Attenuation of arthritis in rodents by a novel orally-available inhibitor of sphingosine kinase.

UNLABELLED: Pro-inflammatory cytokines like TNF-α activate sphingosine kinase (SK). Therefore, inhibition of SK is a potential molecular target for the treatment of rheumatoid arthritis. AIMS: The primary goal of this study was to assess the efficacy of ABC249640 (a selective SK-2 inhibitor) in two models of rodent arthritis. A secondary goal was to evaluate the pharmacological profile of ABC294640, when given in combination with methotrexate. METHODS: The efficacy of ABC294640 was determined by paw diameter/volume measurements, histological evaluations, and micro-CT analyses. RESULTS: ABC294640 attenuated both collagen-induced arthritis in mice, as well as adjuvant-induced arthritis in rats. With the adjuvant arthritis model, the prophylactic efficacy profile of ABC294640 was similar to indomethacin. Of note, ABC294640 reduced the bone and cartilage degradation, associated with adjuvant-induced arthritis. Rats treated with a suboptimal dose of MTX (50 µg/kg/day) in combination with ABC249640 (100 mg/kg/day) had better anti-arthritis effects in the adjuvant model, than treatment with either agent alone. CONCLUSION: Our results suggest that ABC249640 is an orally available drug candidate with a good pre-clinical efficacy profile for the prevention and/or treatment of RA.

Differences in effects on myocardium and mitochondria by angiogenic inhibitors suggest separate mechanisms of cardiotoxicity.

Several multikinase angiogenesis inhibitors demonstrate mitochondrial and/or cardiovascular toxicity, suggesting an on-target pharmacologic effect. To evaluate whether cardiotoxicity is directly related to vascular endothelial growth factor receptor inhibition, we investigated the effects of sunitinib, sorafenib, and pazopanib on myocardial function and structure. We used a rat model to assess myocardial effects of the inhibitors concurrently exposed to the cardiac stressor dobutamine. Echocardiographic abnormalities including premature ventricular contractions, decreases in heart rate, circumferential strain, and radial and circumferential strain rates were noted with sorafenib, but not with sunitinib or pazopanib. Ultrastructural analysis of ventricular cardiomyocytes by transmission electron microscopy revealed mitochondrial swelling, dense deposits, and matrix cavitation in rats given sunitinib and disrupted mitochondrial cristae in rats given sorafenib, but there were no effects with pazopanib. Effects on neonatal rat cardiomyocyte cultures were assessed, which identified decreases in mitochondrial membrane potential with sunitinib treatment, but not with sorafenib or pazopanib. Intracellular adenosine triphosphate depletion was observed with sunitinib and sorafenib, but not pazopanib. Our results show that cardiotoxicity is not necessarily related to a pharmacologic classwide effect of vascular endothelial growth factor receptor inhibition, and the rat myocardial structural and functional changes identified in this study may be instead a result of inhibition of other kinase pathways, the mechanism of which may be associated with mitochondrial toxicity.

Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.

Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase.

A critical step in the mechanism of action of inflammatory cytokines is the stimulation of sphingolipid metabolism, including activation of sphingosine kinase (SK), which produces the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). We have developed orally bioavailable compounds that effectively inhibit SK activity in vitro in intact cells and in cancer models in vivo. In this study, we assessed the effects of these SK inhibitors on cellular responses to tumor necrosis factor alpha (TNFalpha) and evaluated their efficacy in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice. Using several cell systems, it was shown that the SK inhibitors block the ability of TNFalpha to activate nuclear factor kappa B (NFkappaB), induce expression of adhesion proteins, and promote production of prostaglandin E(2) (PGE(2)). In an acute model of DSS-induced ulcerative colitis, SK inhibitors were equivalent to or more effective than Dipentum in reducing disease progression, colon shortening, and neutrophil infiltration into the colon. The effects of SK inhibitors were associated with decreased colonic levels of inflammatory cytokines TNFalpha, interleukin (IL)-1beta, interferon gamma (IFN)-gamma, IL-6, and reduction of S1P levels. A similar reduction in disease progression was provided by SK inhibitors in a chronic model of ulcerative colitis in which the mice received 3-week-long cycles of DSS interspaced with week-long recovery periods. In the chronic model, immunohistochemistry for SK showed increased expression in DSS-treated mice (compared with water-treated controls) that was reduced by drug treatment. S1P levels were also elevated in the DSS group and significantly reduced by drug treatment. Together, these data indicate that SK is a critical component in inflammation and that inhibitors of this enzyme may be useful in treating inflammatory bowel diseases.

Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases.

PURPOSE: The increased vascular permeability and pathogenic angiogenesis observed in diabetic retinopathy are induced, at least in part, by local inflammation and vascular endothelial growth factor (VEGF). Therefore, inhibition of signaling from VEGF and tumor necrosis factor-alpha (TNFalpha) is a promising approach to the treatment of this disease, as well as ocular diseases with similar etiologies, including age-related macular degeneration. A growing body of evidence demonstrates that sphingosine kinase (SK) plays an important role in cellular proliferation and angiogenesis. This study was undertaken to examine the effects of SK inhibitors on the responses of retinal endothelial cells (RECs) to VEGF and TNFalpha and their therapeutic efficacy in a diabetic retinopathy model. METHODS: The expression and function of SK in bovine and human RECs were examined by immunoblot analysis. The involvement of SK in mediating responses to VEGF and TNFalpha was examined by using pharmacologic inhibitors of SK in cellular and in vivo assays, including a 3-month streptozotocin-induced diabetic retinopathy model in rats. RESULTS: SK was present and active in human and bovine RECs, and SK activity in these cells was stimulated by VEGF. Inhibitors of SK blocked VEGF-induced production of sphingosine 1-phosphate and markedly attenuated VEGF-induced proliferation and migration of RECs. In addition, SK inhibitors were shown to block TNFalpha-induced expression of adhesion proteins, suppress VEGF-induced vascular leakage in an in vivo mouse model, and reduce retinal vascular leakage in the rat diabetic retinopathy model. CONCLUSIONS: Overall, these studies demonstrate that inhibitors of SK attenuate the effects of proliferative and inflammatory stimuli on RECs both in vitro and in vivo, and so could be significant therapeutics in the treatment of diabetic retinopathy.

Antitumor activity of sphingosine kinase inhibitors.

Sphingosine kinase (SK) is an oncogenic sphingolipid-metabolizing enzyme that catalyzes the formation of the mitogenic second messenger sphingosine-1-phosphate (S1P) at the expense of proapoptotic ceramide. Thus, SK is an attractive target for cancer therapy because blockage of S1P formation leads to inhibition of proliferation, as well as the induction of apoptosis in cancer cells. We have recently identified novel SK inhibitors with nanomolar to low micromolar potencies toward recombinant human SK. This study describes the continuing analysis of these inhibitors through in vitro and in vivo experiments. All three structurally diverse SK inhibitors tested showed antitumor activity in mice without exhibiting toxicity. Blood and tumor inhibitor concentrations exceeded in vitro potency levels. Cell signaling analyses in vitro revealed mixed inhibition of mitogen-activated protein kinase kinase and Akt phosphorylation by the SK inhibitors. Importantly, 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) is orally bioavailable, detected in the blood for at least 8 h, and showed a significant inhibition of tumor growth in mice. These compounds are the first examples of nonlipid selective inhibitors of SK with in vivo antitumor activity and provide leads for further development of inhibitors of this important molecular target.

Two N-myristoyltransferase isozymes play unique roles in protein myristoylation, proliferation, and apoptosis.

N-myristoyltransferases (NMT) add myristate to the NH(2) termini of certain proteins, thereby regulating their localization and/or biological function. Using RNA interference, this study functionally characterizes the two NMT isozymes in human cells. Unique small interfering RNAs (siRNA) for each isozyme were designed and shown to decrease NMT1 or NMT2 protein levels by at least 90%. Ablation of NMT1 inhibited cell replication associated with a loss of activation of c-Src and its target FAK as well as reduction of signaling through the c-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays showed that depletion of either NMT isozyme induced apoptosis, with NMT2 having a 2.5-fold greater effect than NMT1. Western blot analyses revealed that loss of NMT2 shifted the expression of the BCL family of proteins toward apoptosis. Finally, intratumoral injection of siRNA for NMT1 or for both NMT1 and NMT2 inhibited tumor growth in vivo, whereas the same treatment with siRNA for NMT2 or negative control siRNA did not. Overall, the data indicate that NMT1 and NMT2 have only partially overlapping functions and that NMT1 is critical for tumor cell proliferation.

