Chemical Sciences Division, Saha Institute of Nuclear Physics 1/AF, Bidhannagar, Kolkata-700064, India.
J Phys Chem B. 2009 Dec 24;113(51):16323-31. doi: 10.1021/jp9069527.
Membrane fusion is a critical step in many biological events. The fusion process is always induced by different fusogenic agents of which proteins and peptides form the largest group. The mechanistic details of the fusion process vary depending on the nature of the fusogenic agents. However, membrane fusion induced by small drug molecules at physiologically relevant concentration has not been observed. Only recently our group has shown that three painkillers, namely, meloxicam, piroxicam, and tenoxicam, belonging to the oxicam group of non-steroidal anti-inflammatory drugs (NSAIDs) share this property. In this work, we present the effect of drug concentration and temperature on the kinetics of the fusion process. Small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage whereas transmission electron microscopy (TEM) was used to image the fusion process and to calculate the average diameter of the vesicles. The results show that, in this fusion process, lipid mixing and content mixing are two sequential events and can occur even at a very low drug to lipid ratio (D/L) of 0.018. For a D/L ratio greater than 0.045, leakage of the vesicles leading to rupture compete with the fusion thereby inhibiting it. Temperature variation in the presence of drugs gives linear Arrhenius plots and is used to calculate the activation energies for the lipid mixing and content mixing, which are less compared to that seen in SUVs with a smaller diameter of 45 nm. Thermodynamic parameters of the transition state are calculated. The fusogenic property of the drugs has been interpreted in terms of the ability of the drugs to introduce membrane perturbation even at such low D/L ratios as studied here.
膜融合是许多生物事件中的一个关键步骤。融合过程总是由不同的融合剂诱导的，其中蛋白质和肽形成最大的一组。融合过程的机制细节因融合剂的性质而异。然而，在生理相关浓度下，尚未观察到小分子药物诱导的膜融合。直到最近，我们的研究小组才表明，三种属于非甾体抗炎药（NSAIDs）的昔布类的止痛药，即美洛昔康、吡罗昔康和替诺昔康，具有这种特性。在这项工作中，我们展示了药物浓度和温度对融合过程动力学的影响。我们使用由二肉豆蔻酰磷脂酰胆碱（DMPC）形成的平均直径为 50-60nm 的小单层囊泡（SUV）作为模型膜。荧光分析用于探测脂质混合、内容混合和泄漏的时间依赖性，而透射电子显微镜（TEM）用于成像融合过程并计算囊泡的平均直径。结果表明，在这个融合过程中，脂质混合和内容混合是两个连续的事件，即使在药物与脂质的比例（D/L）非常低的情况下（0.018）也能发生。对于 D/L 比大于 0.045，导致囊泡破裂的泄漏与融合竞争，从而抑制融合。药物存在时的温度变化给出线性阿伦尼乌斯图，并用于计算脂质混合和内容混合的活化能，与直径较小的 45nm SUV 相比，这些活化能较小。还计算了过渡态的热力学参数。根据药物在如此低的 D/L 比下仍能引起膜扰动的能力，解释了药物的融合特性。
