the first examples of which are in press, and we now need to be able to understand structure versus biological effect in order to design the next generations of more-specific small molecules. Of particular importance is the ability to design compounds which do not just act as analogues of either ATRA or the other RA isomers, but which are benign in terms of the building blocks used and do not elicit toxic effects if used for future tissue-regeneration applications. We propose to carry out molecular modelling studies to examine structure-activity relationships of synthetic retinoids compared to natural systems. Sufficient biological data are available, based on over 30 synthetic structures and 3 natural RA compounds. The competitive binding properties of many of these compounds to nuclear receptors are known, with further compounds being evaluated regularly. This provides the data required for a CASE project which will develop a molecular-level understanding of the mode of action of each of the synthetic compounds, from which new series of compounds will be designed. ATRA is a relatively flexible ligand which binds with high affinity to all RARs. Similarly, the relative flexibility of 9-cis-RA allows it to bind to RXRs as well as to RARs which results in low specificity of receptor targeting in therapeutic applications, giving side effects that limit the therapeutic potential of existing retinoids. Synthetic ligands can be designed to be conformationally rigid, allowing considerable scope for targeting specific RARs with higher specificity. The structure of RARs, particularly the ligand binding pocket of RARgamma and its interactions with a range of natural and synthetic ligands, has been elucidated at high resolution. These data permit the ligand-binding domains of RARalpha and RARbeta and their interactions with RARalpha and RARbeta-specific agonists to be modelled, facilitating the design of novel compounds with subtype specificity. This would be achieved by modelling the interactions between the ligands and the binding domain of different RARs, particularly with respect to the conformation of the carboxy-terminal helix 12 which caps the binding pocket on the receptor and the conformation of which is essential for co-activator/co-repressor recruitment and dissociation. The project will involve: a) all compounds will be modelled using molecular mechanics, molecular dynamics, DFT and/or MP2 calculations, and compared with each other, and experimental structures determined by single-crystal X-ray diffraction; b) each structure will subjected to virtual binding studies with RAR and RXR systems, to obtain an approximate rank order of estimated binding capability; c) these data will be compared with actual SAR screening data to look for correlations between virtual and actual binding. Second generation systems will be designed for further synthesis and screening.