Identification of the Sites of Action of Inhibitors of Mammalian Phospholipase D2 (PLD2) and the Role of Interacting Protein Partners
Gerald Alter (Committee Member), Nancy Bigley (Committee Member), Julian Gomez-cambronero (Advisor)
Master of Science (MS)
Phospholipase D (PLD) is a key enzyme for the remodeling of phospholipids in the cell membrane. PLD has been implicated in many physiological functions such as chemotaxis and phagocytosis as well as pathological functions such as ischemia/reperfusion and cancer metastasis. Several small molecule inhibitors of PLD have been recently developed to overcome these pathological effects. We have concentrated on 5-Fluoro-2-Indolyl des-Chlorohalopemide (FIPI) which is an indole derivative of halopemide that inhibits the two mammalian isoforms PLD1 and PLD2. We also concentrate on N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT) and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide (NOPT) that are specific inhibitors of the PLD2 isoform. In spite of knowing their specificity derived from in vitro assays, the mechanism of action of these inhibitors has never been studied. Here for the first time, we show the site of action of FIPI and NFOT on PLD as well as a mechanism of enzymatic action that explains in vivo actions. Our studies showed that FIPI is a non-reversible inhibitor of PLD that binds to the HKD, whereas NFOT shows mixed type of inhibition, which suggests that inhibitor binding site might be anywhere in the regulatory PX or PH domains of PLD2 or the catalytic region HKD. We performed extensive mutational analysis on PLD2 and our results clearly indicate that FIPI binds to S757 residue and S648 and a PIP2 binding site are critical for NFOT to inhibit PLD2, since mutating these key amino acid(s) resulted in partial resistance of the enzyme towards the inhibitor. Inhibitor dose dependent assays of the resistant PLD2 mutants F244N/L245A/L246A and S648A confirmed the same. Also, the mutants exhibited resistance against the inhibitors unlike wild-type, in performing chemotaxis further confirming physiological relevance. In addition, we uncovered the effect of PLD2 binding partners, Grb2 and Ras on the efficacy of inhibitors. Mechanistically it was found that Grb2 positively regulates PLD2, while Ras has a negative effect on PLD2 and that the small molecule inhibitor's action on PLD2 is affected by the absence or presence of Grb2 or Ras. In conclusion, we consider the results obtained in this study regarding the key residues of PLD2 are crucial in order to develop more potent and efficient inhibitors that can be used in vivo for pathological processes such as chronic inflammation and cancer metastasis.
Department or Program
Microbiology and Immunology
Year Degree Awarded
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