Amidine-based Sphingosine Kinase Inhibitors: Optimization of Selectivity, Solubility, and Metabolism

Soon cancer will be the leading cause of death in the United States; unfortunately, the treatments available to patients have severe side effects. Chemotherapies target dividing cells with the goal to kill the rapidly dividing cancer cells before the patient. Researchers have been focusing on determining the cause of the disease and developing better modes of treatment. This has been difficult due to cancer developing resistance to drugs over time and the vast differences among the different types of the disease. One strategy is to develop targeted therapies that inhibit a particular pathway cancer cells rely on to survive. One such pathway is the “Sphingolipid Rheostat” and targeting either the production and/or signaling of sphingosine 1-phosphate (S1P). The research herein describes the design and synthesis of amidine-based inhibitors of the sphingosine kinases (SphKs) to limit the production of S1P since they are the sole producers of the molecule.
Signaling by S1P leads to cell migration, proliferation, and survival. There are two isoforms of the SphKs, SphK1 and SphK2. Of the two, SphK1 has been studied much more since higher concentrations of this enzyme have been found in a variety of cancer cell lines. Our lab has been very successful in selectively targeting SphK1 using amidine-based inhibitors. These molecules lower S1P levels in vitro as well as in vivo. Only recently has SphK2 been more intensely investigated and found to play a more complex role in cell fate. S1P produced by this enzyme, depending on its cellular location, will either lead to cell migration and survival or apoptosis. These contradicting roles of the same endogenous molecule have left researchers needing further understanding of SphK2’s roles in cell fate. The development of potent dual and sub-type selective inhibitors will be useful in elucidating the cellular roles of each of these kinases. Herein is described an attempt to selectively target SphK2 over SphK1 using amidine-based inhibitors. Structure-activity relationship (SAR) studies were performed on these molecules and it was found that these molecules bind to SphK2 at approximately equal strength. These studies lead us to believe that SphK2 is a promiscuous protein that will bind molecules with a variety of functionalization at similar strength.

The Macdonald laboratory has developed some of the most potent SphK1-selective inhibitors in the chemical literature. In order to use these molecules in whole animal studies, they must be long-lived. Unfortunately, these amidine-based inhibitors have a short half-life due to their quick metabolism. The amide linkage of these molecules has been replaced with more metabolically stable 5-membered heterocycles to circumvent this issue. SAR studies were performed on these heterocyclic inhibitors to determine the optimal heterocycle to increase potency, selectivity, and half-life. These heterocyclic inhibitors were found to increase the half-life of the amidine-based SphK inhibitors and are equipotent to their amide counterparts. These potent, longer-lived inhibitors will be useful to scientists to determine the role of the SphKs in cancer cells and other diseases.