Dr. Grattan's research interest involves the synthesis and development of improved enzyme inhibitors for cancer research.

SC-INBRE Research

Design of Novel Inhibitors of Human Sphingosine Kinases 1 and 2

Sphingolipids are a family of compounds that, in addition to being structural constituents of cell membranes, play key roles as signaling molecules. In particular two of these sphingolipid metabolites, ceramide and sphingosine 1-phosphate (S1P), have recently received considerable attention as integral mediators of cell death and survival. The regulator of the ceramide/S1P equilibrium is sphingosine kinase-1 which phosphorylates sphingosine to form S1P. Sphingosine kinase-1 has been identified as an oncogene and is, therefore, of considerable interest in the treatment of cancer. To this end, a number of novel inhibitors of sphingosine kinase-1 have recently been identified and evaluated by Smith et al. These inhibitors show promising chemotherapeutic results in vitro, but are simply a starting point in the eventual optimization of in vivo activity. Work has recently begun, in collaboration with Smith's lab, on developing a synthetic route to produce one of these inhibitor compounds as a template molecule. The design and ultimate completion of this synthetic scheme will allow for numerous derivatives to be synthesized quickly and concisely in effort to evaluate and increase the therapeutic effect of sphingosine kinase-1 inhibition. Considered the central molecule in sphingolipid metabolism, ceramide controls the programmed cell death response to a wide array of anticancer treatments through de-novo synthesis and/or the hydrolysis of sphingomyelin.¹ Typical treatments, such as chemotherapy and radiation, elicit an increase in the intracellular ceramide level occurring before the first biochemical signs of apoptosis.^1a The addition of extracellular short-chain ceramides to cell culture results in apoptosis for a number of cancer cell lines.^1a In contrast to ceramide, S1P promotes cell survival in response to the apoptotic stresses that typically induce ceramide generation in vitro, ex vivo, and in vivo.² The opposing directions of ceramide-mediated and S1P-mediated signaling led to the concept of a ceramide/S1P biostat, and the assumption that the ratio between these two lipids ultimately determines the fate of the cell.^2a Both of these metabolites, ceramide and sphingosine, have been associated with apoptosis and growth arrest in response to multiple stress signals, while simultaneously increasing sphingosine kinase activity as a prosurvival response. This increase in S1P levels may also be regulated through enhanced S1P phosphatase and S1P lyase activities as shown in Figure 1.

Since the discovery that S1P regulates cell growth³ and suppresses apoptosis⁴, there have been numerous important physiological and pathophysiological processes reported to be managed by S1P in higher organisms. To further highlight its importance as a signaling molecule, S1P has also been shown to regulate biological responses in lower organisms. The activity of sphingosine kinase, which exclusively catalyzes the ATP-dependent phosphorylation of sphingosine, is stimulated by many pathways. Sphingosine kinases, SphK1 and SphK2, have been recently found to be expressed in humans, mice, yeast and plants with homologues in worms and flies and each with five conserved domains (Figure 2). The distinctive catalytic domain contained with C1-C3, and the ATP binding site being identified within C2. These two isoforms do exhibit differences in terms of the presence of transmembrane (TM) regions, SphK1 has none while SphK2 has four TM regions. The sequence differences between these two proteins have led researchers to conclude that they are the result of separate gene-duplication events. SphK1 and SphK2 have been cloned and characterized in mammals.⁵ Diverse external stimuli, particularly growth and survival factors, stimulate SphK1, generating S1P that has been implicated in their mitogenic and anti-apoptotic effects.^6,7 In contrast to SphK1, rather than promoting growth and survival, overexpression of SphK2 suppressed growth and enhanced apoptosis,^8,9 implying that they have distinct physiological functions, likely due to their different subcellular localizations. Northern blot analysis has shown that SphKs have different tissue distributions ¹⁰: SphK1 expression is highest in lung and spleen, while SphK2 is more abundant in liver and heart. SphK1 has also been identified as the key enzyme in modulating ceramide and sphingosine 1-phosphate levels, as shown in Figure 1, and is therefore the focal point of our project.

