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The transcription factor Nrf2 determines the ability to adapt and survive under conditions of electrophilic, oxidative, and inflammatory stress by regulating the expression of elaborate networks comprising nearly 500 genes encoding proteins with versatile cytoprotective functions. In mice, disruption of Nrf2 increases susceptibility to carcinogens and accelerates disease pathogenesis. Paradoxically, Nrf2 is upregulated in established human tumors, but whether this upregulation drives carcinogenesis is not known. Here we show that the incidence, multiplicity, and burden of solar-simulated UV radiation–mediated cutaneous tumors that form in SKH-1 hairless mice in which Nrf2 is genetically constitutively activated are lower than those that arise in their wild-type counterparts. Pharmacologic Nrf2 activation by topical biweekly applications of small (40 nmol) quantities of the potent bis(cyano enone) inducer TBE-31 has a similar protective effect against solar-simulated UV radiation in animals receiving long-term treatment with the immunosuppressive agent azathioprine. Genetic or pharmacologic Nrf2 activation lowers the expression of the pro-inflammatory factors IL6 and IL1β, and COX2 after acute exposure of mice to UV radiation. In healthy human subjects, topical applications of extracts delivering the Nrf2 activator sulforaphane reduced the degree of solar-simulated UV radiation–induced skin erythema, a quantifiable surrogate endpoint for cutaneous damage and skin cancer risk. Collectively, these data show that Nrf2 is not a driver for tumorigenesis even upon exposure to a very potent and complete carcinogen and strongly suggest that the frequent activation of Nrf2 in established human tumors is a marker of metabolic adaptation. Cancer Prev Res; 8(6); 475–86. ©2015 AACR.

PDF - 16K, Tri- and Penta-cyclic compounds to not quench pyrene actin florescence intensity. To ascertain that the addition of TBE-31 or CDDO-Im would not quench pyrene-actin fluorescence intensity, pyrene actin polymerization was carried out as described in the Materials section, measured (-) and then DMSO, TBE-31 or CDDO-Im were added to the samples and re-measured (+) for fluorescence intensity. Note that the fluorescence intensity did not appreciably change after vehicle or drug addition.

AVI - 10062K, Confluent monolayers of H1299 lung tumor cells were scratched and incubated at 37 degrees C in media containing either vehicle (DMSO), 1 microM TBE-31 or 1 microM CDDO-Im for 18 h. Representative bright field microscopy images were taken at 10x magnification every 15 min using an Olympus IX81 microscope to create a movie.

Supplemental Figure S1. Spectral characteristics of UVA 340 lamps. Figure S2. Nrf2 disruption leads to an increase in the mRNA of Nrf3, but not Nrf1, in skin of SKH-1 hairless mice. Figure S3. TBE-31 treatment induces NQO1 in dermis and epidermis of WT SKH-1 hairless mice, but has no effect in Nrf2-KO animals. Figure S5. The architecture of the skin is similar among WT, Nrf2-KO and Keap1-KD SKH-1 hairless mice. Figure S6. Dose optimization for the use of TBE-31 as a pharmacological agent for protection against cutaneous carcinogenesis mediated by solar-simulated UV radiation. Figure S7. Optimization of azathioprine treatment in SKH-1 hairless mice. Figure S8. HPLC analysis of the sulforaphane (SF, active)- and glucoraphanin (GR, placebo) extracts used in the human study. Figure S9. NQO1 inducer activity of the sulforaphane (SF, active)- and glucoraphanin (GR, placebo) extracts used in the human study.

PDF - 8699K, Single cell tracking of NIH 3T3 cells was carried out using time-lapse microscopy with an Olympus IX81 inverted microscope equipped with a custom chamber which allowed us to maintain a temperature of 37 degrees C and an atmosphere containing 5% CO2. Bright field images were collected from cells incubated with vehicle (DMSO), 1 microM TBE-31 or 0.5 microM CDDO-Im at 10 min intervals over 18 h. Distance of migration was determined by tracking the positions of cell nuclei over 18 h using the MtrackJ plugin (32) for imageJ software (33).

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non–small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non–small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Cancer Prev Res; 7(7); 727–37. ©2014 AACR.

