Ki16425, a Subtype-Selective Antagonist for EDG-Family Lysophosphatidic Acid Receptors

Abstract

Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA 1 , LPA 2 , and LPA 3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA 4 /GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA 1 , LPA 2 , or LPA 3 , we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhibited the LPA-induced response in the decreasing order of LPA 1 Ն LPA 3 Ͼ Ͼ LPA 2 , whereas DGPP 8:0 preferentially inhibited the LPA 3 -induced actions. Ki16425 inhibited LPA-induced guanosine 5Ј-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced longterm responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA 1 and LPA 3 ; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.Lysophosphatidic acid (LPA) has been shown to elicit diverse biological actions, including Ca 2ϩ mobilization, change in cAMP accumulation, change in cell shape and motility in association with actin rearrangement, and proliferation in a variety of cell types (Moolenaar, 1999;Contos et al., 2000;Ye et al., 2002). Extracellular LPA has also been shown to be involved in certain diseases, such as atherosclerosis and cancer (Xu et al., 1995(Xu et al., , 2001Siess et al., 1999;Maschberger et al., 2000). LPA was first thought to be released from activated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called autotaxin (Sano et al., 2002;Tokumura et al., 2002;Umezu-Goto et al., 2002). The concentration of plasma LPA is about 100 nM, and its serum concentration can be as high as 5 M (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., This work was supported in part by a research grant grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by research gr...

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation

“…Likewise ketoconazole (Tocris, Bristol, UK) was prepared as a 5mM stock in ethanol and stored at -20°C. The LPA1/3 receptor antagonist, Ki16425 [29], was a very generous gift from the Kirin Brewery Company Ltd. (Tokyo, Japan) and was reconstituted at 10mM in DMSO. The preferential LPA3 receptor antagonist, diacylglycerol pyrophosphate [30] as the dioctanoyl form (DGPP 8:0, INstruchemie BV, Zwet 26, The Netherlands), was reconstituted in chloroform to a stock concentration of 25mg/ml and stored at -20°C.…”

Cytoskeletal reorganisation, 1α,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation

“…We recently discovered that the principal serum factor co-operating with D3 in promoting human osteoblast maturation is lysophosphatidic acid (Gidley et al, 2006); in isolation D3 was unable to induce osteoblast differentiation but when used in conjunction with human serum a stark, synergistic maturation response occurred. We subsequently found that osteoblast maturation in response to D3 and serum could be blocked by Ki16425, a lysophosphatidic acid (LPA) receptor antagonist (Ohta et al, 2003). Furthermore the application of commercially available LPA in combination with D3 to MG63 osteoblasts led to striking, synergistic increases in both osteocalcin and alkaline phosphatase (ALP), proteins expressed by the differentiated phenotype (Gidley et al, 2006).…”

α‐Substituted Phosphonate Analogues of Lysophosphatidic Acid (LPA) Selectively Inhibit Production and Action of LPA

“…Several groups have reported the characterization of the LPA agonists and antagonists. [20][21][22][23][24][25][26][27][28][29] Appropriately validated compounds are essential to advance in vivo studies, particularly in view of potential off-target effects. The development of more selective, more stable, more potent, and more druglike agonists and antagonists is eagerly awaited, and is the bottleneck in therapeutic exploration.…”