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An efficient method for regioselective synthesis of C-7 Mannich bases of 6-hydroxyaurones was accomplished by the N,N-dialkylaminomethylation using aminals prepared from dimethylamine, dipropylamine, bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and 1-methylpiperazine. Further transformation of 7-(N,N-dialkylamino)methyl group in these aurones led to formation of C-7 acetoxymethyl and methoxymethyl derivatives of 6-hydroxyaurones, some of which showed promising inhibition of PC-3 prostate cancer cell proliferation in the high nanomolar to low micromolar range that exceeded that of cisplatin. Compound 12c (R3 = Ac, Ar = 3,4-OMePh) displays 75% inhibition of PC-3 prostate cancer cells proliferation at 300 nM concentration.

Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQ), such as N7,N7-dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy–homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc–driven cancers.

Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 μM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.

Background Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related death worldwide. Adequate treatment options for patients with advanced HCC are currently limited. Materials and methods We studied the anti-HCC effect of FH535 and a novel derivative Y3, on proliferation, mitochondrial function and cellular metabolism focusing on the three key substrates, glutamine, glucose, and fatty acids. Results FH535 and Y3 disrupted mitochondrial redox control in HCC cells that resulted from uncoupling mechanisms that increased proton leakage and decreased ATP production leading to apoptosis. The uncoupling effects of the sulfonamides in HCC cells were supported by the loss of activity of the methylated analogs. The accumulation of ROS significantly contributed to cell damage after the impaired autophagic machinery. These sulfonamides, FH535 and Y3, targeted glutamine and fatty acid metabolism and caused HCC cell reprograming towards the preferential use of glucose and the glycolytic pathway. Conclusions FH535, and Y3, demonstrated potent anti-HCC activity by targeting OXPHOS, increasing dangerous levels of ROS and reducing ATP production. These sulfonamides target glutamine and FA metabolic pathways significantly increasing the cellular dependency on glycolysis.

The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast glycogen synthase 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the UDP-glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure-activity relationship (SAR) profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Androgen-deprivation therapy (ADT) is only a palliative measure, and prostate cancer invariably recurs in a lethal, castration-resistant form (CRPC). Prostate cancer resists ADT by metabolizing weak, adrenal androgens to growth-promoting 5α-dihydrotestosterone (DHT), the preferred ligand for the androgen receptor (AR). Developing small-molecule inhibitors for the final steps in androgen metabolic pathways that utilize 17-oxidoreductases required probes that possess fluorescent groups at C-3 and intact, naturally occurring functionality at C-17. Application of the Pictet-Spengler condensation to substituted 4-(2-aminoethyl)coumarins and 5α-androstane-3-ones furnished spirocyclic, fluorescent androgens at the desired C-3 position. Condensations required the presence of activating C-7 amino or N,N-dialkylamino groups in the 4-(2-aminoethyl)coumarin component of these condensation reactions. Successful Pictet-Spengler condensation, for example, of DHT with 9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one led to a spirocyclic androgen, (3R,5S,10S,13S,17S)-17-hydroxy-10,13-dimethyl-1,2,2’,3’,4,5,6,7,8,8’,9,9’,10,11,12,12’,13,13’,14,15,16,17-docosahydro-7’H,11’H-spiro-[cyclopenta[a]phenanthrene-3,4’-pyrido[3,2,1-ij]pyrido[4’,3’:4,5]pyrano[2,3-f]quinolin]-5’(1’H)-one. Computational modeling supported the surrogacy of the C-3 fluorescent DHT analog as a tool to study 17-oxidoreductases for intracrine, androgen metabolism.

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

N,N′-Diaryl-bishydrazones of [1,1′-biphenyl]-3,4′-dicarboxaldehyde, [1,1′-biphenyl]-4,4′-dicarboxaldehyde, and 4,4′-bisacetyl-1,1-biphenyl exhibited excellent antifungal activity against a broad spectrum of filamentous and non-filamentous fungi. These N,N′-diaryl-bishydrazones displayed no antibacterial activity in contrast to previously reported N,N′-diamidino-bishydrazones and N-amidino-N′-aryl-bishydrazones. The leading candidate, 4,4′-bis((E)-1-(2-(4-fluorophenyl)hydrazono)ethyl)-1,1′-biphenyl, displayed less hemolysis of murine red blood cells at concentrations at or below that of a control antifungal agent (voriconazole), was fungistatic in a time-kill study, and possessed no mammalian cytotoxicity and no toxicity with respect to hERG inhibition.