The transcription factor BACH1 regulates the expression of a variety of genes including genes involved in oxidative stress responses, inflammation, cell motility, cancer cell invasion and cancer metabolism. Based on this, BACH1 has become a promising therapeutic target in cancer (as anti-metastatic target) and also in chronic conditions linked to oxidative stress and inflammation, where BACH1 inhibitors share a therapeutic space with activators of transcription factor NRF2. However, while there is a growing number of NRF2 activators, there are only a few described BACH1 inhibitors/degraders. The synthetic acetylenic tricyclic bis(cyanoenone),(±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3.4b,7,8,8a,9,10, 10a-octahydrophenanthrene-2,6-dicarbonitrile, TBE31 is a potent activator of NRF2 without any BACH1 activity. Herein we found that biotinylation of TBE31 greatly reduces its potency as NRF2 activator (50-75-fold less active) while acquiring a novel activity as a BACH1 degrader (100-200-fold more active). We demonstrate that TBE56, the biotinylated TBE31, interacts and promotes the degradation of BACH1 via a mechanism involving the E3 ligase FBXO22. TBE56 is a potent and sustained BACH1 degrader (50-fold more potent than hemin) and accordingly a powerful HMOX1 inducer. TBE56 degrades BACH1 in lung and breast cancer cells, impairing breast cancer cell migration and invasion in a BACH1-dependent manner, while TBE31 has no significant effect. Altogether, our study identifies that the biotinylation of TBE31 provides novel activities with potential therapeutic value, providing a rationale for further characterisation of this and related compounds.

Funder: Stony Brook Foundation Reata Pharmaceuticals, Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism. Nrf2 activation is protective in models of human disease and has benefits in clinical trials. Consequently, the Keap1/Nrf2 protein complex is a drug target. However, in cancer Nrf2 plays a dual role, raising concerns that Nrf2 activators may promote growth of early neoplasms. To address this concern, we examined the role of Nrf2 in development of colorectal adenomas by employing genetic, pharmacological, and metabolomic approaches. We found that colorectal adenomas that form in Gstp−/−: ApcMin/+ mice are characterized by altered one-carbon metabolism and that genetic activation, but not disruption of Nrf2, enhances these metabolic alterations. However, this enhancement is modest compared to the magnitude of metabolic differences between tumor and peri-tumoral tissues, suggesting that the metabolic changes conferred by Nrf2 activation may have little contribution to the early stages of carcinogenesis. Indeed, neither genetic (by Keap1 knockdown) nor pharmacological Nrf2 activation, nor its disruption, affected colorectal adenoma formation in this model. We conclude that pharmacological Nrf2 activation is unlikely to impact the early stages of development of colorectal cancer.

The mitochondrial protein LonP1 is an ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Biallelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its -methyl and -imidazole derivatives reversibly inhibit LonP1 by a noncompetitive mechanism, blocking ATP-hydrolysis and thus proteolysis. By contrast, we found that CDDO-anhydride inhibits the LonP1 ATPase competitively. Docking of CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the ATP-binding site. The binding site of CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (CODAS) syndrome, which ablated inhibition. CDDO failed to inhibit the ATPase activity of the purified 26S proteasome, which like LonP1 belongs to the AAA

This account exemplifies our recent progress on the strategic incorporation of fluorine and organofluorine groups to (i) taxoid anticancer agents, (ii) acylhydrazone-based antifungal agents and (iii) inhibitors of matrix metalloproteinase 9 (MMP9) for medicinal chemistry and chemical biology studies. In the case study (i), a series of next-generation fluorotaxoids, bearing m-OCF3 or m-OCF2H group in the C2-benzoate moiety was designed, synthesized and examined for their potencies. A number of these fluorotaxoids possess two orders of magnitude greater potency in different drug-resistant cancer cell lines as compared to paclitaxel. One of these next-generation fluorotaxoids, SB-T-121205 was selected for detailed mechanistic study against highly paclitaxel-resistant human breast cancer cell line, MCF-7/PTX, which disclosed a unique mechanism of action. Recently, glucosylceramide (GlcCer) synthesis emerged as a promising target for next-generation antifungal agents, especially against cryptococcosis, candidiasis and pulmonary aspergillosis. The HTP screening of compound libraries identified several acylhydrazones as hit compounds. In the case study (ii), fluoro-acylhydrazones containing F, OCF3, OCHF2, o-F/p-OCF3, as well as o-F/p-CF3 functional groups in the ring A and ring B were designed based on these hit compounds, synthesized and examined for their potencies against C. neoformans. A number of those novel fluoro-acylhydrazones exhibited high potency and excellent killing properties. The hemopexin-like domain of matrix metalloproteinases (MMPs) is a highly promising target to circumvent the critical issue in the development of MMP inhibitors for the treatment of various cancers. In the case study (iii), a small optimization library of compounds, based on the OCHF2-containing hit compound, SB-M-001, was generated and evaluated, which identified a fluorine-containing new lead compound, SB-M-103. SB-M-103 was found to inhibit tumor cell growth, migration, and invasion by effectively disrupting the MMP-9 homodimerization.

