Fluorogenic Phospholipid Substrate to Detect Lysophospholipase D/Autotaxin Activity

Abstract

[reaction: see text] Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target.

α‐Substituted Phosphonate Analogues of Lysophosphatidic Acid (LPA) Selectively Inhibit Production and Action of LPA

“…These cell lines have been used extensively for the characterization of LPA GPCR ligands because RH7777 cells are intrinsically unresponsive to LPA, and CHO cells show minimal endogenous responses to LPA unless transfected with LPA 4 . [14,24,44] The oleoyl chain-containing methylene phosphonate LPA analogue 5a, in which a methylene unit replaces the oxygen atom, is a selective, full agonist for LPA 2 with an EC 50 value of 281 nM. Interestingly, replacement of the oleoyl chain in 5a with the palmitoyl chain in 5b switched the activity of this partially selective agonist to that of modest antagonist.…”

“…Analogue 5b had antagonist activity on all LPA receptor subtypes 1-3, with the relatively higher antagonist activity observed toward the LPA 2 receptor (IC 50 = 2.59 μM, K i = 1 296 nM). The oleoyl α-hydroxymethylene phosphonate analogue 10a is a selective and potent LPA 3 agonist (Table 1), with an EC 50 value close to that of LPA (18:1) toward LPA 3 . In contrast, the palmitoyl analogue 10b exhibited a switch in activity to that of a weak partial antagonist of LPA [1][2][3] .…”

“…ATX activity was measured by the hydrolysis of the fluorogenic lysoPC analogue FS-3, which has a K M value of 6.3 μM. [50] The results showed that all of the analogues, the unsubstituted as well as each of the α-substituted phosphonates, inhibited ATX at a concentration of 10 μM. Although doseresponse data remain to be determined, in this preliminary screen, three compounds (5a, 19a, and 19b) were revealed as potent inhibitors of > 90% enzyme activity.…”

“…Each test was performed in quadruplicate. EC 50 , IC 50 , and K i values were calculated as described. [24] PPARγ activation assay PPARγ activation was performed using CV-1 cells transfected with an acyl-coenzyme A oxidase-luciferase (PPRE-Acox-Rluc) reporter gene construct as previously reported.…”

“…This assay uses FS-3 [50] (Echelon Biosciences, Inc., Salt Lake City, UT, USA) as substrate and recombinant ATX-HA (generously provided by Dr. Tim Clair (NCI)). For analysis, 50 μL of ATXHA (0.25 μg) in assay buffer (Tris 50 mM, NaCl 140 mM, KCl 5 mM, CaCl 2 1 mM, MgCl 2 1 mM, pH 8.0) was mixed with 25 μL of FS-3 (1 μM final concentration in assay buffer) and 25 μL of test compound dissolved in assay buffer containing 1:1.5 bovine serum albumin in a 96-well plate.…”