Abstract 2470: Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta.
Authors: Blaskovich Michelle A. , Gevariya Harsukh , Lawrence Nicholas J. , Sebti Saïd , Springett Gregory 
Published: August 2014

Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-β) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-ζ. LPAAT-β expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that knockdown of LPAAT-β by siRNA led to a significant inhibition of both anchorage-dependent and -independent growth in pancreatic cancer cell lines. Having thus established LPAAT-β as a valid therapeutic target in pancreatic cancer, we screened small molecule libraries for compounds which block LPAAT-β enzymatic activity in vitro.

Methods: We utilized a 96-well plate screening assay for LPAAT-β enzymatic activity modified from Aguado and Campbell, J Biol Chem 273(7):4096-4105, 1998. Briefly, compounds were introduced into a mixture containing full-length, recombinant, baculovirus-produced LPAAT-β (Blue Sky Biotechnology), oleoyl-LPA, and oleoyl coenzyme A. The ability of the compound to inhibit the enzyme activity was assayed by the measurement of the reaction of the thiol group of the released coenzyme A with the DTNB, resulting in an increase in the optical density of the sample when read at an absorbance of 413 nm. To rule out non-specific compounds identified from the DTNB plate assay, we confirmed activity using a secondary thin layer chromatography (TLC) assay that directly measures the ability of LPAAT-β to catalyze the synthesis of PA from oleoyl-LPA and a fluorochrome-labeled (NBD) palmitoyl coenzyme A.

Results: We screened a total of ten libraries containing 11204 compounds, and achieved a 1.2% hit rate for inhibition of &gt;50% at the tested concentration (100μM for nine libraries; 10μM for one library). Further testing identified several compounds with IC50s ranging from 0.1-15 μM. One hit identified with an IC50 of 100 nM was chosen for further investigation and for synthesis of analogs to optimize potency and undertake SAR. We verified the activity of this hit and its analogs against LPAAT-β by determining their inhibitory activity of the synthesis of NBD-labeled PA in a TLC assay. Finally, we demonstrated that the best analog from this group inhibited the proliferation of several pancreatic cancer cell lines.

Conclusions: We have developed a library of compounds which show inhibitory activity against LPAAT-β in a screening assay, and have confirmed their ability to inhibit LPAAT-β directly in a TLC assay which monitors the production of PA. These compounds show low micromolar activity against LPAAT-β in vitro, and micromolar activity against the proliferation of pancreatic cancer cell lines in culture.

Citation Format: Michelle A. Blaskovich, Harsukh Gevariya, Nicholas J. Lawrence, Saïd Sebti, Gregory Springett. Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2013-2470