The curious effects of fluorine in therapeutic and diagnostic agents has been extensively investigated and successfully applied to the drug discovery and development in the last three decades. As a result, in the current drug design, fluorine is considered the second “favorite heteroatom” only after nitrogen. The incorporation of fluorine(s) into biologically active compounds often brings about pharmacologically beneficial properties. In this chapter, the critical significance of the strategic incorporation of fluorine(s) into therapeutic agents, which exerts “curious effects” of fluorine on potency and pharmacological properties is demonstrated by relevant examples and rationales are presented in each case. Those examples include fluorine-containing therapeutic drugs and agents, which have recently been approved by the US Food and Drug Administration or evaluated in Phase III clinical trials for the unmet medical needs, for example, Alzheimer’s disease, nonsmall cell lung cancer, hepatitis C, BASF mutant melanoma, diabetes, multidrug resistance tuberculosis, and malaria.

Inorganic arsenic (iAs) is a naturally occurring metalloid present in drinking water and polluted air exposing millions of people globally. Epidemiological studies have linked iAs exposure to the development of numerous diseases including cognitive impairment, cardiovascular failure and cancer. Despite intense research, an effective therapy for chronic arsenicosis has yet to be developed. Laboratory studies have been of great benefit in establishing the pathways involved in iAs toxicity and providing insights into its mechanism of action. However, the in vivo analysis of arsenic toxicity mechanisms has been difficult by the lack of reliable in vivo biomarkers of the effects of iAs. To resolve this issue we have applied the use of our recently developed stress reporter models to study iAs toxicity. The reporter mice Hmox1 (oxidative stress/inflammation; HOTT) and p21 (DNA damage) were exposed to iAs at acute and chronic, environmentally relevant, doses. We observed induction of the oxidative stress reporters in several cell types and tissues, which was largely dependent on the activation of transcription factor NRF2. We propose that our HOTT reporter model can be used as a surrogate biomarker of iAs-induced oxidative stress, and it constitutes a first-inclass platform to develop treatments in arsenicosis. Indeed, in a proof of concept experiment, the HOTT reporter mice were able to predict the therapeutic utility of the antioxidant N-acetyl cysteine in the prevention of iAs associated toxicity.

A unique asymmetric bow-tie poly(amidoamine) (PAMAM) dendrimer (ABTD) scaffold was designed and developed as a well-defined macromolecular carrier for tumor-targeted drug delivery. The ABTD scaffold in this study consists of a G3-half-dendron (G3-HD) unit and a G1-half-dendron (G1-HD) unit, bearing thiol moiety in each unit and a bis(maleimide) linker unit, which undergo sequential thiol–maleimide coupling to assemble the scaffold. This assembly methodology is applicable to all other combinations of different generations of PAMAM dendrimers. In the prototype ABTD in this study, 16 biotin moieties were tethered to the G3-HD unit and 4 payloads (new-generation taxoid) to the G1-HD via a self-immolative linker to form an ABTD-tumor-targeting conjugate (ABTD-TTC-1). Two other ABTD-TTCs were synthesized, wherein the G1-HD unit was tethered to a fluorescence-labeled taxoid or to a fluorescent probe. These three ABTD-TTCs were constructed by using a common key ABTD 6 bearing a terminal acetylene group in the G1-HD unit, which was fully characterized as a single molecule by high-resolution mass spectrometry and NMR despite its high molecular weight (Mw: 12 876). Then, the click reaction was employed to couple ABTD 6 with a small-molecule payload or fluorescence probe unit bearing a terminal azide moiety. ABTD-TTC-3, as a surrogate of ABTD-TTC-2, showed substantially enhanced internalization into two cancer cell lines via receptor-mediated endocytosis, attributed to multibinding effect. ABTD-TTC-1 exhibited a remarkable selectivity to cancer cells (1400–7500 times) compared to human normal cells, which demonstrates the salient feature and bright prospect of the ABTD-based tumor-targeted drug-delivery system.

