AS2717638

Analgesic effects of novel lysophosphatidic acid receptor 5 antagonist AS2717638 in rodents

Lysophosphatidic acid (LPA) is a bioactive lipid that acts via at least six G protein-coupled receptors, LPA receptors 1-6 (LPA1-6), for various physiological functions. We examined (1) whether LPA5 is involved in pain signaling in the spinal cord; and (2) the pharmacological effects of a novel LPA5 antagonist on intrathecal prostaglandin (PG)- and (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced allodynia, and neuropathic and inflammatory pain in rodents. Intrathecal injection of a selective LPA5 agonist, geranylgeranyl diphosphate, and a non-selective agonist, LPA, induced allodynia in wild type, but not in LPA5 knockout mice. These novel results suggest that LPA5 is important for pain signal transmission in the spinal cord. AS2717638 (6,7-dimethoxy-2-(5-methyl-1,2-benzoxazol-3-yl)-4-(piperidin-1-ylcarbonyl)isoquinolin-1(2H)-one) bound to the LPA-binding site on LPA5 and selectively inhibited LPA-induced cyclic adenosine monophosphate accumulation in human LPA5-but not LPA1-, 2-, or 3-expressing cells. Further, oral administration of AS2717638 inhibited LPA5 agonist-induced allodynia in mice. AS2717638 also significantly improved PGE2-, PGF2α-, and AMPA-induced allodynia, while both pregabalin and duloxetine alleviated only PGE2-induced allodynia in mice. Similarly, AS2717638 significantly ameliorated static mechanical allodynia and thermal hyperalgesia in rat models of chronic constriction injury (CCI)-induced neuropathic pain. AS2717638 also showed analgesic effects in a rat model of inflammatory pain. These findings suggest that LPA5 antagonists elicit broad analgesic effects against both neuropathic and inflammatory pain. Accordingly, pharmacological LPA5 antagonists are attractive development candidates for potential novel pain therapies.

Isoquinolone derivatives as lysophosphatidic acid receptor 5 (LPA5) antagonists: Investigation of structure-activity relationships, ADME properties and analgesic effects

Blockade of lysophosphatidic acid receptor 5 (LPA5) by a recently reported antagonist AS2717638 (2) attenuated inflammatory and neuropathic pains, although it showed moderate in vivo efficacy and its structure-activity relationships and the ADME properties are little studied. We therefore designed and synthesized a series of isoquinolone derivatives and evaluated their potency in LPA5 calcium mobilization and cAMP assays. Our results show that substituted phenyl groups or bicyclic aromatic rings such as benzothiophenes or benzofurans are tolerated at the 2-position, 4-substituted piperidines are favored at the 4-position, and methoxy groups at the 6- and 7-positions are essential for activity. Compounds 65 and 66 showed comparable in vitro potency, excellent selectivity against LPA1-LPA4 and >50 other GPCRs, moderate metabolic stability, and high aqueous solubility and brain permeability. Both 65 and 66 significantly attenuated nociceptive hypersensitivity at lower doses than 2 and had longer-lasting effects in an inflammatory pain model, and 66 also dose-dependently reduced mechanical allodynia in the chronic constriction injury model and opioid-induced hyperalgesia at doses that had no effect on the locomotion in rats. These results suggest that these isoquinolone derivatives as LPA5 antagonists are of promise as potential analgesics.

Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

Increasing evidence suggests that systemic inflammation triggers a neuroinflammatory response that involves sustained microglia activation. This response has deleterious consequences on memory and learning capability in experimental animal models and in patients. However, the mechanisms connecting systemic inflammation and microglia activation remain poorly understood. Here, we identify the autotaxin (ATX)/lysophosphatidic acid (LPA)/LPA-receptor axis as a potential pharmacological target to modulate the LPS-mediated neuroinflammatory response in vitro (the murine BV-2 microglia cell line) and in vivo (C57BL/6J mice receiving a single i.p. LPS injection). In LPS-stimulated (20 ng/mL) BV-2 cells, we observed increased phosphorylation of transcription factors (STAT1, p65, and c-Jun) that are known to induce a proinflammatory microglia phenotype. LPS upregulated ATX, TLR4, and COX2 expression, amplified NO production, increased neurotoxicity of microglia conditioned medium, and augmented cyto-/chemokine concentrations in the cellular supernatants. PF8380 (a type I ATX inhibitor, used at 10 and 1 ?M) and AS2717638 (an LPA5 antagonist, used at 1 and 0.1 ?M) attenuated these proinflammatory responses, at non-toxic concentrations, in BV-2 cells. In vivo, we demonstrate accumulation of PF8380 in the mouse brain and an accompanying decrease in LPA concentrations. In vivo, co-injection of LPS (5 mg/kg body weight) and PF8380 (30 mg/kg body weight), or LPS/AS2717638 (10 mg/kg body weight), significantly attenuated LPS-induced iNOS, TNFα, IL-1β, IL-6, and CXCL2 mRNA expression in the mouse brain. On the protein level, PF8380 and AS2717638 significantly reduced TLR4, Iba1, GFAP and COX2 expression, as compared to LPS-only injected animals. In terms of the communication between systemic inflammation and neuroinflammation, both inhibitors significantly attenuated LPS-mediated systemic TNFα and IL-6 synthesis, while IL-1β was only reduced by PF8380. Inhibition of ATX and LPA5 may thus provide an opportunity to protect the brain from the toxic effects that are provoked by systemic endotoxemia.

Small-Molecule Lysophosphatidic Acid Receptor 5 (LPAR5) Antagonists: Versatile Pharmacological Tools to Regulate Inflammatory Signaling in BV-2 Microglia Cells

Lysophosphatidic acid (LPA) species in the extracellular environment induce downstream signaling via six different G protein-coupled receptors (LPAR1-6). These signaling cascades are essential for normal brain development and function of the nervous system. However, in response to acute or chronic central nervous system (CNS) damage, LPA levels increase and aberrant signaling events can counteract brain function. Under neuro-inflammatory conditions signaling along the LPA/LPAR5 axis induces a potentially neurotoxic microglia phenotype indicating the need for new pharmacological intervention strategies. Therefore, we compared the effects of two novel small-molecule LPAR5 antagonists on LPA-induced polarization parameters of the BV-2 microglia cell line. AS2717638 is a selective piperidine-based LPAR5 antagonist (IC₅₀ 0.038 μM) while compound 3 is a diphenylpyrazole derivative with an IC₅₀ concentration of 0.7 μM in BV-2 cells. Both antagonists compromised cell viability, however, at concentrations above their IC₅₀ concentrations. Both inhibitors blunted LPA-induced phosphorylation of STAT1 and STAT3, p65, and c-Jun and consequently reduced the secretion of pro-inflammatory cyto-/chemokines (IL-6, TNFα, IL-1β, CXCL10, CXCL2, and CCL5) at non-toxic concentrations. Both compounds modulated the expression of intracellular (COX-2 and Arg1) and plasma membrane-located (CD40, CD86, and CD206) polarization markers yet only AS2717638 attenuated the neurotoxic potential of LPA-activated BV-2 cell-conditioned medium towards CATH.a neurons. Our findings from the present in vitro study suggest that the two LPAR5 antagonists represent valuable pharmacological tools to interfere with LPA-induced pro-inflammatory signaling cascades in microglia.