Thioredoxin reductase is a major regulator of metabolism in leukemia cells.

Despite the fact that AML is the most common acute leukemia in adults, patient outcomes are poor necessitating the development of novel therapies. We identified that inhibition of Thioredoxin Reductase (TrxR) is a promising strategy for AML and report a highly potent and specific inhibitor of TrxR, S-250. Both pharmacologic and genetic inhibition of TrxR impairs the growth of human AML in mouse models. We found that TrxR inhibition leads to a rapid and marked impairment of metabolism in leukemic cells subsequently leading to cell death. TrxR was found to be a major and direct regulator of metabolism in AML cells through impacts on both glycolysis and the TCA cycle. Studies revealed that TrxR directly regulates GAPDH leading to a disruption of glycolysis and an increase in flux through the pentose phosphate pathway (PPP). The combined inhibition of TrxR and the PPP led to enhanced leukemia growth inhibition. Overall, TrxR abrogation, particularly with S-250, was identified as a promising strategy to disrupt AML metabolism.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

Potent suppression of both spontaneous and carcinogen-induced colitis-associated colorectal cancer in mice by dietary celastrol supplementation.

Celastrol is an anti-inflammatory natural triterpenoid, isolated from the herb Tripterygium wilfordii or thunder god vine. Here, we define mechanisms mediating anti-inflammatory activity of celastrol and demonstrate efficacy of a dietary celastrol supplement for chemoprevention of inflammation-driven carcinogenesis in mice. Dietary celastrol (31.25 ppm in rodent diet from 8 weeks to 25 weeks of age) is well tolerated and protects against LPS-induced acute inflammation in C57BL/6 mice, potently suppressing LPS-induction of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, Interleukin (IL)-6 and IL-1ß. To test whether dietary celastrol suppresses inflammation-driven colorectal cancer (CRC), we employed a unique model of spontaneous, inflammation-driven CRC in mice harboring a germ line deletion of the p27Kip1 gene and a T cell-specific deletion of Smad4 gene (Smad4co/co;Lck-crep27Kip1-/-or DKO), which develop severe intestinal inflammation and carcinogenesis as early as 3 months of age. Exposure of DKO mice to daily dietary celastrol (12.5 ppm in diet) from 6 weeks of age significantly suppressed development of colitis-associated CRC (CAC). Celastrol chemoprevention of CAC in this new model of intestinal neoplasia was associated with significant suppression of iNOS at 4 months of age, and iNOS, COX-2 and NFκB at 6 months of age, with significant reduction in inflammatory cytokines, IL-6 and IL-1ß. Chemoprevetion of CAC by dietary celastrol was further confirmed in the model of azoxymethane (AOM) plus dextran sodium sulfate (DSS)-induced carcinogenesis in C57BL/6 mice. These data suggest the potential for celastrol as a safe and effective dietary supplement in the chemoprevention of CAC in humans.

Structure and Spectroscopy of Alkene-Cleaving Dioxygenases Containing an Atypically Coordinated Non-Heme Iron Center.

Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of ∼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (∼4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe-NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe-O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.

Identification of a negative feedback loop in biological oxidant formation fegulated by 4-hydroxy-2-(E)-nonenal.

4-Hydroxy-2-(E)-nonenal (4-HNE) is one of the major lipid peroxidation product formed during oxidative stress. At high concentrations, 4-HNE is cytotoxic and exerts deleterious effects that are often associated with the pathology of oxidative stress-driven disease. Alternatively, at low concentrations it functions as a signaling molecule that can activate protective pathways including the antioxidant Nrf2-Keap1 pathway. Although these biphasic signaling properties have been enumerated in many diseases and pathways, it has yet to be addressed whether 4-HNE has the capacity to modulate oxidative stress-driven lipid peroxidation. Here we report an auto-regulatory mechanism of 4-HNE via modulation of the biological oxidant nitric oxide (NO). Utilizing LPS-activated macrophages to induce biological oxidant production, we demonstrate that 4-HNE modulates NO levels via inhibition of iNOS expression. We illustrate a proposed model of control of NO formation whereby at low concentrations of 4-HNE a negative feedback loop maintains a constant level of NO production with an observed inflection at approximately 1 µM, while at higher 4-HNE concentrations positive feedback is observed. Further, we demonstrate that this negative feedback loop of NO production control is dependent on the Nrf2-Keap1 signaling pathway. Taken together, the careful regulation of NO production by 4-HNE argues for a more fundamental role of this lipid peroxidation product in normal physiology.

Glutathionylated 4-hydroxy-2-(E)-alkenal enantiomers in rat organs and their contributions toward the disposal of 4-hydroxy-2-(E)-nonenal in rat liver.