Next-generation taxoids, such as SB-T-1214, are highly potent cytotoxic agents that exhibit remarkable efficacy against drug-resistant tumors in vivo, including those that overexpress the P-glycoprotein (Pgp) efflux pump. As SB-T-1214 is not a substrate for Pgp-mediated efflux, it may exhibit a markedly different biodistribution and tumor-accumulation profile than paclitaxel or docetaxel, which are both Pgp substrates. To investigate the biodistribution and tumor-accumulation levels of SB-T-1214 using positron emission tomography (PET), a new synthetic route has been developed to allow the incorporation of 11C, a commonly employed positron-emitting radionucleide, via methyl iodide at the last step of chemical synthesis. This synthetic route features a highly stereoselective chiral ester enolate–imine cyclocondensation, regioselective hydrostannation of the resulting β-lactam, and the Stille coupling of the novel vinylstannyl taxoid intermediate with methyl iodide. Conditions have been established to allow...

Theranostics will play a significant role in the next-generation chemotherapy. Two novel tumor-targeting theranostic drug conjugates, bearing imaging arms, were designed and synthesized. These theranostic conjugates consist of biotin as the tumor-targeting moiety, a second generation taxoid, SB-T-1214, as a potent anticancer drug, and two different imaging arms for capturing 99mTc for SPECT (single photon emission computed tomography) and 64Cu for PET (positron emission tomography). To explore the best reaction conditions for capturing radionuclides and work out the chemistry directly applicable to "hot" nuclides, cold chemistry was investigated to capture 185Re(I) and 63Cu(II) species as surrogates for 99mTc and 64Cu, respectively.

Background & Aims Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. Methods Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. Results TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. Conclusions Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress., Graphical abstract

The Nuclear factor (erythroid 2-derived)-like 2 (Nrf2) plays a key role in inflammation which is implicated in the pathophysiology of depression. The Nrf2 activators have antidepressant effects in animal models of depression. The present study was undertaken to examine whether TBE-31 [(±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile] and MCE-1 [(±)-3-ethynyl-3-methyl-6-oxocyclohexa-1,4-dienecarbonitrile], the novel Nrf2 activators, could show antidepressant effects in inflammation model of depression. We found that TBE-31 and MCE-1 significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration dependent manner. The Nrf2 siRNA, but not negative control of siRNA, significantly blocked the potentiating effects of TBE-31 and MCE-1 on neurite outgrowth in PC12 cells. Furthermore, oral administration of TBE-31 or MCE-1 significantly attenuated an increase in serum levels of tumor necrosis factor-α (TNF-α) after administration of lipopolysaccharide (LPS: 0.5mg/kg). In the tail-suspension test and forced swimming test, oral administration of TBE-31 or MCE-1 significantly attenuated an increase in the immobility time after LPS (0.5mg/kg) administration. These findings suggest that the novel Nrf2 activators such as TBE-31 and MCE-1 might be potential therapeutic drugs for inflammation-related depression.

