Mallipeddi Srikrishnan, Zvonok Nikolai, Makriyannis Alexandros
Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, 02115, USA.
Center for Drug Discovery, Northeastern University, Boston, MA, 02115, USA.
Sci Rep. 2018 Feb 13;8(1):2935. doi: 10.1038/s41598-018-19749-5.
The human cannabinoid 1 receptor (hCB1) is involved in numerous physiological processes and therefore provides a wide scope of potential therapeutic opportunities to treat maladies such as obesity, cardio-metabolic disorders, substance abuse, neuropathic pain, and multiple sclerosis. Structure-based drug design using the current knowledge of the hCB1 receptor binding site is limited and requires purified active protein. Heterologous expression and purification of functional hCB1 has been the bottleneck for ligand binding structural studies using biophysical methods such as mass spectrometry, x-ray crystallography and NMR. We constructed several plasmids for in-cell or in vitro Escherichia coli (E. coli) based expression of truncated and stabilized hCB1 receptor (hΔCB1 and hΔCB1) variants and evaluated their competency to bind the CP-55,940 ligand. MALDI-TOF MS analysis of in vitro expressed and purified hΔCB1his6 variants, following trypsin digestion, generated ~80% of the receptor sequence coverage. Our data demonstrate the feasibility of a cell-free expression system as a promising part of the strategy for the elucidation of ligand binding sites of the hCB1 receptor using a "Ligand Assisted Protein Structure" (LAPS) approach.
人类大麻素1受体（hCB1）参与众多生理过程，因此为治疗肥胖症、心脏代谢紊乱、药物滥用、神经性疼痛和多发性硬化症等疾病提供了广泛的潜在治疗机会。利用目前对hCB1受体结合位点的了解进行基于结构的药物设计存在局限性，且需要纯化的活性蛋白。功能性hCB1的异源表达和纯化一直是使用质谱、X射线晶体学和核磁共振等生物物理方法进行配体结合结构研究的瓶颈。我们构建了几种质粒，用于基于大肠杆菌（E. coli）在细胞内或体外表达截短和稳定的hCB1受体（hΔCB1和hΔCB1）变体，并评估它们与CP-55,940配体结合的能力。对体外表达和纯化的hΔCB1his6变体进行胰蛋白酶消化后的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析，产生了约80%的受体序列覆盖率。我们的数据证明了无细胞表达系统作为使用“配体辅助蛋白质结构”（LAPS）方法阐明hCB1受体配体结合位点策略中一个有前景部分的可行性。
