Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology.

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

Sterol metabolism controls T(H)17 differentiation by generating endogenous RORγ agonists.

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

Structure-based parallel medicinal chemistry approach to improve metabolic stability of benzopyran COX-2 inhibitors.

Combination of the structure-based design and solid-phase parallel synthesis provided an integrated approach to rapidly develop the structure-activity relationship of benzopyran COX-2 inhibitors. Binding free energies predicted by free energy perturbation theory yielded good agreement with experimental results. New potent and selective lead compounds with improved metabolic properties were identified.

A novel autotaxin inhibitor reduces lysophosphatidic acid levels in plasma and the site of inflammation.

Autotaxin is the enzyme responsible for the production of lysophosphatidic acid (LPA) from lysophosphatidyl choline (LPC), and it is up-regulated in many inflammatory conditions, including but not limited to cancer, arthritis, and multiple sclerosis. LPA signaling causes angiogenesis, mitosis, cell proliferation, and cytokine secretion. Inhibition of autotaxin may have anti-inflammatory properties in a variety of diseases; however, this hypothesis has not been tested pharmacologically because of the lack of potent inhibitors. Here, we report the development of a potent autotaxin inhibitor, PF-8380 [6-(3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one] with an IC(50) of 2.8 nM in isolated enzyme assay and 101 nM in human whole blood. PF-8380 has adequate oral bioavailability and exposures required for in vivo testing of autotaxin inhibition. Autotaxin's role in producing LPA in plasma and at the site of inflammation was tested in a rat air pouch model. The specific inhibitor PF-8380, dosed orally at 30 mg/kg, provided >95% reduction in both plasma and air pouch LPA within 3 h, indicating autotaxin is a major source of LPA during inflammation. At 30 mg/kg PF-8380 reduced inflammatory hyperalgesia with the same efficacy as 30 mg/kg naproxen. Inhibition of plasma autotaxin activity correlated with inhibition of autotaxin at the site of inflammation and in ex vivo whole blood. Furthermore, a close pharmacokinetic/pharmacodynamic relationship was observed, which suggests that LPA is rapidly formed and degraded in vivo. PF-8380 can serve as a tool compound for elucidating LPA's role in inflammation.

Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.

Discovery of (3S,3aR)-2-(3-chloro-4-cyanophenyl)-3-cyclopentyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazole-7-carboxylic acid (PF-3882845), an orally efficacious mineralocorticoid receptor (MR) antagonist for hypertension and nephropathy.

We have discovered a novel class of nonsteroidal pyrazoline antagonists of the mineralocorticoid receptor (MR) that show excellent potency and selectivity against other nuclear receptors. Early analogues were poorly soluble and had a propensity to inhibit the hERG channel. Remarkably, both of these challenges were overcome by incorporation of a single carboxylate moiety. Structural modification of carboxylate-containing lead R-4g with a wide range of substituents at each position of the pyrazoline ring resulted in R-12o, which shows excellent activity against MR and reasonable pharmacokinetic profile. Introduction of conformational restriction led to a novel series characterized by exquisite potency and favorable steroid receptor selectivity and pharmacokinetic profile. Oral dosing of 3S,3aR-27d (PF-3882845) in the Dahl salt sensitive preclinical model of salt-induced hypertension and nephropathy showed blood pressure attenuation significantly greater than that with eplerenone, reduction in urinary albumin, and renal protection. As a result of these findings, 3S,3aR-27d was advanced to clinical studies.