Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany.
Institute of Bioinformatics, Johannes Gutenberg University, 55131 Mainz, Germany.
Int J Mol Sci. 2023 Jun 16;24(12):10240. doi: 10.3390/ijms241210240.
Cancer therapy with clinically established anticancer drugs is frequently hampered by the development of drug resistance of tumors and severe side effects in normal organs and tissues. The demand for powerful, but less toxic, drugs is high. Phytochemicals represent an important reservoir for drug development and frequently exert less toxicity than synthetic drugs. Bioinformatics can accelerate and simplify the highly complex, time-consuming, and expensive drug development process. Here, we analyzed 375 phytochemicals using virtual screenings, molecular docking, and in silico toxicity predictions. Based on these in silico studies, six candidate compounds were further investigated in vitro. Resazurin assays were performed to determine the growth-inhibitory effects towards wild-type CCRF-CEM leukemia cells and their multidrug-resistant, P-glycoprotein (P-gp)-overexpressing subline, CEM/ADR5000. Flow cytometry was used to measure the potential to measure P-gp-mediated doxorubicin transport. Bidwillon A, neobavaisoflavone, coptisine, and z-guggulsterone all showed growth-inhibitory effects and moderate P-gp inhibition, whereas miltirone and chamazulene strongly inhibited tumor cell growth and strongly increased intracellular doxorubicin uptake. Bidwillon A and miltirone were selected for molecular docking to wildtype and mutated P-gp forms in closed and open conformations. The P-gp homology models harbored clinically relevant mutations, i.e., six single missense mutations (F336Y, A718C, Q725A, F728A, M949C, Y953C), three double mutations (Y310A-F728A; F343C-V982C; Y953A-F978A), or one quadruple mutation (Y307C-F728A-Y953A-F978A). The mutants did not show major differences in binding energies compared to wildtypes. Closed P-gp forms generally showed higher binding affinities than open ones. Closed conformations might stabilize the binding, thereby leading to higher binding affinities, while open conformations may favor the release of compounds into the extracellular space. In conclusion, this study described the capability of selected phytochemicals to overcome multidrug resistance.
癌症的临床治疗通常会受到肿瘤耐药性和正常组织器官严重副作用的阻碍。因此，人们迫切需要研发高效低毒的药物。植物化学物质是药物研发的重要资源，通常比合成药物的毒性更小。生物信息学可以加速和简化高度复杂、耗时且昂贵的药物研发过程。在这里，我们使用虚拟筛选、分子对接和计算机毒性预测分析了 375 种植物化学物质。基于这些计算机研究，我们进一步在体外研究了 6 种候选化合物。使用 Resazurin 测定法来确定对野生型 CCRF-CEM 白血病细胞及其多药耐药、P-糖蛋白 (P-gp) 过表达亚系 CEM/ADR5000 的生长抑制作用。流式细胞术用于测量潜在的 P-gp 介导的阿霉素转运。Bidwillon A、新巴西黄酮、黄连碱和 Z-芝麻脂素均显示出生长抑制作用和适度的 P-gp 抑制作用，而密特龙和蓝蓟素则强烈抑制肿瘤细胞生长并强烈增加细胞内阿霉素摄取。Bidwillon A 和密特龙被选择用于分子对接野生型和突变型 P-gp 形式的封闭和开放构象。P-gp 同源模型包含临床相关的突变，即六个单错义突变（F336Y、A718C、Q725A、F728A、M949C、Y953C）、三个双突变（Y310A-F728A；F343C-V982C；Y953A-F978A）或一个四突变（Y307C-F728A-Y953A-F978A）。与野生型相比，突变体的结合能没有明显差异。封闭的 P-gp 形式通常比开放形式具有更高的结合亲和力。封闭构象可能会稳定结合，从而导致更高的结合亲和力，而开放构象可能有利于化合物向细胞外空间释放。总之，本研究描述了选定植物化学物质克服多药耐药性的能力。