Aberrant hyperactivation of NRF2 is a common event in many tumour types and associates with resistance to therapy and poor patient prognosis. The identification of ways to overcome the protection provided by NRF2 and selectively kill cancer cells addicted to NRF2 is a desirable goal. Exploiting the CRISPR/Cas9 technology, we generated colorectal cancer cell lines with hyperactive NRF2, and used them to perform a drug screen. We identified AT9283, an Aurora kinase inhibitor, for its selectivity towards killing cancer cells with hyperactive NRF2 as a consequence to either genetic or pharmacological activation. Our results show that hyperactivation of NRF2 presents a potential vulnerability that could be therapeutically exploited, and further suggest that AT9283, a drug that is currently in clinical trials, holds promise for the treatment of tumours with hyperactive NRF2.HighlightsWe present a new model for NRF2 hyperactivation in colorectal cancer cells.AT9283 selectively kills cancer cells with hyperactive NRF2Both genetic and pharmacological activation of NRF2 sensitise cells to AT9283

Aryl trifluoromethyl ethers (OCF3), aryl difluoromethyl ethers (OCHF2), and aromatic sulfur pentafluorides (SF5) are of particular interest in drug design due to their favorable properties including high chemical and metabolic stability, suitable lipophilicity, unique electronic properties, definitive conformational characteristics, and their roles in molecular recognition in binding sites of biological targets. The deployments of these functional groups are relatively rare, although these compounds are increasingly found in pharmaceutical agents. This chapter describes a number of FDA-approved drugs and drug candidates that contain OCF3, OCHF2, and SF5 groups: Riluzole for amyotrophic lateral sclerosis Delamanid and Pretomanid for tuberculosis, Pantoprazole for erosive esophagitis, Roflumilast for chronic obstructive pulmonary disease ELQ-300 and DSM-265 for malaria, BMS-665053 for depression, and a fluoro-analog of Rimonabant for obesity. The successful case studies substantiate the prominence of their utilities and synthetic enablement in drug discovery research.

Inorganic arsenic (iAs) is a naturally occurring metalloid present in drinking water and polluted air exposing millions of people globally. Epidemiological studies have linked iAs exposure to the development of numerous diseases including cognitive impairment, cardiovascular failure and cancer. Despite intense research, an effective therapy for chronic arsenicosis has yet to be developed. Laboratory studies have been of great benefit in establishing the pathways involved in iAs toxicity and providing insights into its mechanism of action. However, the in vivo analysis of arsenic toxicity mechanisms has been difficult by the lack of reliable in vivo biomarkers of iAs’s effects. To address this issue we have applied the use of our recently developed stress reporter models to study iAs toxicity. The reporter mice Hmox1 (oxidative stress/inflammation; HOTT) and p21 (DNA damage) were exposed to iAs at acute and chronic, environmentally relevant, doses. We observed induction of the oxidative stress reporters in several cell types and tissues, which was largely dependent on the activation of transcription factor NRF2. We propose that our HOTT reporter model can be used as a surrogate biomarker of iAs-induced oxidative stress, and it constitutes a first-in-class platform to develop treatments aimed to counteract the role of oxidative stress in arsenicosis. Indeed, in a proof of concept experiment, the HOTT reporter mice were able to predict the therapeutic utility of the antioxidant N-acetyl cysteine in the prevention of iAs associated toxicity.

Tricyclic, bicyclic, and monocyclic compounds containing cyanoenones induce various anti-inflammatory and cytoprotective enzymes through activation of the Keap1/Nrf2/ARE (antioxidant response element) pathway. The potency of these compounds as Nrf2 activators was determined using a prototypic cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in Hepa1c1c7 murine hepatoma cells. The electron affinity (EA) of the compounds, expressed as the energy of their lowest unoccupied molecular orbital [E (LUMO)], was evaluated using two types of quantum mechanical calculations: the semiempirical (AM1) and the density functional theory (DFT) methods. We observed striking linear correlations [r = 0.897 (AM1) and 0.936 (DFT)] between NQO1 inducer potency of these compounds and their E (LUMO) regardless of the molecule size. Importantly and interestingly, this finding demonstrates that the EA is the essentially important factor that determines the reactivity of the cyanoenones with Keap1.

Nitrous oxide, one of the earth-warming and ozone-destructing gases, is produced through either nitrification or denitrification depending on the O2 availability in soil. Aerobically denitrifying bacteria express denitrification tract even under the gas phase containing O2 at the ambient air level. The net reduction of exogenous N2O by novel aerobically denitrifying bacteria were studied. We carried out two different isolation strategies in the primary screening. One was to select isolates of interest out of periplasmic nitrate reductase-dependent denitrifying bacteria in a eutrophic condition. The other was to use diluted nutrient agar to allow the formation of colonies of diverse bacteria. Among aerobically denitrifying bacteria, those which showed net aerobic N2O reduction were only minor populations. As a result, eight isolates belonging to Proteobacteria were obtained from soil and cow manure. The denitrification and net N2O reduction properties of the three representative isolates, Pseudomon...

