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1.  Ligand-Based Autotaxin Pharmacophore Models Reflect Structure-Based Docking Results 
The autotaxin (ATX) enzyme exhibits lysophospholipase D activity responsible for the conversion of lysophosphatidyl choline to lysophosphatidic acid (LPA). ATX and LPA have been linked to the initiation of atherosclerosis, cancer invasiveness, and neuropathic pain. ATX inhibition therefore offers currently unexploited therapeutic potential, and substantial interest in the development of ATX inhibitors is evident in the recent literature. Here we report the performance-based comparison of ligand-based pharmacophores developed on the basis of different combinations of ATX inhibitors in the training sets against an extensive database of compounds tested for ATX inhibitory activity, as well as with docking results of the actives against a recently reported ATX crystal structure. In general, pharmacophore models show better ability to select active ATX inhibitors binding in a common location when the ligand-based superposition shows a good match to the superposition of actives based on docking results. Two pharmacophore models developed on the basis of competitive inhibitors in combination with the single inhibitor crystallized to date in the active site of ATX were able to identify actives at rates over 40%, a substantial improvement over the <10% representation of active site-directed actives in the test set database.
doi:10.1016/j.jmgm.2011.09.004
PMCID: PMC3224989  PMID: 21967734
Autotaxin; pharmacophore; docking
2.  Autotaxin Structure Activity Relationships Revealed through Lysophosphatidylcholine Analogs 
Bioorganic & medicinal chemistry  2009;17(9):3433-3442.
Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating it can bind to both the enzyme and the enzyme-substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.
doi:10.1016/j.bmc.2009.03.030
PMCID: PMC2705928  PMID: 19345587
3.  Benzyl and Naphthalene-Methyl Phosphonic Acid Inhibitors of Autotaxin with Anti-invasive and Anti-metastatic Actions 
ChemMedChem  2011;6(5):922-935.
Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) via a lysophospholipase D activity that leads to the generation of the growth factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Here we report the synthesis and pharmacological characterization of inhibitors of ATX based on the 4-tetradecanoylaminobenzyl phosphonic acid scaffold that was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzyl phosphonic acid and 6-substituted naphthalen-2-yl-methyl phosphonic acid analogs blocked ATX with Ki values in the low-micromolar-nanomolar range against FS-3, LPC, and nucleotide substrates through a mixed-mode mechanism of inhibition. None of the compounds tested inhibited the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor subtypes. Analogs 22 and 30b, the two most potent ATX inhibitors, dose-dependently inhibited the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro. The average terminal half-life for compound 22 was 10h ± 5.4h and it caused a long-lasting reduction plasma LPA levels. Compounds 22 and 30b significantly reduced lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The described 4-substituted benzyl phosphonic acids and 6-substituted naphthalen-2-yl-methyl phosphonic acids represent new lead compounds that effectively inhibit the ATX-LPA-LPA receptor axis both in vitro and in vivo.
doi:10.1002/cmdc.201000425
PMCID: PMC3517046  PMID: 21465666
ATX inhibitors; LPA receptors; 4-substituted benzyl phosphonic acids; 6-substituted naphthalen-2-yl-methyl phosphonic acids; structure-activity relationships
4.  Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis 
The Journal of biological chemistry  2006;281(32):22786-22793.
Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2, NPP2) is an autocrine motility factor initially characterized from A2058 melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.
doi:10.1074/jbc.M512486200
PMCID: PMC3505596  PMID: 16782709
5.  Phospholipase D2-dependent Inhibition of the Nuclear Hormone Receptor PPARγ by Cyclic Phosphatidic Acid 
Molecular cell  2010;39(3):421-432.
Summary
Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature’s simplest phospholipids, is found in cells from slime mold to humans and has largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus coupled-manner by Phospholipase D2 (PLD2), and binds to and inhibits the nuclear hormone receptor PPARγ with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARγ target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARγ inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARγ, revealing that PPARγ is regulated by endogenous agonists as well as by antagonists.
doi:10.1016/j.molcel.2010.07.022
PMCID: PMC3446787  PMID: 20705243
6.  (S)-FTY720-Vinylphosphonate, an Analogue of the Immunosuppressive Agent FTY720, Is a Pan-antagonist of Sphingosine 1-Phosphate GPCR Signaling and Inhibits Autotaxin Activity 
Cellular signalling  2010;22(10):1543-1553.
FTY720 (Fingolimod™), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P1 receptor internalization and targeting to the proteasomal degradative pathway, and thus acts as a functional antagonist of S1P1 by depleting the functional S1P1 receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)- and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P1 (EC50 20 ± 3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P1,3,4 (Ki 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P2, and S1P5. Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with Ki values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)- and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P2 transcripts and traces of S1P5. (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. The biological effects of the (R)- and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets.
doi:10.1016/j.cellsig.2010.05.023
PMCID: PMC3446790  PMID: 20566326
FTY720; sphingosine 1-phosphate; lysophosphatidic acid; autotaxin; lysophospholipase D; lymphocyte egress; EDG receptor; inhibitor
7.  Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7 
BMC Biochemistry  2011;12:65.
Background
Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM.
Results
The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding.
Conclusions
Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.
doi:10.1186/1471-2091-12-65
PMCID: PMC3282672  PMID: 22177013
8.  Synthesis and Pharmacological Evaluation of the Stereoisomersof 3-Carba Cyclic-Phosphatidic Acid 
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Here we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA5 GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA5 compared to the R stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.
doi:10.1016/j.bmcl.2010.09.115
PMCID: PMC3040411  PMID: 21051230
lysophosphatidic acid; NPP2; autotaxin; GPR92; lysophospholipase D
9.  Autotaxin Inhibitors: A Perspective on Initial Medicinal Chemistry Efforts 
Expert opinion on therapeutic patents  2010;20(12):1619-1625.
The lysophospholipase D enzyme, autotaxin (ATX), has been linked to numerous human diseases including cancer, neurophatic pain, obesity, and Alzheimer’s disease. Although the ATX protein was initially purified and characterized in 1992, a link to bioactive lipid metabolism was not made until 2002. In the past decade, metal chelators, lysophospholipid product analogs, and more recently small non-lipid inhibitors of the enzyme were successfully identified. The majority of these inhibitors have been characterized using recombinant purified ATX in vitro, with very few examples studied in more complex systems. Translation of ATX inhibitors from the hands of medicinal chemists to clinical use will require substantially expanded characterization of ATX inhibitors in vivo.
doi:10.1517/13543776.2010.533658
PMCID: PMC3058224  PMID: 21047298
Autotaxin; lysophosphatidic acid; lysophospholipase D; NPP2; cancer
10.  Synthesis of Photoactivatable Analogues of Lysophosphatidic Acid and Covalent Labeling of Plasma Proteins 
The Journal of organic chemistry  2006;71(2):629-635.
Lysophosphatidic acid bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyl-type ester or oleyl-type ether chain and 32P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked LPA 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin.
doi:10.1021/jo052030w
PMCID: PMC2533437  PMID: 16408973
11.  Lysophosphatidic Acid Induces Neointima Formation Through PPARγ Activation 
Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor–like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662 and mimicked by PPARγ agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARα agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARγ activation in vitro and disparate from that of LPA G protein–coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARγ ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARγ is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.
doi:10.1084/jem.20031619
PMCID: PMC2212723  PMID: 15007093
neointima; LPA; PPAR; atherogenesis; lipid mediator

Results 1-11 (11)