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 α-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.

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 Ͼ Ͼ 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.Lysophosphatidic acid (LPA, 2 1-radyl-2-hydroxy-sn-3-glycero phosphate) specifically interacts with several protein targets that regulate physiological and pathophysiological processes (1-3). LPA targets include specific G-protein-coupled receptors (GPCRs) that mediate a wide variety of biological effects, including cell proliferation (4), cell survival (5), cell migration (6), and platelet aggregation (7,8). GPCRs are the largest family of transmembrane receptors and represent targets of many therapeutics (9). Eight LPA-specific mammalian GPCRs, LPA 1-8 , have been identified to date (10 -12). Among the eight LPA receptors, LPA 1 , LPA 2 , and LPA 3 are members of the endothelial differentiation gene (EDG) family (13), and the transmembrane domains of human LPA 1-3 show 81% homology with each other (14). The five other members of the EDG family are specific for the related lysophospholipid sphingosine 1-phosphate (S1P). The structural foundation for LPA selectivity over S1P has been linked to a single amino acid at position 3.29, a conserved glutamine in the LPA-specific and glutamate in the S1P-specific members of the EDG family (14 -16). However, the recently identified non-EDG family LPA receptors, LPA 4 /p2y9 (13), LPA 5 /GPR92 (17, 18), LPA 6 /GPR87 (19), LPA 7 /p2y5 (12), and LPA 8 /p2y10 (10), are more closely related to the purinoreceptor gene cluster and share less than 20% amino acid...

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 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. Lysophosphatidic acid (LPA)2 and sphingosine 1-phosphate (S1P) are structurally related lysophospholipid growth factors that mediate a variety of cellular effects, including regulation of cellular proliferation, survival, migration, and morphology (1-3). LPA has been shown to play an important role in a variety of diseases including ovarian cancer, prostate cancer, breast cancer, and cardiovascular disease (4 -14). Many of the biological effects of LPA are mediated through cell surface receptors of the endothelial differentiation gene (EDG) family of G protein-coupled receptors (GPCRs).The EDG family of GPCRs includes eight closely related genes that show the conserved GPCR topology of an extracellular amino terminus followed by seven ␣-helical transmembrane domains (TMs) (15). Three of these genes (LPA 1-3 ) are cellular receptors for LPA and share 55% overall homology in humans. The other five (S1P 1-5 ) are cellular receptors for S1P and share 50% homology in humans. The two subclusters are 35% homologous with each other. The transmembrane domains of human LPA 1-3 where ligand binding takes place show 81% homology with each other. LPA has also been shown to elicit cellular responses through binding to three non-EDG family GPCRs, p2y9/LPA 4 , GPR92/LPA 5 , and GPR87/LPA 6 , which are more closely related to the purinoreceptor clus...

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC 50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 59-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a targetbinding site for inhibitors of enzymatic activity.

Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonists of the type 2 GPCR for lysophosphatidic acid (LPA2) 2-[4-(1,3-Dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay up to 72 hours reduced mortality of C57BL/6 mice but not in LPA2 KO mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ–H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34+ hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering of the hematopoietic acute radiation syndrome after total body irradiation. DBIBB represents the first drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system.

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.