Argiriadi Maria A, Goedken Eric R, Banach David, Borhani David W, Burchat Andrew, Dixon Richard W, Marcotte Doug, Overmeyer Gary, Pivorunas Valerie, Sadhukhan Ramkrishna, Sousa Silvino, Moore Nigel St John, Tomlinson Medha, Voss Jeffrey, Wang Lu, Wishart Neil, Woller Kevin, Talanian Robert V
Department of Molecular & Cellular Pharmacology, Abbott Laboratories, Worcester, MA, USA.
BMC Struct Biol. 2012 Sep 20;12:22. doi: 10.1186/1472-6807-12-22.
Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds.
A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods.
Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
基于结构的药物设计（SBDD）可加速抑制剂先导物的设计与优化，而快速、迭代的结构测定需要包括蛋白质纯化、表征、结晶和高分辨率衍射在内的高效方法。Janus激酶是适合基于结构的药物设计的重要靶点。在此，我们展示了首个与3-氨基吲唑化合物复合的小鼠Tyk2晶体结构。
全面的构建体设计工作包括N端和C端变异、激酶失活突变以及多个物种的直系同源物。高通量克隆和表达方法与简化的纯化方案相结合，以优化蛋白质的溶解性和稳定性。总共生成了50个Tyk2构建体。许多构建体表现出表达不佳、溶解性不足或亲和标签处理不完全的问题。一种与ATP竞争性3-氨基吲唑抑制剂复合的激酶失活小鼠Tyk2构建体提供了衍射分辨率达2.5 - 2.6 Å的晶体。该结构揭示了基于结构的药物设计的初始“热点”区域，并为配体浸泡实验提供了一个强大的平台。与先前报道的人Tyk2抑制剂晶体结构（Chrencik等人，（2010）《分子生物学杂志》400:413）相比，我们的结构揭示了抑制剂诱导的富含甘氨酸环构象的关键差异。配体结合还赋予了对嗜热菌蛋白酶降解的抗性。由于未结合配体的酶无法获得晶体，这种增强的稳定性可能对成功的结晶和抑制剂浸泡方法很重要。
构建体性能和优先级的实用标准、优化纯化方案以提高蛋白质产量和稳定性以及使用高通量构建体探索能够在药物发现过程的早期实现结构测定方法。此外，特定配体可稳定Tyk2蛋白，从而可能实现结晶。
