title: Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
authors.forename: Ramya
authors.initials: R
authors.lastname: Ganesan
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Mahankali
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA.
authors.forename: Gerald
authors.initials: G
authors.lastname: Alter
authors.affiliation: Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435, USA. Electronic address: julian.cambronero@wright.edu.
authors.forename: Julian
authors.initials: J
authors.lastname: Gomez-Cambronero
mesh_terms: D000495:Allosteric Site; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D002522:Chlorocebus aethiops; D018920:Coculture Techniques; D004294:Domperidone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology*; D008024:Ligands; D062105:Molecular Docking Simulation; D009281:Naphthalenes / Q000737:chemistry / Q000494:pharmacology*; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: C546435:5-fluoro-2-indolyldeschlorohalopemide; D004791:Enzyme Inhibitors; D007211:Indoles; D008024:Ligands; D009281:Naphthalenes; D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D004294:Domperidone; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1
keywords: Cancer; Catalysis; Lipid signaling; Phosphoinositide; Protein structure