Substrate-dependent differences in the extent of product inhibition of Autotaxin by lysophosphatidic acid

Sarah Caunt, Lucy Elphick, Viral Patel, Lilya Sviridenko, Mark Dowling. Novartis, Wimblehurst Road, Horsham, West Sussex, RH12 5AB, UK

Autotaxin (ATX) is a secreted lysophospholipase D (lysoPLD) that hydrolyses lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). LPA binds to G protein-coupled receptors evoking a wide variety of cellular responses such as migration, stimulation and proliferation which may lead to pathological conditions including cancer and fibrosis. Product inhibition by LPA would prevent excessive LPA accumulation, however reports vary into the extent of LPA based inhibition of ATX (Albers, et al 2010). This may be partly due to the different substrates from which ATX activity is observed, such as the non-native fluorescent LPC analogue, FS-3 (Ferguson, et al 2006).

In this study we aimed firstly to investigate the degree of inhibition towards ATX by different LPA species, and secondly whether LPA inhibition differed when using native and non-native substrates. The FS-3 assay was compared to a double-coupled enzyme assay utilizing native LPC species to investigate LPA inhibition and inhibitor modality.

Michaelis-Menten curves were performed in the choline release assay (n = 3) to determine Kcat/Km ratios for each LPC species (14:0, 16:0, 17:0, 18:0, 18:1, 20:0 and 20:4), varying in acyl chain length and degree of unsaturation. The highest turnover was observed with the unsaturated LPCs 18:1 and 20:4 (0.8 and 0.7min-1μM-1), whilst the saturated isoforms vary in turnover rate based on their respective acyl chain lengths, values ranging from 0.71min -1μM-1 for 14:0 LPC to 0.27min-1μM-1 with 20:0 LPC. FS-3 gave the lowest Kcat/Km ratio 0.2min-1μM-1. LPA dose-response curves were obtained in the choline release using native LPC species and compared to those obtained from the FS-3 assay. LPA-induced inhibition of ATX activity was observed in the FS-3 assay by 20:4 LPA (Ki 0.05μM), and to a varying degree with all other LPA isoforms. In contrast, LPA failed to inhibit ATX activity in the choline release assay, except for 20:4 LPA (Ki 9.8μM). The known ATX inhibitor PF-8380 showed inhibition towards ATX in both assays, with a Ki of 0.1nM in the FS-3 and Ki of 1nM in the choline release assay. Investigation into the mode of 20:4 LPA inhibition between assay formats revealed differences between native and non-native substrates, with the lipid showing competitive inhibition against LPC but a mixed mode of inhibition in the FS-3 assay.

These results show significant differences in the level of LPA inhibition exhibited between the assay formats depending on the substrate utilized. Caution should be applied when using non-native substrates to report ATX inhibition profiles, as they may not accurately reflect inhibition of native LPC substrates.

Albers HM, et al (2010) J.Med.Chem.; 53 (13):4958-4967.

Ferguson CG, et al (2006) Org.Lett.; 8 (10):2023-2026.