The major route for elimination of 4-hydroxy-2-(E)-nonenal (4-HNE) has long been considered to be through glutathionylation and eventual excretion as a mercapturic acid conjugate. To better quantitate the glutathionylation process, we developed a sensitive LC-MS/MS method for the detection of glutathione (GSH) conjugates of 4-hydroxy-2-(E)-alkenal enantiomers having a carbon skeleton of C5 to C12. The newly developed method enabled us to quantify 4-hydroxy-2-(E)-alkenal-glutathione diastereomers in various organs, i.e., liver, heart, and brain. We identified the addition of iodoacetic acid as a critical step during sample preparation to avoid an overestimation of glutathione-alkenal conjugation. Specifically, we found that in the absence of a quenching step reduced GSH and 4-hydroxy-2-(E)-alkenals react very rapidly during the extraction and concentration steps of sample preparation. Rat liver perfused with d11-4-hydroxy-2-(E)-nonenal (d11-4-HNE) revealed enantioselective conjugation with GSH and transportation out of the liver. In the d11-4-HNE-perfused rat livers, the amount of d11-(S)-4-HNE-GSH released from the rat liver was higher than that of d11-(R)-4-HNE-GSH, and more d11-(R)-4-HNE-GSH than d11-(S)-4-HNE-GSH remained in the perfused liver tissues. Overall, the glutathionylation pathway was found to account for only 8.7% of the disposition of 4-HNE, whereas catabolism to acetyl-CoA, propionyl-CoA, and formate represented the major detoxification pathway.

Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis.

The fatty acid-binding protein FABP5 shuttles ligands from the cytosol to the nuclear receptor PPARß/δ (encoded for by Pparδ), thereby enhancing the transcriptional activity of the receptor. This FABP5/PPARδ pathway is critical for induction of proliferation of breast carcinoma cells by activated epidermal growth factor receptor (EGFR). In this study, we show that FABP5 is highly upregulated in human breast cancers and we provide genetic evidence of the pathophysiologic significance of FABP5 in mammary tumorigenesis. Ectopic expression of FABP5 was found to be oncogenic in 3T3 fibroblasts where it augmented the ability of PPARδ to enhance cell proliferation, migration, and invasion. To determine whether FABP5 is essential for EGFR-induced mammary tumor growth, we interbred FABP5-null mice with MMTV-ErbB2/HER2 oncomice, which spontaneously develop mammary tumors. FABP5 ablation relieved activation of EGFR downstream effector signals, decreased expression of PPARδ target genes that drive cell proliferation, and suppressed mammary tumor development. Our findings establish that FABP5 is critical for mammary tumor development, rationalizing the development of FABP5 inhibitors as novel anticarcinogenic drugs.

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by ß oxidation of fatty acids in the isolated rat heart.

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on ß-oxidation pathways. The hypothesis driving this report is that perturbations of ß oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1mM octanoate). During ischemia, HNE concentrations doubled and the glutathione-HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through ß oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to ß oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.

A small molecule agonist of EphA2 receptor tyrosine kinase inhibits tumor cell migration in vitro and prostate cancer metastasis in vivo.

During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo.

Bryonolic acid transcriptional control of anti-inflammatory and antioxidant genes in macrophages in vitro and in vivo.

Bryonolic acid (BA) (1) is a naturally occurring triterpenoid with pleiotropic properties. This study characterizes the mechanisms mediating the anti-inflammatory and antioxidant activities of BA and validates the utility of BA as a tool to explore the relationships between triterpenoid structure and activity. BA reduces the inflammatory mediator NO by suppressing the expression of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 macrophage cells. In addition, BA robustly induces the antioxidant protein heme oxygenase-1 (HO-1) in vitro and in vivo in an Nrf2-dependent manner. Further analyses of Nrf2 target genes reveal selectivity for the timing and level of gene induction by BA in treated macrophages with distinct patterns for Nrf2-regulated antioxidant genes. Additionally, the distinct expression profile of BA on Nrf2 target genes relative to oleanolic acid suggests the importance of the triterpenoid scaffold in dictating the pleiotropic effects exerted by these molecules.

Synthesis and biological evaluation of analogues of a novel inhibitor of beta-amyloid secretion.

A drug library of 17,200 compounds was screened to select small molecules that inhibit the secretion of amyloid beta peptide (Abeta), the major component of Alzheimer disease senile plaques, from a human neuronal cell line. Twenty-nine hits were validated that decreased Abeta secretion by >40% at 10 microM, for a 0.17% hit rate. A lead hit was selected for further study based on its activity and low cytotoxicity, and it was found to inhibit Abeta secretion through activation of the alpha-secretase pathway. Twenty-four commercially available and 53 synthesized analogues were analyzed for activity. Selected analogues were evaluated for biological stability by incubation with hepatoma cells and for transcellular permeability using Caco-2 cell monolayers. The analogue with the best permeability was evaluated in 2-month old amyloid precursor protein transgenic mice and found to acutely reduce cerebral Abeta levels by 40% after a single iv administration.

Bryonolic acid: a large-scale isolation and evaluation of heme oxygenase 1 expression in activated macrophages.

Bryonolic acid (BA) is a triterpenoid found in the Cucurbitaceae family of plants. Our interests in the immunomodulatory effects of this class of natural products led us to discover that BA induces a marked increase in the expression of a phase 2 response enzyme, heme oxygenase 1 (HO-1), in a dose-dependent manner. This phenotype has translational implications in malarial disease progression, and consequently we developed a large-scale isolation method for BA that will enable future in vitro and in vivo analyses. We have determined ideal growth conditions and time scale for maximizing BA content in the roots of Cucurbita pepo and analyzed BA production by HPLC. Large-scale extraction yielded 1.34% BA based on dry weight, allowing for the isolation of BA on a multigram scale.

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain.

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.