Ganesan Ramya, Mahankali Madhu, Alter Gerald, Gomez-Cambronero Julian
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Biochim Biophys Acta. 2015 Mar;1851(3):261-72. doi: 10.1016/j.bbalip.2014.12.007. Epub 2014 Dec 20.
Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.
磷脂酶D（PLD）参与了许多生理功能，如趋化作用和吞噬作用，以及病理功能，如癌细胞侵袭和转移。已有新的抑制剂被报道，它们能阻碍PLD在这些病理过程中的作用，但作用位点尚不清楚。我们已对PLD1/2双重抑制剂5-氟-2-吲哚基去氯卤培米特（FIPI）以及特异性PLD2抑制剂N-[2-[1-(3-氟苯基)-4-氧代-1,3,8-三氮杂螺[4.5]癸-8-基]乙基]-2-萘甲酰胺（NFOT）的生化和生物学行为进行了表征，发现FIPI和NFOT均为混合动力学抑制剂。诱变研究表明，FIPI结合于PLD2的S757位点，该位点位于该酶的HKD2催化位点内，而NFOT在两个不同位点与PLD2结合，一个位点在S757/S648，另一个位点在一个变构位点，该变构位点是PIP2（R210/R212）占据的天然位点。后一个位点与F244/L245/L246一起在PH结构域中形成一个疏水口袋。FIPI的作用机制是对催化位点产生直接影响（因此抑制PLD1和PLD2两种亚型），而NFOT既影响催化位点（正构）又阻断PIP2与PLD2的结合（变构），这消除了PIP2的天然增强作用。此外，NFOT可阻止癌细胞的侵袭，而在过表达PLD2-F244A/L245A/L246A、PLD2-R210A/R212A或PLD2-S757/S648突变体的细胞中则不会发生这种情况。这项研究为PLD2的酶调节以及激活和抑制机制提供了新的具体知识，这对于理解其在细胞信号传导中的作用以及开发用于癌细胞侵袭和转移的新抑制剂是必要的。
