Truc Chi T. Pham

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 Ki 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.

(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.

Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 A (1 A=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

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.

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.

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.

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.

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.

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

BackgroundNucleotide 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.ResultsThe 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.ConclusionsSubstrate 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.

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.