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.

Long non-coding RNA RAMS11 promotes metastatic colorectal cancer progression.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.

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.

Synthesis of potent leukotriene A(4) hydrolase inhibitors. Identification of 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid.

Leukotriene B(4) (LTB(4)) is a potent, proinflammatory mediator involved in the pathogenesis of a number of diseases including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. The enzyme LTA(4) hydrolase represents an attractive target for pharmacological intervention in these disease states, since the action of this enzyme is the rate-limiting step in the production of LTB(4). Our previous efforts focused on the exploration of a series of analogues related to screening hit SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) and resulted in the identification of potent, orally active inhibitors such as 2. Additional structure-activity relationship studies around this structural class resulted in the identification of a series of alpha-, beta-, and gamma-amino acid analogues that are potent inhibitors of the LTA(4) hydrolase enzyme and demonstrated good oral activity in a mouse ex vivo whole blood LTB(4) production assay. The efforts leading to the identification of clinical candidate SC-57461A (8d, 3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid) are described.

High-throughput screening assay for sphingosine kinase inhibitors in whole blood using RapidFire® mass spectrometry.

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire(®) mass spectrometry system.

Synthesis of imidazopyridines and purines as potent inhibitors of leukotriene A4 hydrolase.

The synthesis and biological evaluation of a series of heterocyclic analogues of the previously reported LTA(4) hydrolase inhibitor 1b are described. Imidazopyridine and purine analogues are specifically highlighted with several demonstrating excellent potency in our in vitro assays, as well as good oral activity in a mouse ex vivo assay.

Pyrrolidine and piperidine analogues of SC-57461A as potent, orally active inhibitors of leukotriene A(4) hydrolase.

The synthesis and biological evaluation of a series of functionalized pyrrolidine- and piperidine-containing analogues of our lead LTA(4) hydrolase inhibitor, SC-57461A, is described. A number of compounds showed excellent potency in our in vitro screens and several demonstrated good oral activity in a mouse ex vivo assay. These efforts led to the identification of SC-56938 (14) as a potent, orally active inhibitor of LTA(4) hydrolase.