Gorai Sukhamoy, Bagdi Prasanta Ray, Borah Rituparna, Paul Debasish, Santra Manas Kumar, Khan Abu Taleb, Manna Debasis
Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039, India.
National Center for Cell Science, Pune 411007, Maharashtra, India.
Biochem Biophys Rep. 2015 May 27;2:75-86. doi: 10.1016/j.bbrep.2015.05.007. eCollection 2015 Jul.
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] is an important regulator of several cellular processes and a precursor for other second messengers which are involved in cell signaling pathways. Signaling proteins preferably interact with PI(4,5)P through its pleckstrin homology (PH) domain. Efforts are underway to design small molecule-based antagonist, which can specifically inhibit the PI(4,5)P/PH-domain interaction to establish an alternate strategy for the development of drug(s) for phosphoinositide signaling pathways.
Surface plasmon resonance, molecular docking, circular dichroism, competitive Förster resonance energy transfer, isothermal titration calorimetric analyses and liposome pull down assay were used.
In this study, we employed 1,2,3-triazol-4-yl methanol containing small molecule (CIPs) as antagonists for PI(4,5)P/PH-domain interaction and determined their inhibitory effect by using competitive-surface plasmon resonance analysis (IC ranges from 53 to 159 nM for PI(4,5)P/PLCδ1-PH domain binding assay). We also used phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P], PI(4,5)P specific PH-domains to determine binding selectivity of the compounds. Various physicochemical analyses showed that the compounds have weak affect on fluidity of the model membrane but, strongly interact with the phospholipase C δ1 (PLCδ1)-PH domains. The 1,2,3-triazol-4-yl methanol moiety and nitro group of the compounds are essential for their exothermic interaction with the PH-domains. Potent compound can efficiently displace PLCδ1-PH domain from plasma membrane to cytosol in A549 cells.
Overall, our studies demonstrate that these compounds interact with the PIP-binding PH-domains and inhibit their membrane recruitment.
These results suggest specific but differential binding of these compounds to the PLCδ1-PH domain and emphasize the role of their structural differences in binding parameters. These triazole-based compounds could be directly used/further developed as potential inhibitor for PH domain-dependent enzyme activity.
磷脂酰肌醇 -4,5- 二磷酸 [PI(4,5)P₂] 是多种细胞过程的重要调节因子，也是参与细胞信号通路的其他第二信使的前体。信号蛋白通常通过其普列克底物蛋白同源性（PH）结构域与 PI(4,5)P₂相互作用。目前正在努力设计基于小分子的拮抗剂，其可以特异性抑制 PI(4,5)P₂/PH 结构域的相互作用，从而为开发用于磷酸肌醇信号通路的药物建立一种替代策略。
使用表面等离子体共振、分子对接、圆二色性、竞争性福斯特共振能量转移、等温滴定量热分析和脂质体下拉分析。
在本研究中，我们使用含 1,2,3 - 三唑 -4- 基甲醇的小分子（CIPs）作为 PI(4,5)P₂/PH 结构域相互作用的拮抗剂，并通过竞争性表面等离子体共振分析确定它们的抑制作用（对于 PI(4,5)P₂/PLCδ1 - PH 结构域结合测定，IC₅₀范围为 53 至 159 nM）。我们还使用磷脂酰肌醇 -3,4,5- 三磷酸 [PI(3,4,5)P₃]、磷脂酰肌醇 -3,4- 二磷酸 [PI(3,4)P₂]、PI(4,5)P₂特异性 PH 结构域来确定这些化合物的结合选择性。各种物理化学分析表明，这些化合物对模型膜的流动性影响较弱，但与磷脂酶 Cδ1（PLCδ1）- PH 结构域强烈相互作用。化合物的 1,2,3 - 三唑 -4- 基甲醇部分和硝基对于它们与 PH 结构域的放热相互作用至关重要。强效化合物可以有效地将 PLCδ1 - PH 结构域从 A549 细胞的质膜转移到细胞质中。
总体而言，我们的研究表明这些化合物与 PIP 结合的 PH 结构域相互作用并抑制它们的膜募集。
这些结果表明这些化合物与 PLCδ1 - PH 结构域具有特异性但不同的结合，并强调了它们结构差异在结合参数中的作用。这些基于三唑的化合物可直接用作/进一步开发为 PH 结构域依赖性酶活性的潜在抑制剂。
