Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics.

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/ phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC 50 ∼ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.A utotaxin (ATX or NPP2) is a secreted nucleotide pyrophosphatase/phosphodiesterase (NPP) originally isolated as an autocrine motility factor from melanoma cells (1). ATX, a ∼120 kDa glycoprotein, is unique amongst the NPPs in that it functions as a lysophospholipase D (lysoPLD) that converts extracellular lysophosphatidylcholine (LPC) into the lipid mediator lysophosphatidic acid (LPA; mono-acyl-sn-glycero-3-phosphate) (2-5). LPA acts on specific G protein-coupled receptors and thereby stimulates the migration, proliferation, and survival of many cell types (6 and 7) (Fig. 1). ATX is produced by various tissues and is the major LPA-producing enzyme in the circulation. Newly produced LPA is subject to degradation by membranebound lipid phosphate phosphatases (LPPs) (8 and 9). However, little is known about the dynamic regulation of steady-state LPA levels in vivo.ATX is essential for vascular development (10 and 11) and is found overexpressed in various human cancers (12). Forced overexpression of ATX or individual LPA receptors promotes tumor progression in mouse models (13-16), while LPA receptor deficiency protects from colon carcinogenesis (17). In addition to its role in cancer, ATX-LPA signaling has been implicated in lymphocyte homing and (chronic) inflammation (18), fibrotic diseases (19 and 20), and thrombosis (21). Therefore, the ATX-LPA axis qualifies as an attractive target for therapies.Potent and selective ATX inhibitors are now needed as a starting point for the development of targeted anti-ATX/LPA therapy. Direct targeting of LPA receptors seems to be a less attractive strategy, since LPA acts on multiple receptors that show overlapping activities (2 and 6). Since the initial finding that ATX is subject to product inhibition by LPA and sphingosine 1-phosphate (S1P) (22), various synthetic phospho-and phosphonate lipids have been explored as ATX inhibitors (23-26). However, s...

Autotaxin (ATX) is an extracellular enzyme that hydrolyzes lysophosphatidylcholine (LPC) to produce the lipid mediator lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in diverse physiological and pathological processes, including vascular development, inflammation, fibrotic disease, and tumor progression. Therefore, targeting ATX with small molecule inhibitors is an attractive therapeutic strategy. We recently reported that 2,4-thiazolidinediones inhibit ATX activity in the micromolar range. Interestingly, inhibitory potency was dramatically increased by introduction of a boronic acid moiety, designed to target the active site threonine in ATX. Here we report on the discovery and further optimization of boronic acid based ATX inhibitors. The most potent of these compounds inhibits ATX-mediated LPC hydrolysis in the nanomolar range (IC(50) = 6 nM). The finding that ATX can be targeted by boronic acids may aid the development of ATX inhibitors for therapeutic use.

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.

Lymphocyte extravasation from the high endothelial venules (HEVs) of lymph nodes is crucial for the maintenance of immune homeostasis, but its molecular mechanism remains largely unknown. In this article, we report that lymphocyte transmigration across the basal lamina of the HEVs is regulated, at least in part, by autotaxin (ATX) and its end-product, lysophosphatidic acid (LPA). ATX is an HEV-associated ectoenzyme that produces LPA from lysophosphatidylcholine (LPC), which is abundant in the systemic circulation. In agreement with selective expression of ATX in HEVs, LPA was constitutively and specifically detected on HEVs. In vivo, inhibition of ATX impaired the lymphocyte extravasation from HEVs, inducing lymphocyte accumulation within the endothelial cells (ECs) and sub-EC compartment; this impairment was abrogated by LPA. In vitro, both LPA and LPC induced a marked increase in the motility of HEV ECs; LPC’s effect was abrogated by ATX inhibition, whereas LPA’s effect was abrogated by ATX/LPA receptor inhibition. In an in vitro transmigration assay, ATX inhibition impaired the release of lymphocytes that had migrated underneath HEV ECs, and these defects were abrogated by LPA. This effect of LPA was dependent on myosin II activity in the HEV ECs. Collectively, these results strongly suggest that HEV-associated ATX generates LPA locally; LPA, in turn, acts on HEV ECs to increase their motility, promoting dynamic lymphocyte–HEV interactions and subsequent lymphocyte transmigration across the basal lamina of HEVs at steady state.

Autotaxin (ATX) is a secreted phosphodiesterase that hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been implicated in inflammation, fibrosis, and tumor progression, rendering ATX an attractive drug target. We recently described a boronic acid-based inhibitor of ATX, named HA155 (1). Here, we report the design of new inhibitors based on the crystal structure of ATX in complex with inhibitor 1. Furthermore, we describe the syntheses and activities of these new inhibitors, whose potencies can be explained by structural data. To understand the difference in activity between two different isomers with nanomolar potencies, we performed molecular docking experiments. Intriguingly, molecular docking suggested a remarkable binding pose for one of the isomers, which differs from the original binding pose of inhibitor 1 for ATX, opening further options for inhibitor design.

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Objective. Lysophosphatidic acid (LPA) is a bioactive lipid that binds to a group of cell surface G protein-coupled receptors (LPA receptors 1-6 [LPA 1-6 ]) and has been implicated as an important mediator of angiogenesis, inflammation, and cancer growth. This study was undertaken to analyze the effects of LPA 1 on the development of arthritis.Methods. Expression of LPA receptors on synovial tissue was analyzed by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. The effects of abrogation of LPA 1 on collageninduced arthritis (CIA) were evaluated using LPA 1 -deficient mice or LPA 1 antagonist. Migrating fluorescence-labeled CD11b؉ splenocytes, which were transferred into the synovium of mice with CIA, were counted. CD4؉ naive T cells were incubated under Th1-, Th2-, or Th17-polarizing conditions, and T helper cell differentiation was assessed. Osteoclast formation from bone marrow cells was examined.Results. LPA 1 was highly expressed in the synovium of patients with rheumatoid arthritis (RA) compared with that of patients with osteoarthritis. LPA 1 -deficient mice did not develop arthritis following immunization with type II collagen (CII). LPA 1 antagonist also ameliorated murine CIA. Abrogation of LPA 1 was associated with reductions in cell infiltration, bone destruction in the joints, and interleukin-17 production from CII-stimulated splenocytes. Infiltration of transferred CD11b؉ macrophages from LPA 1 -deficient mice into the synovium was suppressed compared with infiltration of macrophages from wild-type mice. LPA 1 antagonist inhibited the infiltration of macrophages from wild-type mice. Differentiation into Th17, but not Th1 or Th2, and osteoclast formation were also suppressed under conditions of LPA 1 deficiency or LPA 1 inhibition in vitro.Conclusion. Collectively, these results indicate that LPA/LPA 1 signaling contributes to the development of arthritis via cellular infiltration, Th17 differentiation, and osteoclastogenesis. Thus, LPA 1 may be a promising target molecule for RA therapy.Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by inflammatory cell infiltration and bone destruction at multiple joints. The inflammation process in RA leads to synovial hyperplasia with proliferation of fibroblast-like synoviocytes (FLS), angiogenesis, and infiltration of inflammatory cells, including lymphocytes and macrophages (1,2).