Antibody-assisted Investigations into allosteric regulation of Wnt signalling: building the sclerostin franchise

骨质疏松症影响全球数亿人，发生在骨骼强度和完整性丧失后。尽管全世界的患者越来越多，但目前的治疗方案提供的好处有限，大多数抑制了骨的吸收，而对已经丢失的骨的修复几乎没有效果。经典的Wnt信号通路已成为骨稳态的关键调节因子，该通路的激活受到一些分泌型抑制蛋白的严格调控，如硬化素。对一系列骆驼VHH抗体片段提升到细胞因子的开创性研究发现了以前未知的变构位点，其中特定VHH的结合导致远距离功能位点的调节。对VHH与细胞因子结合的结构的测定确定了负责变构调节的特定相互作用，并强调了识别类似调节活性的合成小分子的可能性。基于核磁共振的研究表明，与细胞因子一样，硬化素的特征是具有显著灵活性的区域。这些柔性区域对应于硬化素与其结合伙伴之间相互作用所涉及的位点。抗体辅助的结构方法可能识别硬化素上未知的变构位点和/或稳定与LRP共同受体结合所需的关键区域。将确定功能修饰的VHH，并对其结构和相互作用进行表征。了解硬化素的变构潜力以及如何将其转化为蛋白质功能的调节，将对这种高度动态的蛋白质的作用机制提供有价值的见解。此外，抗体辅助药物发现技术的进一步发展，以及对调节硬化素的潜力的进一步了解，将有助于未来识别抑制硬化素的小分子。

Osteoporosis affects hundreds of millions of people worldwide occurring after a loss of bone strength and integrity. Despite a growing number of patients worldwide current therapeutic options offer limited benefit with the majority inhibiting resorption of bone while having little effect on the restoration of bone that has already been lost. The canonical Wnt signalling pathway has emerged as a key regulator of bone homeostasis with activation of this pathway being tightly regulated by a number of secreted inhibitory proteins such as sclerostin. Pioneering studies of a range of camelid VHH antibody fragments raised to a cytokine identified previously unknown allosteric sites, where binding of specific VHHs results in modulation of distant functional sites. Determination of structures for VHHs bound to the cytokine identified specific interactions responsible for the allosteric regulation and highlighted the potential to identify synthetic small molecules that would similarly modulate activity.NMR based studies have shown that sclerostin, like the cytokine, is characterised by regions that show significant flexibility. These flexible regions correspond to sites involved in the interactions between sclerostin and its binding partners. Antibody-assisted structural approaches are likely to identify unknown allosteric sites on sclerostin and/or stabilize key regions required for binding to LRP co-receptors. Function modifying VHHs will be identified and have their structures and interactions characterized. Gaining an understanding of the allosteric potential of sclerostin and how this translates into regulation of protein function will give valuable insights into the mechanism of action of this highly dynamic protein. In addition, further development of antibody-assisted drug discovery technology, together with enhanced understanding of the potential to regulate sclerostin, will assist future identification of small molecules that inhibit sclerostin.