Jeffrey A. Scholten

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.

Synthesis and biological evaluation of substituted benzoxazoles as inhibitors of mPGES-1: use of a conformation-based hypothesis to facilitate compound design.

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is a novel therapeutic target for the treatment of inflammation and pain. In the preceding letter, we detailed the discovery of clinical candidate PF-04693627, a potent mPGES-1 inhibitor possessing a novel benzoxazole structure. While PF-04693627 was undergoing further preclinical profiling, we sought to identify a back-up mPGES-1 inhibitor that differentiated itself from PF-04693627. The design, synthesis, mPGES-1 activity and in vivo PK of a novel set of substituted benzoxazoles are described herein. Also described is a conformation-based hypothesis for mPGES-1 activity based on the preferred conformation of the cyclohexane ring within this class of inhibitors.

Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl)-2H-tetrazol-5-yl)-2-methylpyrimidine-4-carboxamide. A Highly Selective and Orally Bioavailable Matrix Metalloproteinase-13 Inhibitor for the Potential Treatment of Osteoarthritis

Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.

Discovery of a Potent and Selective Sphingosine Kinase 1 Inhibitor through the Molecular Combination of Chemotype-Distinct Screening Hits

Sphingosine kinase (SphK) is the major source of the lipid mediator and G protein-coupled receptor agonist sphingosine-1-phosphate (S1P). S1P promotes cell growth, survival, and migration and is a key regulator of lymphocyte trafficking. Inhibition of S1P signaling has been proposed as a strategy for treatment of inflammatory diseases and cancer. Two different formats of an enzyme-based high-throughput screen yielded two attractive chemotypes capable of inhibiting S1P formation in cells. The molecular combination of these screening hits led to compound 22a (PF-543) with 2 orders of magnitude improved potency. Compound 22a inhibited SphK1 with an IC50 of 2 nM and was more than 100-fold selective for SphK1 over the SphK2 isoform. Through the modification of tail-region substituents, the specificity of inhibition for SphK1 and SphK2 could be modulated, yielding SphK1-selective, potent SphK1/2 dual, or SphK2-preferential inhibitors.