Christopher J. Trabbic

Bilirubin Binding to PPARα Inhibits Lipid Accumulation

Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin.

Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.

Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.

Nicotinic Acid Adenine Dinucleotide Phosphate Analogues Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on ReceptorMediated Ca2+ Release

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20-500-fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca(2+) with an EC50 of 31 μM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced.

Timcodar (VX-853) Is a Non-FKBP12 Binding Macrolide Derivative That Inhibits PPARγ and Suppresses Adipogenesis

Nutrient overload and genetic factors have led to a worldwide epidemic of obesity that is the underlying cause of diabetes, atherosclerosis, and cardiovascular disease. In this study, we used macrolide drugs such as FK506, rapamycin, and macrolide derived, timcodar (VX-853), to determine their effects on lipid accumulation during adipogenesis. Rapamycin and FK506 bind to FK506-binding proteins (FKBPs), such as FKBP12, which causes suppression of the immune system and inhibition of mTOR. Rapamycin has been previously reported to inhibit the adipogenic process and lipid accumulation. However, rapamycin treatment in rodents caused immune suppression and glucose resistance, even though the mice lost weight. Here we show that timcodar (1 μM), a non-FKBP12-binding drug, significantly (p < 0.001) inhibited lipid accumulation during adipogenesis. A comparison of the same concentration of timcodar (1 μM) and rapamycin (1 μM) showed that both are inhibitors of lipid accumulation during adipogenesis. Importantly, timcodar potently (p < 0.01) suppressed transcriptional regulators of adipogenesis, PPARγ and C/EBPα, resulting in the inhibition of genes involved in lipid accumulation. These studies set the stage for timcodar as a possible antiobesity therapy, which is rapidly emerging as a pandemic.

Synthesis and biological evaluation of isomeric methoxy substitutions on anti-cancer indolyl-pyridinyl-propenones: Effects on potency and mode of activity.

Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption. Ready access to 5-indole substitution has allowed characterization of this position to be important for both types of mechanisms when a simple methoxy group is present. We now report the syntheses and biological effects of isomeric methoxy substitutions on the indole ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl moiety were evaluated for anticancer activity. The results demonstrate that the location of the methoxy group can alter both the potency and the mechanism of cell death. Remarkably, changing the methoxy from the 5-position to the 6-position switched the biological activity from induction of methuosis to disruption of microtubules. The latter may represent a prototype for a new class of mitotic inhibitors with potential therapeutic utility.

Abstract 1244: A novel microtubule-disrupting indole-based chalcone that induces caspase-independent cell death in glioblastoma and melanoma cells

Indolyl-pyridinyl-propenones (a subclass of synthetic indole-based chalcones) have been shown to induce a novel form of cell death termed ‘methuosis’ in glioblastoma and other tumor cell lines. This form of cell death is caspase-independent and involves massive vacuolization of macropinosome and endosome compartments. In the course of structure-activity relationship studies identifying a lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (abbreviated 5-MOMIPP), we discovered that changing the methoxy substitution from the 5-position to the 6-position on the indole ring markedly alters the biological activity of the compound. 6-MOMIPP exhibits potent anti-proliferative and cytotoxic activity in glioblastoma, melanoma and other cancer cell lines at a concentration of 100 nM, nearly two orders of magnitude lower than the maximally effective concentration of 5-MOMIPP. Instead of inducing methuosis, 6-MOMIPP (at > 500 nM) immediately disrupts microtubules, as confirmed by immunofluorescence microscopy. By 24 h the majority of the cells are rounded and arrested in mitosis, and by 48 h cell viability is greatly reduced. The few remaining live cells contain multiple abnormal nuclei. Extensive cell death occurs before activation of caspases 3, 7 and 9 can be detected. Scintillation proximity assays show that 6-MOMIPP competes with 3H-colchicine, but not 3H-vinblastine, for binding to tubulin. Studies aimed at testing the toxicity of 6-MOMIPP in normal cells are in progress, but work with a closely related microtubule-active indole-based chalcone indicates that toxicity is substantially reduced in quiescent human fibroblasts and rat neuronal progenitor cells. Although drugs targeted to microtubules (e.g., Vinca alkaloids and taxanes) are widely used as anti-neoplastic agents, they have shown little efficacy in treating primary or metastatic brain tumors, partly due to limited penetrance of the blood-brain barrier (BBB). Preliminary pharmacokinetic studies in mice show that 6-MOMIPP injected IP can readily cross the BBB, attaining brain concentrations nearly equal to plasma concentrations within 30 min to 1 h. Therefore, these preliminary findings suggest that 6-MOMIPP merits further preclinical evaluation as a potential therapeutic agent for primary and metastatic brain tumors. Supported by NIH grant CA115495 and by the Helen and Harold McMaster Endowment. Citation Format: Shengnan Du, Jeffrey G. Sarver, Christopher J. Trabbic, Paul W. Erhardt, Jean H. Overmeyer, William A. Maltese. A novel microtubule-disrupting indole-based chalcone that induces caspase-independent cell death in glioblastoma and melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1244.