Summary Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH-associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism, allowing adaptation and survival under conditions of oxidative, inflammatory, and metabolic stress. Nrf2 is the principal determinant of redox homeostasis, and contributes to mitochondrial function and integrity and cellular bioenergetics. Using proteomics and lipidomics, we show that genetic downregulation of Keap1 in mice, and the consequent Nrf2 activation to pharmacologically relevant levels, leads to upregulation of carboxylesterase 1 (Ces1) and acyl-CoA oxidase 2 (Acox2), decreases triglyceride levels, and alters the lipidome. This is accompanied by downregulation of hepatic ATP-citrate lyase (Acly) and decreased levels of acetyl-CoA, a trigger for autophagy. These findings suggest that downregulation of Keap1 confers features of a fasted metabolic state, which is an important consideration in the drug development of Keap1-targeting pharmacologic Nrf2 activators., Graphical Abstract, Highlights • Keap1 downregulation in mice increases Ces1 and Acox2 and decreases triglyceride levels • Genetic interference with Keap1/Nrf2 alters the murine lipidome • Reduced expression of Keap1 lowers hepatic levels of acetyl-CoA • Deleting constitutively active Nrf2 decreases tubulin acetylation and autophagic flux, Human Metabolism; Molecular Biology; Omics

"Tropone" is a non-benzenoid aromatic skeleton that can be found in a variety of natural products. This cyclohepta-2,4,6-trien-1-one skeleton appears simple, but there have been no straightforward ways to construct this molecular architecture. It is conceivable that this molecule can be constructed via a higher order cycloaddition of three acetylene units and CO, but such process was not known until we have discovered that the carbonylative [2+2+2+1] cycloaddition of triynes can take place in the presence of a Rh complex catalyst and CO. However, this highly challenging process is naturally accompanied by ordinary [2+2+2] cyclotrimization products, i.e., benzenes, as side products. A mechanistic study led to two competing processes wherein the critical CO insertion occurs either to a rhodacyclopentadiene intermediate (Path A) or a rhodacycloheptatriene intermediate (Path B). The DFT analysis of those two pathways disclosed that the Path A should be the one that yields the carbonylative [2+2+2+1] cycloaddition products, i.e., fused tricyclic tropones. A further substrate design, inspired by colchicine structure, led to the almost exclusive formation of a fused tetracyclic tropone from a triyne bearing 1,2-disubstituted benzene moiety in a single step and excellent yield.

The nonbenzenoid aromatics, tropones and tropolones, are found in various natural products such as colchicine and hinokitol, which possess significant biological activities. The traditional methods to construct the tropone skeletons include oxidation of cycloheptatriene and [4+3] cycloadditions. In addition, the total synthesis of colchicine and its analogues requires laborious organic transformations in the formations of 6-7-7 fused-ring systems. Transition metal-catalyzed carbocyclization and cycloaddition reactions have proven to be among the most efficient methods for constructing complex polycyclic systems. On the basis of our recent discovery of the Rh-catalyzed carbonylative [2+2+2+1] cycloaddition of triynes to the formation of a fused tropone system, we report here the application of this methodology to the one-step formation of the 6-7-7-5 fused tetracyclic scaffold of colchicinoids based on the [2+2+2+1] cycloaddition of o-phenylenetriynes with CO. In addition, the one-step formation of allocolchicinoids bearing the 6-7-6-5 fused tetracyclic system through the Rh-catalyzed [2+2+2] cycloaddition of o-phenylenetriynes is also described.