Fluorinated aryl- and heteroaryl-substituted monohydrazones displayed excellent broad-spectrum activity against various fungal strains, including a panel of clinically relevant Candida auris strains relative to a control antifungal agent, voriconazole (VRC). These monohydrazones displayed less hemolysis of murine red blood cells than that of VRC at the same concentrations, possessed fungicidal activity in a time-kill study, and exhibited no mammalian cell cytotoxicity. In addition, these monohydrazones prevented the formation of biofilms that otherwise block antibiotic effectiveness and did not trigger the development of resistance when exposed to C. auris AR Bank # 0390 over 15 passages.

// Michael V. Fiandalo 1 , John J. Stocking 1 , Elena A. Pop 1 , John H. Wilton 1, 2 , Krystin M. Mantione 2 , Yun Li 1 , Kristopher M. Attwood 3 , Gissou Azabdaftari 4 , Yue Wu 1 , David S. Watt 5 , Elizabeth M. Wilson 6 and James L. Mohler 1 1 Department of Urology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 2 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 3 Department of Biostatistics and Bioinformatics Roswell Park Cancer Institute, Buffalo, NY 14263, USA 4 Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 5 Center for Pharmaceutical Research and Innovation and Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA 6 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA Correspondence to: James L. Mohler, email: James.Mohler@RoswellPark.org Keywords: androstanediol; dihydrotestosterone; dutasteride; 3α-oxidoreductases; androgen deprivation therapy Received: September 06, 2017 Accepted: November 19, 2017 Published: January 10, 2018 ABSTRACT Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth.

Fluorinated, phenylethynyl-substituted heterocycles that possessed either an N-methylamino or N,N-dimethylamino group attached to heterocycles including pyridines, indoles, 1H-indazoles, quinolines, and isoquinolines inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21Wif1/Cip1) served as a readout for antineoplastic activity at a cellular level. On a molecular level, these agents, particularly 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine, bound and inhibited the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A).

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. More than 50% of patients with colorectal cancer will develop local recurrence or distant organ metastasis. Cancer stem cells play a major role in the survival and metastasis of cancer cells. In this study, we examined the effects of novel AMP-activated protein kinase (AMPK) activating compounds on colorectal cancer metastatic and stem cell lines as potential candidates for chemotherapy. We found that activation of AMPK by all fluorinated N,N-diarylureas (FND) compounds at micromolar levels significantly inhibited the cell-cycle progression and subsequent cellular proliferation. In addition, we demonstrated that select FNDs significantly increased apoptosis in colorectal cancer metastatic and cancer stem cells. Therefore, FNDs hold considerable promise in the treatment of metastatic colorectal cancer, through elimination of both regular cancer cells and cancer stem cells. Mol Cancer Ther; 16(5); 831–7. ©2017 AACR.

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional, peroxisomal enzyme, hydroxysteroid 17β-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the D-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.

SUMMARY The induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ), is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth., Graphical abstract

INTRODUCTION AND OBJECTIVES:Men with advanced prostate cancer (CaP) receive androgen deprivation therapy (ADT), which lowers circulating testosterone (T) levels, impairs androgen receptor (AR) acti...

Autophagy targets cellular components for lysosomal-dependent degradation in which the products of degradation may be recycled for protein synthesis and utilized for energy production. Autophagy also plays a critical role in cell homeostasis and the regulation of many physiological and pathological processes and prompts this investigation of new agents to effect abnormal autophagy in hepatocellular carcinoma (HCC). 2,5-Dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide (FH535) is a synthetic inhibitor of the Wnt/β-catenin pathway that exhibits anti-proliferative and anti-angiogenic effects on different types of cancer cells. The combination of FH535 with sorafenib promotes a synergistic inhibition of HCC and liver cancer stem cell proliferation, mediated in part by the simultaneous disruption of mitochondrial respiration and glycolysis. We demonstrated that FH535 decreased HCC tumor progression in a mouse xenograft model. For the first time, we showed the inhibitory effect of an FH535 derivative, FH535-N, alone and in combination with sorafenib on HCC cell proliferation. Our study revealed the contributing effect of Wnt/β-catenin pathway inhibition by FH535 and its derivative (FH535-N) through disruption of the autophagic flux in HCC cells.

