Highly Potent Non-Carboxylic Acid Autotaxin Inhibitors Reduce Melanoma Metastasis and Chemotherapeutic Resistance of Breast Cancer Stem Cells.

Autotaxin (ATX, aka. ENPP2) is the main source of the lipid mediator lysophosphatidic acid (LPA) in biological fluids. This study reports on inhibitors of ATX derived by lead optimization of the benzene-sulfonamide in silico hit compound 3. The new analogues provide a comprehensive structure-activity relationship of the benzene-sulfonamide scaffold that yielded a series of highly potent ATX inhibitors. The three most potent analogues (3a, IC50 ∼ 32 nM; 3b, IC50 ∼ 9 nM; and 14, IC50 ∼ 35 nM) inhibit ATX-dependent invasion of A2058 human melanoma cells in vitro. Two of the most potent compounds, 3b and 3f (IC50 ∼ 84 nM), lack inhibitory action on ENPP6 and ENPP7 but possess weak antagonist action specific to the LPA1 G protein-coupled receptor. In particular, compound 3b potently reduced in vitro chemotherapeutic resistance of 4T1 breast cancer stem-like cells to paclitaxel and significantly reduced B16 melanoma metastasis in vivo.

Targeting the hydrophobic pocket of autotaxin with virtual screening of inhibitors identifies a common aromatic sulfonamide structural motif.

Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small-molecule inhibitors is a promising strategy for blocking the ATX-lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high-throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand-based strategy and its application in virtual screening, which yielded novel high-potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure-activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.

Structural Characterization of an LPA1 Second Extracellular Loop Mimetic with a Self-Assembling Coiled-Coil Folding Constraint.

G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane α-helical domains. The EL of the LPA(1) receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA(1) GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA(1)-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA(1) receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.

Structure and catalytic function of sphingosine kinases: analysis by site-directed mutagenesis and enzyme kinetics.

Sphingosine kinases 1 and 2 (SK1 and SK2) generate the bioactive lipid mediator sphingosine 1-phosphate and as such play a significant role in cell fate and in human health and disease. Despite significant interest in and examination of the role played by SK enzymes in disease, comparatively little is currently known about the three-dimensional structure and catalytic mechanisms of these enzymes. To date, limited numbers of studies have used site directed mutagenesis and activity determinations to examine the roles of individual SK residues in substrate, calmodulin, and membrane binding, as well as activation via phosphorylation. Assays are currently available that allow for both single and bisubstrate kinetic analysis of mutant proteins that show normal, lowered and enhanced activity as compared to wild type controls. Additional studies will be required to build on this foundation to completely understand SK mediated substrate binding and phosphoryl group transfer. A deeper understanding of the SK catalytic mechanism, as well as SK interactions with potential small molecule inhibitors will be invaluable to the future design and identification of SK activity modulators as research tools and potential therapeutics. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7.

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.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity.

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to 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. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4-tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzylphosphonic acid and 6-substituted naphthalen-2-ylmethylphosphonic acid analogues block ATX activity with K(i) values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed-mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose-dependent manner. The average terminal half-life for compound 22 is 10±5.4 h and it causes a long-lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The 4-substituted benzylphosphonic acids and 6-substituted naphthalen-2-ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX-LPA-LPAR axis both in vitro and in vivo.

Synthesis and pharmacological evaluation of the stereoisomers of 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 five-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 LPA(5) 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 LPA(5) 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.

(S)-FTY720-vinylphosphonate, an analogue of the immunosuppressive agent FTY720, is a pan-antagonist of sphingosine 1-phosphate GPCR signaling and inhibits autotaxin activity.

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 S1P(1) receptor internalization and targeting to the proteasomal degradative pathway, and thus functions as an antagonist of S1P(1) by depleting the functional S1P(1) 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 S1P(1) (EC(50) 20+/-3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P(1,3,4) (K(i) 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P(2), and S1P(5). Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K(i) 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 S1P(2) transcripts and traces of S1P(5). (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.

Pharmacophore development and application toward the identification of novel, small-molecule autotaxin inhibitors.

