Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.

摘要: Abstract Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, well pathological cancer cell invasion metastasis. New inhibitors have described that hamper the role of PLD those pathologies but their site action is not known. We characterized biochemical biological behavior PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,­8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), found both FIPI NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate binds at S757 PLD2, which within HKD2 catalytic enzyme, whereas to two different sites, one being S757/S648 another an allosteric a natural occupied by PIP 2 (R210/R212). This latter site, along with F244/L245/L246, forms hydrophobic pocket PH domain. The mechanism direct effect on (and inhibits PLD1 isoforms), affects (orthosteric) blocks binding (allosteric), negates enhancing . Moreover, prevents cells, does occur cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, PLD2-S757/S648 mutants. study provides new knowledge enzyme regulation mechanisms activation inhibition necessary understand its signaling develop for