Huntingtin interacting protein 14 is an oncogenic human protein: palmitoyl acyltransferase.

Protein palmitoyltransferases (PATs) represent an exciting new target for anticancer drug design due to their pivotal roles in the subcellular localization of a number of oncogenes. We show that the Huntingtin interacting protein 14 (HIP14) is a PAT with a preference for the farnesyl-dependent palmitoylation motif found in H- and N-RAS. Characterization of HIP14 in mouse cells has revealed that it has the ability to induce colony formation in cell culture, anchorage-independent growth, and tumors in mice. Activity of the enzyme and its ability to transform cells is dependent on critical residues in the active site of the enzyme.

Synthesis and evaluation of dihydropyrroloquinolines that selectively antagonize P-glycoprotein.

In a search for improved multiple drug resistance (MDR) modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein (Pgp) without modulating multidrug resistance-related protein 1 (MRP1). These compounds were evaluated for their toxicity toward drug-sensitive tumor cells (i.e. MCF-7, T24) and for their ability to antagonize Pgp-mediated drug-resistant cells (i.e. NCI/ADR) and MRP1-mediated resistant cells (i.e. MCF-7/VP). Cytotoxicity and drug accumulation assays demonstrated that the dihydropyrroloquinolines inhibit Pgp to varying degrees, without any significant inhibition of MRP1. The compound termed PGP-4008 was the most effective at inhibiting Pgp in vitro and was further evaluated in vivo. PGP-4008 inhibited tumor growth in a murine syngeneic Pgp-mediated MDR solid tumor model when given in combination with doxorubicin. PGP-4008 was rapidly absorbed after intraperitoneal administration, with its plasma concentrations exceeding the in vitro effective dose for more than 2 h. PGP-4008 did not alter the plasma distribution of concomitantly administered anticancer drugs and did not cause systemic toxicity as was observed for cyclosporin A. Because of their enhanced selectivity toward Pgp, these substituted dihydropyrroloquinolines may be effective MDR modulators in a clinical setting.

Cyclohexyl-octahydro-pyrrolo[1,2-a]pyrazine-based inhibitors of human N-myristoyltransferase-1.

N-myristoyltransferase (NMT) is an emerging therapeutic target that catalyzes the attachment of myristate to the N terminus of an acceptor protein. We have developed a medium-throughput assay for screening potential small molecule inhibitors of human NMT-1 consisting of recombinant enzyme, biotinylated peptide substrate, and [3H]myristoyl-CoA. Approximately 16,000 diverse compounds have been evaluated, and significant inhibition of NMT was found with 0.8% of the compounds. From these hits, we have identified the cyclohexyl-octahydropyrrolo[1,2-a]pyrazine (COPP) chemotype as inhibitory toward human NMT-1. Thirty-two compounds containing this substructure inhibited NMT-1, with IC(50) values ranging from 6 microM to millimolar concentrations, and a quantitative structure-activity relationship equation (r(2) = 0.72) was derived for the series. The most potent inhibitor (24, containing 9-ethyl-9H-carbazole) demonstrated competitive inhibition for the peptide-binding site of NMT-1 and noncompetitive inhibition for the myristoyl-CoA site. Computational docking studies using the crystal structure of the highly homologous yeast NMT confirmed that 24 binds with excellent complementarity to the peptide-binding site of the enzyme. To evaluate the ability of 24 to inhibit NMT activity in intact cells, monkey CV-1 cells expressing an N-myristoylated green fluorescent protein (GFP) fusion protein were treated with a known NMT inhibitor or with 24. Each compound caused the redistribution of GFP from the plasma membrane to the cytosol. Furthermore, 24 inhibits cancer cell proliferation at doses similar to those that inhibit protein myristoylation. Overall, these studies establish an efficient assay for screening for inhibitors of human NMT and identify a novel family of inhibitors that compete at the peptide-binding site and have activity in intact cells.