During a recent screening of a synthetic compounds library using an assay designed for recombinant human sphingosine kinase activity testing, Smith et al. identified a new panel of SphK inhibitors.^11a These compounds, I-IV, as well as one synthetic derivative Compound V, (Figure 3) have been found to possess selectivity towards SphK in comparison with other lipid and protein kinases and are not competitive inhibitors of the ATP-binding site of SphK. These nonlipid-based compounds demonstrated activity at sub-micromolar concentrations, making them more potent than any previously reported SphK inhibitor. The inhibitors are also antiproliferative toward a panel of human tumor cell lines with simultaneous induction of apoptosis. The compounds inhibit S1P formation in intact cells and maintain activity toward cells that express the drug transport proteins P-glycoprotein (Pgp) or MRP1. Overall, a series of potent, structurally novel lead inhibitors of SphK were identified and due to the antiproliferative potential as drugs a synthetic scheme to design and subsequently evaluate these derivatives was required.

Compounds I, II, III, and IV (at 5 µg/ml) inhibited SphK activity by 99, 85, 99, and 89%, respectively, and serve as template structures for nonlipid inhibitors.^11a Upon evaluation of these structures, the chemotype of compound IV was the most easily synthesized of the four classes of compounds to quickly provide a suitable test derivative. This bioisosteric replacement was expected to produce a compound with comparable activity and resulted in compound V.^11a   

A common problem with known kinase inhibitors is their tendency toward nonselectivity because the majority of these inhibitors interact with the highly conserved nucleotide binding site. Therefore, we performed competition assays in which sphingosine and SK concentrations were held constant, whereas ATP concentrations were varied. For each inhibitor, the K_M for ATP and the V_max for S1P formation was determined. Compounds that are competitive inhibitors for the ATP-binding site would be expected to increase the K_M for ATP without affecting the V_max of the reaction. The data for compounds I–IV are summarized in Table 1.^11a The V_maxs show significant decreases with all of the test compounds versus vehicle alone. In contrast, K_Ms were not increased such that ATP concentrations up to at least 10 times the K_M were unable to overcome inhibition by the compounds. Therefore, these compounds are not competitive inhibitors at the ATP-binding site of SphK.

The effects of compounds I–V were determined at multiple concentrations, and IC₅₀s for each compound were calculated for human isoforms of ERK2, PI3k, and PKC-α. GST-hSK inhibition data is provided for comparison. The compounds demonstrated IC₅₀s in the sub- to low micromolar range, making them more potent inhibitors of SphK than any previously reported compound.^11a Biological evaluations demonstrated that the IC₅₀s for inhibition of SphK and tumor cell proliferation by the newly synthesized compound V, ~2 µM, indicated that it is somewhat less potent than compound IV, ~0.6 µM.

In continuing studies of these SphK inhibitors as cancer therapeutic agents, Smith reported additional in vitro and in vivo properties of three SphK inhibitors (Compounds I, II and V).^11b Their findings show that the antitumor activities mostly correlated well with their concentrations in blood and tumors. When inhibitor concentrations were normalized to equal S1P formation inhibition, the rank order of modulation for both pathways was V > II >> I. The reason for differences in pathway modulation is unknown. From our previous kinase selectivity assays, it was observed that V was the most promiscuous, potently inhibiting PI3k.^11a Therefore, compound V may potently inhibit SphK but most likely inhibits other kinases as well. One of the inhibitors, II, showed promising oral bioavailability and antitumor activity while compounds I and V showed extremely poor oral bioavailability.^11b These findings provide additional validation for sphingosine kinase-1 as a cancer therapeutic target, as well as confirming that further evaluation of these small molecule inhibitors is required.

There are four separate areas of consideration in the currently accepted evaluation of whether a compound with certain pharmacological or biological activity has the properties to become a suitable drug candidate. The rule describes molecular properties important for a drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion ("ADME"). The rule is integral for drug development where a pharmacologically active lead structure is optimized step-wise for increased activity and selectivity, as well as drug-like properties as described by Lipinski's rule.¹² The modification of the molecular structure often leads to drugs with higher molecular weight, more rings, more rotatable bonds, and a higher lipophilicity. However, the rule does not predict if a compound is pharmacologically active.

To better understand the pharmacophoric nature of Compound 1, we will design and synthesize various derivatives in an attempt to improve upon the oral bioavailability of the inhibitors while maintaining the overall activity.This will allow for more of an in-depth examination of this region's role in the binding of these inhibitors to the target enzyme and allow for the development of interesting organic methodology by the undergraduate student.

