Daniel L. Baker

Lysophosphatidic Acid Induces Neointima Formation Through PPARγ Activation

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor–like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662 and mimicked by PPARγ agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARα agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARγ activation in vitro and disparate from that of LPA G protein–coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARγ ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARγ is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.

Identification of Edg1 receptor residues that recognize sphingosine 1-Phosphate

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg120 and Arg292 ion pair with the phosphate, whereas the acidic Glu121 residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [35S]GTPγS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Subtype-Selective Antagonists of Lysophosphatidic Acid Receptors Inhibit Platelet Activation Triggered by the Lipid Core of Atherosclerotic Plaques

Background—Lysophosphatidic acid (LPA) is a platelet-activating component of mildly oxidized LDL (mox-LDL) and lipids isolated from human atherosclerotic plaques. Specific antagonists of platelet LPA receptors could be useful inhibitors of thrombus formation in patients with cardiovascular disease. Methods and Results—Short-chain analogs of phosphatidic acid (PA) were examined for their effect on two initial platelet responses, platelet shape change and Ca2+ mobilization. Dioctylglycerol pyrophosphate [DGPP(8:0)] and dioctylphosphatidic acid [PA(8:0)], recently described selective antagonists of the LPA1 and LPA3 receptors, inhibited platelet activation evoked by LPA but not by other platelet stimuli. DGPP(8:0) was more potent than PA(8:0). DGPP(8:0) also inhibited platelet shape change induced by mox-LDL and lipid extracts from human atherosclerotic plaques. Notably, we demonstrate for the first time that the lipid-rich core isolated from soft plaques was able to directly induce shape change. This effect was completely abrogated by prior incubation of platelets with DGPP(8:0). Moreover, coapplication of the lipid-rich core or LPA together with subthreshold concentrations of ADP or epinephrine synergistically induced platelet aggregation; this effect was inhibited by DGPP(8:0). Analysis by liquid chromatography-mass spectrometry revealed the presence of LPA alkyl- and acyl-molecular species with high platelet-activating potency (16:0-alkyl-LPA, 20:4-acyl-LPA). Conclusions—LPA molecules present in the core region of atherosclerotic plaques trigger rapid platelet activation through the stimulation of LPA1 and LPA3 receptors. Antagonists of platelet LPA receptors might provide a new strategy to prevent thrombus formation in patients with cardiovascular diseases.

Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

Molecular basis for lysophosphatidic acid receptor antagonist selectivity

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.

Phospholipase D2-dependent Inhibition of the Nuclear Hormone Receptor PPARγ by Cyclic Phosphatidic Acid

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of natures simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

Different Residues Mediate Recognition of 1-O-Oleyllysophosphatidic Acid and Rosiglitazone in the Ligand Binding Domain of Peroxisome Proliferator-activated Receptor γ

Here we showed that a naturally occurring ether analog of lysophosphatidic acid, 1-O-octadecenyl-2-hydroxy-sn-glycero-3-phosphate (AGP), is a high affinity partial agonist of the peroxisome proliferator-activated receptor γ (PPARγ). Binding studies using the PPARγ ligand binding domain showed that [32P]AGP and [3H]rosiglitazone (Rosi) both specifically bind to PPARγ and compete with each other. [32P]AGP bound PPARγ with an affinity (Kd(app) 60 nm) similar to that of Rosi. However, AGP displaced ∼40% of bound [3H]Rosi even when applied at a 2000-fold excess. Activation of PPARγ reporter gene expression by AGP and Rosi showed similar potency, yet AGP-mediated activation was ∼40% that of Rosi. A complex between AGP and PPARγ was generated using molecular modeling based on a PPARγ crystal structure. AGP-interacting residues were compared with Rosi-interacting residues identified within the Rosi-PPARγ co-crystal complex. These comparisons showed that the two ligands occupy partially overlapping positions but make different hydrogen bonding and ion pairing interactions. Site-specific mutants of PPARγ were prepared to examine individual ligand binding. H323A and H449A mutants showed reduced binding of Rosi but maintained binding of AGP. In contrast, the R288A showed reduced AGP binding but maintained Rosi binding. Finally, alanine replacement of Tyr-473 abolished binding and activation by Rosi and AGP. These observations indicate that the endogenous lipid mediator AGP is a high affinity ligand of PPARγ but that it binds via interactions distinct from those involved in Rosi binding. These distinct interactions are likely responsible for the partial PPARγ agonism of AGP.

Ustekinumab: Lessons Learned from Targeting Interleukin‐12/23p40 in Immune‐Mediated Diseases

Interleukin (IL)‐12 and IL‐23 are related cytokines that have been implicated in the pathogenesis of several immune‐mediated disorders. IL‐12 and IL‐23 are heterodimers made up of a common p40 subunit complexed to unique p35 (IL‐12) or p19 (IL‐23) subunits. Ustekinumab is a human monoclonal antibody that specifically binds the p40 subunit of IL‐12/23. Ustekinumab prevents IL‐12 and IL‐23 from binding their cell surface receptor complexes, thereby blocking the T helper (Th) 1 (IL‐12) and Th17 (IL‐23) inflammatory pathways. Here, we discuss the preclinical and human translational data supporting a role for IL‐12/23 in the pathogenesis of immune‐mediated disorders, and how that rationale was challenged in the clinic during the course of the ustekinumab development program in several indications including psoriasis, psoriatic arthritis, Crohns disease, and multiple sclerosis. We review the key efficacy and safety data in each of these immune‐mediated diseases and compare and contrast the safety lessons learned from IL‐12/23 genetically‐deficient mice and humans in context of the overall clinical trial experience with ustekinumab.

Gold Nanorods Carrying Paclitaxel for Photothermal-Chemotherapy of Cancer

Nanotechnology-based photothermal therapy has emerged as a promising treatment for cancer during the past decade. However, heterogeneous laser heating and limited light penetration can lead to incomplete tumor cell eradication. Here, we developed a method to overcome these limitations by combining chemotherapy with photothermal therapy using paclitaxel-loaded gold nanorods. Paclitaxel was loaded to gold nanorods with high density (2.0 × 10(4) paclitaxel per gold nanorod) via nonspecific adsorption, followed by stabilization with poly(ethylene glycol) linked with 11-mercaptoundecanoic acid. Paclitaxel was entrapped in the hydrophobic pocket of the polymeric monolayer on the surface of gold nanorods, which allows direct cellular delivery of the hydrophobic drugs via the lipophilic plasma membrane. Highly efficient drug release was demonstrated in a cell membrane mimicking two-phase solution. Combined photothermal therapy and chemotherapy with the paclitaxel-loaded gold nanorods was shown to be highly effective in killing head and neck cancer cells and lung cancer cells, superior to photothermal therapy or chemotherapy alone due to a synergistic effect. The paclitaxel-gold nanorod enabled photothermal chemotherapy has the potential of preventing tumor reoccurrence and metastasis and may have an important impact on the treatment of head and neck cancer and other malignancies in the clinic.

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to 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. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with Ki values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b, the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.