Autotaxin (ATX) is a secreted glycoprotein with lysophospholipase D (LPLD) activity that generates the bioactive lipid lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been linked to the promotion and progression of cancer as well as cardiovascular disease and obesity. Despite the fact that ATX inhibitors have the potential to be useful chemotherapeutics for multiple indications, few examples of potent ATX inhibitors are described in the current literature. Here we describe the development of pharmacophore models for the inhibition of ATX by nonlipids and apply these tools to the discovery of additional ATX inhibitors using the NCI open chemical repository database. From this database of > 250,000 compounds, 168 candidate inhibitors were identified. Of these candidates, 106 were available for testing and 33 were identified as active (those that inhibited ATX activity by > or =50% at a single 10 microM concentration), a 31% hit rate. Five of these compounds had IC(50) < 1.5 microM and the most potent compound possessed a K(i) of 271 nM.

Characterization of non-lipid autotaxin inhibitors.

Autotaxin (ATX) is a member of the ecto-nucleotide pyrophosphatase/phosphodiesterase (NPP) family and is a lysophospholipase D that cleaves the choline headgroup from lysophosphatidylcholine to generate the bioactive lipid lysophosphatidic acid (LPA). Enhanced expression of ATX and specific receptors for LPA in numerous cancer cell types has created an interest in studying ATX as a potential chemotherapeutic target. Likewise, ATX has been linked to several additional human diseases including multiple sclerosis, diabetes, obesity, neuropathic pain, and Alzheimer's disease. ATX inhibitors reported to date consist of metal ion chelators, lipid-like product analogs, and non-lipid small molecules. In the current research, we examined the pharmacology of the best of our previously reported non-lipid small molecule inhibitors. Here, these six inhibitors were studied utilizing the synthetic fluorescent lysophospholipid substrate FS-3, the nucleotide substrate pNP-TMP and the endogenous substrate LPC (16:0). All six compounds inhibited FS-3 hydrolysis >or=50%, whereas only three inhibited the hydrolysis of pNP-TMP to this degree. None of the six compounds blocked LPC 16:0 hydrolysis within the desired 50% inhibition range. The most potent analog (5, H2L 7905958) displayed an IC(50) of 1.6microM (K(i)=1.9microM, competitive inhibition) with respect to ATX-mediated FS-3 hydrolysis and an IC(50) of 1.2microM (K(i)=K(i)(')=6.5microM, non-competitive inhibition) against ATX-mediated pNP-TMP hydrolysis. All six inhibitors were specific for ATX as they were without affect on two additional lipid preferring NPP isoforms.

Molecular recognition in the sphingosine 1-phosphate receptor family.

Computational modeling and its application in ligand screening and ligand receptor interaction studies play important roles in structure-based drug design. A series of sphingosine 1-phosphate (S1P) receptor ligands with varying potencies and receptor selectivities were docked into homology models of the S1P(1-5) receptors. These studies provided molecular insights into pharmacological trends both across the receptor family as well as at single receptors. This study identifies ligand recognition features that generalize across the S1P receptor family, features unique to the S1P(4) and S1P(5) receptors, and suggests significant structural differences of the S1P(2) receptor. Docking results reveal a previously unknown sulfur-aromatic interaction between the S1P(4) C5.44 sulfur atom and the phenyl ring of benzimidazole as well as pi-pi interaction between F3.33 of S1P(1,4,5) and aromatic ligands. The findings not only confirm the importance of a cation-pi interaction between W4.64 and the ammonium of S1P at S1P(4) but also predict the same interaction at S1P(5). S1P receptor models are validated for pharmacophore development including database mining and new ligand discovery and serve as tools for ligand optimization to improve potency and selectivity.

Virtual screening approaches for the identification of non-lipid autotaxin inhibitors.

Autotaxin (ATX, NPP-2) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA), a mitogenic cell survival factor that stimulates cell motility. The high expression of both ATX and receptors for LPA in numerous tumor cell types has produced substantial interest in exploring ATX as an anticancer chemotherapeutic target. ATX inhibitors reported to date are analogs of LPA, a phospholipid, and are more hydrophobic than is typical of orally bioavailable drugs. This study applied both structure-based and ligand-based virtual screening techniques with hit rates of 20% and 37%, respectively, to identify a promising set of non-lipid, drug-like ATX inhibitors. Structure-based virtual screening necessitated development of a homology model of the ATX catalytic domain due to the lack of structural information on any mammalian NPP family member. This model provided insight into the interactions necessary for ATX inhibition, and produced a suitably diverse training set for the development and application of binary QSAR models for virtual screening. The most efficacious compound identified in this study was able to completely inhibit ATX-catalyzed hydrolysis of 1 microM FS-3 (a synthetic, fluorescent LPC analog) at a 10 microM concentration.

Peptide design and structural characterization of a GPCR loop mimetic.

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.