Phospholipase D (PLD) catalyzes the production of the lipid second messenger phosphatidic acid (PtdOH). PLD expression and/or enzymatic activity are both elevated in a variety of human cancers. Inhibition of PLD enzymatic activity, via genetic or biochemical methods, leads to decreased cancer cell invasion and decreased cancer cell survival. The aforementioned evidence provided the impetus for a medicinal chemistry project focused on the development of isoform-selective PLD inhibitors. A group from Novartis published a report in 2007 disclosing halopemide as a hit from a high throughput screen for PLD inhibitors. While the iterative analog synthesis campaign described in this dissertation began with halopemide a broad chemical space was explored through the synthesis of several hundred compounds. This effort yielded the most potent, isoform-selective PLD inhibitors ever described. Halopemide inhibits both PLD isoforms relatively equally; VU0359595 inhibits PLD1 1,700 times more potently than PLD2, and VU0364739 inhibits PLD2 75 times more potently than PLD1. An aryl(trifluoromethyl)diazirine containing photoprobe based on the structure of VU0359595 was prepared and was found to inhibit a truncated form of PLD missing the first 311 amino acids, PLD1c.d311. Subsequently, the ability of the probe to covalently modify PLD1c.cd311 was verified. Tandem mass spectrometry experiments to identify the covalently labeled peptide(s) and potentially amino acid(s) are ongoing. In addition to the chemical synthesis of PLD inhibitors and photolabeling experiments some molecular mechanism of action studies were conducted. The VU series of PLD inhibitors block catalysis of both lipososomal and monomeric substrates. Unfortunately, Michealis-Menten kinetics experiments were unable to be performed, because the truncated form of PLD2, PLD2.d308, displays substrate inhibition kinetics in a monomeric substrate containing enzyme activity assay. This research has provided chemical probes that are facilitating the study, both in vitro and in vivo, of how the two mammalian isoforms of PLD affect diverse physiological and pathological processes.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-11052014-230853 |
| Date | 06 November 2014 |
| Creators | Lavieri, Robert Raymond |
| Contributors | Lawrence J. Marnett, Tina M. Iverson, H. Alex Brown, Craig W. Lindsley, David Cortez |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-11052014-230853/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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