References

1. (a) Ogretman, B.; Hannun, Y. A. "Biologically active sphingolipids in cancer pathogenesis and treatment," Nat. Rev. Cancer 2004, 4, 604. (b) Senechenkov, A.; Litvak, D. A.; Cabot, M. C. "Targeting ceramide metabolism: a strategy for overcoming drug resistance," J. Natl. Cancer Inst. 2001, 93, 347.

2. (a) Kohama, T.; Olivera, A.; Edsall, L. C.; Nagiec, M. M.; Dickson, R.; Spiegel, S. "Molecular cloning and functional characterization of murine sphingosine kinase," J. Biol. Chem. 1998, 273, 23722. (b) Liu, H.; Suguira, M.; Nava, V. E.; Edsall, L. C.; Kono, K.; Poulton, S. et al. "Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform," J. Biol. Chem. 2000, 275, 19513. (c) Johnson, K. R.; Becker, K. P.; Facchinetti, M. M.; Hannun, Y. A.; Obeid, L. M. "PKC-dependent activation of sphingosine kinase 1 and translocation to the plasma membrane. Extracellular release of sphingosine-1-phosphate induced by phrobol 12-myristate 13-acetate (PMA)," J. Biol. Chem. 2002, 277, 35257.

3. (a) Zhang, H. et al. "Sphingosine 1-phosphate, a novel lipid, involved in cellular proliferation," J. Cell Biol. 1991, 114, 155. (b) Olivera, A.; Spiegel, S. "Sphingosine 1-phosphate as a second messenger in cell proliferation induced by PDGF and FCS mitogens," Nature, 1993, 365, 557.

4. Cuvillier, O. et al. "Suppression of ceramide-mediated programmed cell death by sphingosine 1-phosphate," Nature, 1996, 381, 800.

5. Spiegel, S., and Milstien, S. "Sphingosine-1-phosphate: an enigmatic signalling lipid," Nat. Rev. Mol. Cell Biol., 2003, 4, 397–407. 6. Taha, T. A., Hannun, Y. A., and Obeid, L. M. "Sphingosine kinase: biochemical and cellular regulation and role in disease," J. Biochem. Mol. Biol., 2006, 39, 113–131.

7. Milstien, S., and Spiegel, S. "Targeting sphingosine-1-phosphate: A novel avenue for cancer therapeutics," Cancer Cell, 2006, 9, 148–150.

8. Okada, T., Ding, G., Sonoda, H., Kajimoto, T., Haga, Y., Khosrowbeygi, A., Gao, S., Miwa, N., Jahangeer, S., and Nakamura, S. "Involvement of N-terminal-extended Form of Sphingosine Kinase 2 in Serum-dependent Regulation of Cell Proliferation and Apoptosis," J. Biol. Chem., 2005, 280, 36318–36325.

9. Maceyka, M., Sankala, H., Hait, N. C., Le Stunff, H., Liu, H., Toman, R., Collier, C., Zhang, M., Satin, L., Merrill, A. H., Jr., Milstien, S., and Spiegel, S. "SphK1 and SphK2, Sphingosine Kinase Isoenzymes with Opposing Functions in Sphingolipid Metabolism," J. Biol. Chem., 2005, 280, 37118–37129.

10. Liu, H., Chakravarty, D., Maceyka, M., Milstein, S., Spiegel, S. "Sphingosine kinases: a novel family of lipid kinases," Prog. Nucleic Acid Res. Mol. Biol. 2002, 71, 493-511.

11. (a) French, K. J.; Schrecengost, R. S; Lee, B. D.; Zhuang, Y.; Smith, S. N.; Eberly, J. L.; Yun, J. K.; Smith, C. D. "Discovery and evaluation of inhibitors of human sphingosine kinase," Cancer Res. 2003, 63, 5962. (b) French, K. J.; Upson, J. J.; Keller, S. N.; Zhuang, Y.; Yun, J. K.; Smith, C. D. "Antitumor activity of sphingosine kinase inhibitors," J. Pharmacol. Exptl. Ther. 2006, 318, 596.

12. Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J. "Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings," Adv. Drug Del. Rev., 2001, 46, 3-26.

Current Students

• Kevin Mays
• Amber Wallace
• Jaclyn Cika

Former Students

• Matt Wilson
• Erin White Wilson
• Demetrius Miles
• Madalyn McCaulley
• Nicole Quigley
• Valencia Fleming
• Ray Olang