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.
LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX–LPA signalling axis an emerging target for cancer therapy.
Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA 2 G proteincoupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl) carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo- [de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L55-47924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA 2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro.Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA 1&2 double-knockout mice reconstituted with the LPA 2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA 2 , Na ϩ -H ϩ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA 2 -specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.
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.
A rapid method for quantitative chiral analysis of phthaloylglutamic acid and its dimethyl ester by Cook's kinetic method is demonstrated using electrospray ionization (ESI) and matrix-assisted laser desorption techniques. Transition-metal-bound complex ions containing the chiral phthaloylglutamic acid and its dimethyl ester are generated by ESI mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture. A seven-point calibration curve, derived from the kinetic method, allowed rapid quantitation of the enantiomeric excess of drug mixtures. In this paper, matrix-assisted laser desorption/ionization (MALDI) coupled with the linear ion trap (LIT) technique is evaluated for its applicability as a complementary technique to ESI for chiral discrimination and quantitation.
ATX, a lysophospholipase D that generates LPA, is highly up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancers secrete ATX, and it contributes to their invasive properties. LPA produced by ATX activates LPA1-8 G protein-coupled receptors on the cell surface. Many cancers over-express multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. Therefore, ATX-LPA signaling axis is an emerging target for cancer therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and metastasis in vivo. In this study, we sought to understand the roles of the LPA GPCRs both in the tumor cells and tissues targeted by metastatic cancer cells. Using the in vivo B16 melanoma metastasis model syngeneic to the C57BL/6 mouse strain, we found that the incidence of lung metastasis was greatly reduced in the LPA5KO mice compared to WT, LPA1KO, and LPA2KO mice, respectively. In addition, we found that the selective inhibitor of LPA1&3, Ki16425 also reduced lung metastasis, suggesting that inhibition of LPA5 and LPA1 receptors in the target tissues may prevent cancer metastasis. B16 melanoma cells predominantly express LPA5 receptor. These cells do not invade across a matrigel layer in response to LPA. We found that siRNA knockdown of LPA5 receptors in B16 melanoma cells relieves this inhibition and resulted in an increase in invasion. These results suggest that the LPAR, in particular LPA5 receptors in tumor and stromal cells may play different roles in invasion and metastasis. Citation Format: Gabor Tigyi, Yuko Fujiwara, SueChin Lee, Jianxiong Liu, Renukadevi Patil, Renuka Gupte, Duane D. Miller, Tamas Oravecz, Louisa Balazs. Targeting ATX and LPA receptors melanoma invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2013-3884
Radiomitigators attenuate radiation injury when administered after irradiation. Lysophosphatidic acid (LPA) a lipid mediator rescues apoptotically condemned cells. LPA protects IEC‐6 intestinal epithelial cells in vitro and intestinal crypts in vivo from radiation‐induced apoptosis. Using KO mice and cells we established that the LPA2 receptor is required for radiomitigation. Through its C‐terminal PDZ and LIM‐binding motifs LPA2 forms agonist‐dependent macromolecular signaling complexes. LPA2 interacts with the proapoptotic protein Siva‐1 and targets it for proteasomal degradation. Full activation of the NFkB and ERK1/2 prosurvival pathway requires formation of an LPA2, NHERF2, and TRIP‐6 ternary macromolecular complex. These signals inhibit the mitochondrial apoptosis pathway. Reconstitution of the LPA2 receptor into MEFs derived from LPA1/2 KO mice restores radioprotection. Conditioned medium from irradiated U937 cells induces bystander apoptosis in non‐irradiated IEC‐6 cultures and LPA2‐transfected MEFs. Using in silico drug discovery we found a novel LPA2‐selective agonist GRI977143 that protects cells from radiation‐ and chemotherapy‐induced apoptosis. GRI977143 also increases the survival of mice exposed to lethal levels of radiation. Supported by AI08405.
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