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1.  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 LPA1 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 LPA1 GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA1-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 LPA1 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.
PMCID: PMC3588015  PMID: 23434648
G protein-coupled receptor; GPCR; lysophosphatidic acid; LPA; NMR; GPCR segment model
2.  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.
PMCID: PMC3517046  PMID: 21465666
ATX inhibitors; LPA receptors; 4-substituted benzyl phosphonic acids; 6-substituted naphthalen-2-yl-methyl phosphonic acids; structure-activity relationships
3.  (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.
PMCID: PMC3446790  PMID: 20566326
FTY720; sphingosine 1-phosphate; lysophosphatidic acid; autotaxin; lysophospholipase D; lymphocyte egress; EDG receptor; inhibitor
4.  Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7 
BMC Biochemistry  2011;12:65.
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.
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.
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.
PMCID: PMC3282672  PMID: 22177013

Results 1-4 (4)