PurposeTriple negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer. AMP-activated protein kinase (AMPK) is a major energy regulator that suppresses tumor growth, and 1-(3-chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) is a novel AMPK activator that inhibits growth and induces apoptosis in colon cancer. The purpose of this project was to test the effects of FND-4b on AMPK activation, proliferation, and apoptosis in breast cancer with a particular emphasis on TNBC.Materials and methods(i) Estrogen-receptor positive breast cancer (ER+BC; MCF-7, and T-47D), TNBC (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND-4b for 24h. Immunoblot analysis assessed AMPK, acetyl-CoA carboxylase (ACC), ribosomal protein S6, cyclin D1, and cleaved PARP. (ii) Sulforhodamine B growth assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. Proliferation was also assessed by counting cells after 72h of FND-4b treatment. (iii) Cell death ELISA assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h.Results(i) FND-4b increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in all subtypes. (ii) FND-4b decreased proliferation in all cells, while dose-dependent growth decreases were found in ER+BC and TNBC. (iii) Increases in apoptosis were observed in ER+BC and the MDA-MB-231 cell line with FND-4b treatment.ConclusionsOur findings indicate that FND-4b decreases proliferation for a variety of breast cancers by activating AMPK and has notable effects on TNBC. The growth reductions were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+BC cells were more susceptible to FND-4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose treatment. Further development of FND compounds could result in a novel therapeutic for TNBC.

An improved pre-clinical cardiac chemical exchange saturation transfer (CEST) pulse sequence (cardioCEST) was used to selectively visualize paramagnetic CEST (paraCEST)-labeled cells following intramyocardial implantation. In addition, cardioCEST was used to examine the effect of diet-induced obesity upon myocardial creatine CEST contrast. CEST pulse sequences were designed from standard turbo-spin-echo and gradient-echo sequences, and a cardiorespiratory-gated steady-state cine gradient-echo sequence. In vitro validation studies performed in phantoms composed of 20 mM Eu-HPDO3A, 20 mM Yb-HPDO3A, or saline demonstrated similar CEST contrast by spin-echo and gradient-echo pulse sequences. Skeletal myoblast cells (C2C12) were labeled with either Eu-HPDO3A or saline using a hypotonic swelling procedure and implanted into the myocardium of C57B6/J mice. Inductively coupled plasma mass spectrometry confirmed cellular levels of Eu of 2.1 × 10(-3) ng/cell in Eu-HPDO3A-labeled cells and 2.3 × 10(-5) ng/cell in saline-labeled cells. In vivo cardioCEST imaging of labeled cells at ±15 ppm was performed 24 h after implantation and revealed significantly elevated asymmetric magnetization transfer ratio values in regions of Eu-HPDO3A-labeled cells when compared with surrounding myocardium or saline-labeled cells. We further utilized the cardioCEST pulse sequence to examine changes in myocardial creatine in response to diet-induced obesity by acquiring pairs of cardioCEST images at ±1.8 ppm. While ventricular geometry and function were unchanged between mice fed either a high-fat diet or a corresponding control low-fat diet for 14 weeks, myocardial creatine CEST contrast was significantly reduced in mice fed the high-fat diet. The selective visualization of paraCEST-labeled cells using cardioCEST imaging can enable investigation of cell fate processes in cardioregenerative medicine, or multiplex imaging of cell survival with imaging of cardiac structure and function and additional imaging of myocardial creatine.

The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485 000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Therea...

