Graduate Institute of Pharmaceutical Chemistry, Luliang University, Shanxi 033001, PR China.
Department of Bioscience, Luliang University, Shanxi 033001, PR China.
Eur J Pharm Sci. 2017 Nov 15;109:262-268. doi: 10.1016/j.ejps.2017.08.024. Epub 2017 Aug 20.
A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min, together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in itself.
构建了一个苦瓜调控蛋白系统的变构体，通过体外蛋白磷酸化作用，试图评估抗真菌的多效性及其对白色念珠菌的剂量依赖性敏感性。通过抗真菌药敏试验，在实验室中制备了对酮康唑、依康唑、咪康唑、5-氟胞嘧啶、制霉菌素和两性霉素 B 敏感的真菌株系。通过细胞周期蛋白依赖性激酶 1、蛋白激酶 A 和蛋白激酶 C 分别催化 5'-腺嘌呤、鸟嘌呤、胞嘧啶和尿嘧啶和环磷酸腺苷的反应进行蛋白磷酸化。酶反应的生化分析表明，表观米氏常数和最大速度值分别为 16.57-91.97mM 和 55.56-208.33μM·min，反应物的近似比例为 1:1。通过激酶磷酸化工具理论预测 Thr255、Thr102 和 Thr24 三个主要的蛋白磷酸化位点。此外，圆二色性光谱表明，磷酸化后，蛋白质折叠结构在无规卷曲、β6-片层和α1-螺旋部分区域减少。McFarland 等价标准测试显示了浓度依赖性的抑制模式，同时在萨布罗琼脂上培养真菌。游离蛋白和磷酸化蛋白的最小抑菌浓度（MIC）分别为 0.16-0.51μM（响应率为 50%）。在 3 次循环药敏试验方案中，以 0.14μM/循环的剂量依次给予总蛋白形式。对于酮康唑敏感的白色念珠菌生长，相对抑制率保持在初始值的 66.13-81.04%。由于调节多效性，提出了一种抑制蛋白系统，它可以降低对多种抗真菌药物的敏感性，而自身对敏感性的贡献很小。