Next-generation taxoids, such as SB-T-1214, are highly potent cytotoxic agents that exhibit remarkable efficacy against drug-resistant tumors in vivo, including those that overexpress the P-glycoprotein (Pgp) efflux pump. As SB-T-1214 is not a substrate for Pgp-mediated efflux, it may exhibit a markedly different biodistribution and tumor-accumulation profile than paclitaxel or docetaxel, which are both Pgp substrates. To investigate the biodistribution and tumor-accumulation levels of SB-T-1214 using positron emission tomography (PET), a new synthetic route has been developed to allow the incorporation of

2 p.- Correction to: Scientific Reports https://doi.org/10.1038/s41598-017-01678-4, published online 10 May 2017

The transcription factor Nrf2 determines the ability to adapt and survive under conditions of electrophilic, oxidative, and inflammatory stress by regulating the expression of elaborate networks comprising nearly 500 genes encoding proteins with versatile cytoprotective functions. In mice, disruption of Nrf2 increases susceptibility to carcinogens and accelerates disease pathogenesis. Paradoxically, Nrf2 is upregulated in established human tumors, but whether this upregulation drives carcinogenesis is not known. Here we show that the incidence, multiplicity, and burden of solar-simulated UV radiation–mediated cutaneous tumors that form in SKH-1 hairless mice in which Nrf2 is genetically constitutively activated are lower than those that arise in their wild-type counterparts. Pharmacologic Nrf2 activation by topical biweekly applications of small (40 nmol) quantities of the potent bis(cyano enone) inducer TBE-31 has a similar protective effect against solar-simulated UV radiation in animals receiving long-term treatment with the immunosuppressive agent azathioprine. Genetic or pharmacologic Nrf2 activation lowers the expression of the pro-inflammatory factors IL6 and IL1β, and COX2 after acute exposure of mice to UV radiation. In healthy human subjects, topical applications of extracts delivering the Nrf2 activator sulforaphane reduced the degree of solar-simulated UV radiation–induced skin erythema, a quantifiable surrogate endpoint for cutaneous damage and skin cancer risk. Collectively, these data show that Nrf2 is not a driver for tumorigenesis even upon exposure to a very potent and complete carcinogen and strongly suggest that the frequent activation of Nrf2 in established human tumors is a marker of metabolic adaptation. Cancer Prev Res; 8(6); 475–86. ©2015 AACR.

The extraordinary potential of fluorine in therapeutic and diagnostic agents has been extensively studied and successfully exploited for the drug discovery and development in the past three decades. Accordingly, in the current drug design, fluorine is considered the second “favorite heteroatom” only after nitrogen. The strategic incorporation of fluorine(s) into biologically active compounds often brings about pharmacologically beneficial properties. 18 F-positron emission tomographic (PET) imaging enables rapid and accurate assessment of biodistribution and metabolic profiles of drug candidates. Also, 18 F-PET imaging can be used for diagnosing neurological disorders such as Alzheimer disease. In this chapter, selected new fluorine-containing drugs, which have recently been approved by the US Food and Drug Administration or evaluated in phase III clinical trials for the unmet medical needs, are described, wherein the significance of the strategic incorporation of fluorine(s) in these drugs, as well as the modern synthetic processes employed for the production of these drugs, is discussed.

Tricyclic bis(cyanoenone), TBE-31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti-inflammatory and cytoprotective agent. (13) C2 (15) N2 -labeled TBE-31 ([(13) C2 (15) N2 ]-TBE-31), which has two (13) C and two (15) N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE-31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope-labeled internal standard. [(13) C2 (15) N2 ]-TBE-31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two (13) C atoms with ethyl [(13) C]formate and two (15) N atoms with hydroxyl[(15) N]amine. The stable isotope dilution liquid chromatography-mass spectrometry method for quantification of TBE-31 was successfully developed using [(13) C2 (15) N2 ]-TBE-31 to compensate for any variables encountered during sample processing and analysis.

An improved synthesis of hydroxymethyl tricyclic ketone, (±)-(4aS,8aS)-8a-(hydroxymethyl)-1,1,4a-trimethyl-3,4,4a,6,7,8,8a,9,10,10a-decahydrophenanthren-2(1H)-one, in five steps (34% yield) from cyclohexanone has been successfully established. Accordingly, 10 grams of a highly potent anti-inflammatory and cytoprotective agent, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile (TBE-31), was obtained in 15 steps (9.2% overall yield) via the hydroxymethyl tricyclic ketone from 32 grams of cyclohexanone.