Development of a syngeneic in vivo tumor model and its use in evaluating a novel P-glycoprotein modulator, PGP-4008.

Multidrug resistance (MDR) is a phenomenon by which tumor cells develop reduced sensitivity to anticancer drugs, which often leads to the failure of cancer chemotherapy. A prominent mechanism of MDR is the overexpression of the multidrug efflux pump, P-glycoprotein (P-gp), that decreases the intracellular accumulation of many anticancer drugs, leading to increased tumor growth. Intensive efforts are under way to develop clinically useful MDR modulators that inhibit the function of P-gp for use in combination with established anticancer drugs. Our goal was to develop an improved in vivo solid tumor model utilizing immunocompetent animals to examine the efficacy of P-gp-specific MDR modulators. Using in vitro cytotoxicity and drug accumulation assays, two transformed murine cell lines, JC and TIB-75, were found to demonstrate the P-gp-mediated MDR phenotype. In contrast, two similar lines did not express functional P-gp. Western blot analyses confirmed the expression of P-gp and the lack of expression of the closely related drug efflux protein MRP1 in the JC and TIB-75 cell lines. The JC cell line displayed excellent tumorigenicity and consistent growth kinetics when implanted into immune-competent Balb/c mice. Animals treated with a combination of a known MDR modulator, cyclosporin A, and a cytotoxic drug, doxorubicin, exhibited significantly reduced tumor growth compared with untreated controls or animals treated with either cyclosporin A or doxorubicin alone. Similarly, a novel P-gp-specific MDR modulator, PGP-4008, in combination with doxorubicin showed inhibition of tumor growth. However, in contrast with the significant loss of body weight observed in the animals treated with the combination of cyclosporin A and doxorubicin, those treated with PGP-4008 plus doxorubicin did not experience weight loss. Therefore, this syngeneic solid tumor model provides a new in vivo system that can be used to evaluate the efficacy of P-gp inhibitors in an immune-competent host. This should allow improved prediction of the clinical utility of these compounds.

Discovery and evaluation of inhibitors of human sphingosine kinase.

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of bioactive lipids, including the proapoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate. Accumulating evidence indicates that sphingosine kinase (SK) plays a pivotal role in regulating tumor growth and that SK can act as an oncogene. Despite the importance of SK for cell proliferation, pharmacological inhibition of SK is an untested means of treating cancer because of the current lack of nonlipid inhibitors of this enzyme. To further assess the involvement of SK in human tumors, levels of RNA for SK in paired samples of cDNA prepared from tumors and normal adjacent tissue were analyzed. Expression of SK RNA was significantly elevated in a variety of solid tumors, compared with normal tissue from the same patient. To identify and evaluate inhibitors of SK, a medium throughput assay for recombinant human SK fused to glutathione S-transferase was developed, validated, and used to screen a library of synthetic compounds. A number of novel inhibitors of human SK were identified, and several representative compounds were characterized in detail. These compounds demonstrated activity at sub- to micromolar concentrations, making them more potent than any other reported SK inhibitor, and were selective toward SK compared with a panel of human lipid and protein kinases. Kinetic studies revealed that the compounds were not competitive inhibitors of the ATP-binding site of SK. The SK inhibitors were antiproliferative toward a panel of tumor cell lines, including lines with the multidrug resistance phenotype because of overexpression of either P-glycoprotein or multidrug resistance phenotype 1, and were shown to inhibit endogenous human SK activity in intact cells. Furthermore, each inhibitor induced apoptosis concomitant with tumor cell cytotoxicity. Methods for the synthesis of a series of aurone inhibitors of SK were established, and a prototypical dihydroxyaurone was found to have moderate antitumor activity in vivo in the absence of overt toxicity to the mice. These compounds are the first examples of nonlipid inhibitors of SK with in vivo antitumor activity and so provide leads for additional development of inhibitors of this important molecular target.

Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol.

Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.