Assessing differences in toxicity and teratogenicity of three phthalates, Diethyl phthalate, Di-n-propyl phthalate, and Di-n-butyl phthalate, using Xenopus laevis embryos.

Phthalates, compounds used to add flexibility to plastics, are ubiquitous in the environment. In particular, the diethyl (DEP), di-n-propyl (DnPP), and di-n-butyl (DBP) phthalates were found to exert detrimental effects in both mammalian and non-mammalian studies, with toxic effects varying according to alkyl chain length. Embryos of Xenopus laevis, the African clawed frog, have been used to assess toxicity and teratogenicity of several compounds and serves as a model for assessing adverse and teratogenic effects of ortho-phthalate esters. The purpose of this study was to develop a model for comparison of developmentally toxic effects of ortho-phthalate esters using Xenopus embryos. In this study developing Xenopus laevis embryos were exposed to increasing concentrations of DEP, DnPP, and DBP using the 96-h Frog Embryo Teratogenesis Assay-Xenopus (FETAX), with 96-h lethal concentrations, effective concentrations to induce malformations, teratogenic indices, and concentrations to inhibit growth determined. DEP, DnPP, and DBP showed enhanced toxicity with increasing ester length. Developing Xenopus laevis exposed to DEP, DnPP, and DBP showed similar malformations that also occurred at lower concentrations with increasing alkyl chain length. Teratogenic risk did not change markedly with alkyl chain length, with data showing only DBP to be teratogenic.

Dual activity lysophosphatidic acid receptor pan-antagonist/autotaxin inhibitor reduces breast cancer cell migration in vitro and causes tumor regression in vivo.

Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric alpha-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein-coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.

Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation.

Lysophosphatidic acid (LPA) is a ligand for LPA(1-3) of the endothelial differentiation gene family G-protein-coupled receptors, and LPA(4-8) is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA(5) is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA(5) residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA(5) were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA(5) using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates >> N-arachidonoylglycine. These results confirm LPA(5) to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA(5) agonists. A pharmacophore model of LPA(5) binding requirements was developed for in silico screening, which identified two non-lipid LPA(5) antagonists. Because LPA(5) transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA(5) antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA(5) may provide a basis for future anti-thrombotic therapies.

Identification of non-lipid LPA3 antagonists by virtual screening.

In the present study, we utilized virtual screening to identify LPA(3) antagonists. We have developed a three-point structure-based pharmacophore model based on known LPA(3) antagonists. This model was used to mine the NCI database. Docking, pharmacophore development, and database mining produced new, non-lipid leads. Experimental testing of seven computationally selected pharmacophore hits produced one potentiator and three antagonists, one of which displays both LPA(3) selectivity and nanomolar potency. Similarity searching in the ChemBridge database using the most promising lead as the search target produced four additional LPA(3) antagonists and a potent dual LPA(1&2) antagonist.

Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors.

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.