The microwave heating drastically accelerates Diels–Alder cycloadditions between Danishefsky's diene and derivatives of ethyl α-(hydroxymethyl)acrylate whose hydroxyl group is protected with various protective groups to give previously unknown adducts, which are necessary as intermediates for the synthesis of a biotin conjugate of a monocyclic cyanoenone with high anti-inflammatory activity. The reaction time is only 1 h and the average yield is approximately 80%. Compared to the traditional thermal conditions this method requires 1/48th to 1/14th of the time and the yields are 2–7 times more.

Chronic treatment with azathioprine, a highly effective anti-inflammatory and immunosuppressive agent, profoundly increases the risk for development of unusually aggressive cutaneous squamous cell carcinoma. Its ultimate metabolite, 6-thioguanine (6-TG) nucleotide, is incorporated in DNA of skin cells, and upon exposure to UVA radiation, causes oxidative stress, followed by damage of DNA and associated proteins. The acetylenic tricyclic bis(cyano enone) TBE-31 is a strong inhibitor of inflammation and a potent inducer of the Keap1/Nrf2/ARE pathway, which orchestrates the expression of a large network of cytoprotective genes. We now report that long-term (five days per week for four weeks) topical daily applications of small (200 nmol) quantities of TBE-31 cause a robust systemic induction of the Keap1/Nrf2/ARE pathway and decreases the 6-TG incorporation in DNA of skin, blood, and liver of azathioprine-treated mice, indicating extraordinary bioavailability and efficacy. In addition, TBE-31, at nanomolar concentrations, protects cells with 6-TG in their genomic DNA against oxidative stress caused by UVA radiation through induction of the Keap1/Nrf2/ARE pathway. At the same 6-TG DNA levels, Keap1-knockout cells, in which the pathway is constitutively upregulated, are highly resistant to UVA radiation–induced oxidative stress. The protective effects of both the Keap1-knockout genotype and TBE-31 are completely lost in the absence of transcription factor Nrf2. Our findings suggest that compounds of this kind are excellent candidates for mechanism-based chemoprotective agents against conditions in which oxidative stress and inflammation underlie disease pathogenesis. Moreover, their potential skin patch incorporation for transdermal delivery is an exciting possibility. Cancer Prev Res; 5(7); 973–81. ©2012 AACR.

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1β with potencies that are higher than those of bardoxolone methyl and TBE-31.

A novel acetylenic tricyclic bis-(cyano enone), TBE-31, is a lead compound in a series of tricyclic compounds with enone functionalities in rings A and C. Nanomolar concentrations of this potent multifunctional molecule suppress the induction of the inflammatory protein, inducible nitric oxide synthase, activate phase 2 cytoprotective enzymes in vitro and in vivo, block cell proliferation, and induce differentiation and apoptosis of leukemia cells. Oral administration of TBE-31 also significantly reduces formation of aflatoxin-DNA adducts and decreases size and number of aflatoxin-induced preneoplastic hepatic lesions in rats by >90%. Because of the two cyano enones in rings A and C, TBE-31 may directly interact with DTT and protein targets such as Keap1 that contain reactive cysteine residues. The above findings suggest that TBE-31 should also be tested for chemoprevention and chemotherapy in relevant models of cancer and against other chronic, degenerative diseases in which inflammation and oxidative stress contribute to disease pathogenesis. [Cancer Res 2008;68(16):6727–33]

Inducible phase 2 enzymes constitute a primary line of cellular defense. The oleanane dicyanotriterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile (TP-225) is a very potent inducer of these systems. Topical application of TP-225 to SKH-1 hairless mice increases the levels of NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) and protects against UV radiation-induced dermal thickening. Daily topical treatments of 10 nmol of TP-225 to the backs of mice that were previously subjected to low-level chronic UVB radiation (30 mJ/cm(2)/session, twice a week for 17 weeks), led to 50% reduction in multiplicity of skin tumors. In addition, the total tumor burden of squamous cell carcinomas was reduced by 5.5-fold. The identification of new agents for protection against UV radiation-induced skin cancer and understanding of their mechanism(s) of action is especially important in view of the fact that human skin cancers represent a significant source of increasing morbidity and mortality.