Standard paclitaxel-platinum-based chemotherapies often lead to relapses and chemoresistant diseases. New therapeutic strategies are urgently needed for improving the clinical outcomes of ovarian caner patients. Our goal is to identify dysfunctional cellular pathways that are critical for tumor progression and drug resistance and to design novel therapeutic interventions that affect these altered cellular functions. The dysregulation of unfolded protein response (UPR) pathway is often found in cancer cells and contributes to cancer cell survival and their resistance to stress caused by chemotherapies, hypoxia, and nutrition deprivation. The objective of our research is to develop new therapeutic agents to target UPR in ovarian cancer cells in order to overcome chemoresistance. Sulfonamides (SFs) have been used to synthesize antibacterial drugs. We have recently discovered a family of new SFs with anticancer activity. Based on preliminary study, we hypothesize that these new SFs induce apoptosis in ovarian cancer cells through targeting UPR. Using one of the SFs, namely SF-Y3, we compared its effects on epithelial ovarian cancer (EOC) cell lines and immortalized normal fallopian tube (FT) cell lines by performing luminescent CellTiter assay. Phospho-S6 ribosomal protein (P-S6) staining and Annexin V-FITC/PI staining assays were performed to evaluate the effects of SF-Y3 on cell health. Human transcriptome array (HTA) was used to identify the gene expression changes in SF-Y3-treated EOC cells, which results were confirmed by quantitative real-time PCR (qPCR). Western blot and XBP1 RNA splicing PCR were performed to assess the activation of proteins in UPR pathway. Using 4u8c, an inhibitor of the ER transmembrane protein IRE1, we determined whether inhibiting UPR could rescue cancer cells from the SF-Y3-induced apoptosis. Co-immunoprecipitation (co-PI) was used to determine the effects of SF-Y3 on the ER membrane protein-protein interaction. Moreover, we encapsulated SF-Y3 with nanoparticle to improve its bioavailability for evaluating the in vivo efficacy in EOC mouse model as a single treatment and in combination with platinum-based chemotherapy. The cell viability data demonstrated that SF-Y3 significantly reduced the viability of EOC cells expressing high levels of Bip1, a key chaperone protein in the endoplasmic. SF-Y3 was less effective in EOC cells with low levels of Bip1 and has no effects on normal FT cells. P-S6 and Annexin V staining assays demonstrated that SF-Y3 inhibited EOC cell proliferation and induced apoptosis. HTA and qPCR data showed that the UPR genes were unregulated by SF-Y3. Western blot and XBP1 RNA splicing PCR results indicated that SF-Y3 activated proteins in the UPR pathway, including ATF6, PERK, eIF2α, XBP1, and CHOP. SF-Y3 interrupted the interaction between Bip1 and three ER membrane-associated sensors, supporting that Bip1 is a possible target of SF-Y3. UPR inhibitor 4u8c partially rescued the apoptosis induced by SF-Y3. These data support that SF-Y3 has anticancer activity in EOC models possibly through inhibiting Bip1 and inducing UPR-induced apoptosis. Further investigation of how SFs interact with Bip1 and UPR pathway in vitro and in vivo may lead to new approaches to overcome drug resistance and a significant therapeutic advance for EOC. Citation Format: Wonmin Park, Tobias MP. Hartwich, Kay Y. Chong, Chunming Liu, David S. Watt, Dongin Kim, Yang Yang-Hartwich. TARGETING UNFOLDED PROTEIN RESPONSE FOR OVARIAN CANCER THERAPY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-116.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US with the majority of deaths due to metastatic disease. Current chemotherapeutic regimens involve highly toxic agents, which limits their utility; therefore, more effective and less toxic agents are required to see a reduction in CRC mortality. Novel fluorinated N,N'-diarylureas (FND) were developed and characterized by our group as potent activators of adenosine monophosphate-activated kinase (AMPK) that inhibit cell cycle progression. The purpose of this study was to determine the effect of a lead FND compound, FND-4b, either alone or combined with PI-103 (a dual PI3K/mTOR inhibitor) or SN-38 (active metabolite of irinotecan) on cell cycle arrest and apoptosis of CRC cell lines (both commercially-available and novel lines established from our patient population). Treatment with FND-4b for 24h resulted in a marked induction of phosphorylated AMPK expression and a concomitant reduction in markers of cell proliferation, such as cyclin D1, in all CRC cell lines. Apoptosis was also notably increased in CRC cells treated with FND-4b. Regardless of the genetic profile of the CRC cells, FND-4b treatment alone resulted in decreased cell proliferation. Moreover, the combination of FND-4b with PI-103 resulted in increased cell death in all cell lines, while the combination of FND-4b with SN-38 resulted in increased cell death in select cell lines. Our findings identify FND-4b, which activates AMPK at micromolar concentrations, as a novel and effective inhibitor of CRC growth either alone or in combination with PI-103 and SN-38.

Nitrosation and cyclization of 4-(3-aminothieno[2,3-b]pyridine-2-yl)-2H-chromen-2-ones 1 afforded substituted 6H-chromeno[3,4-c]pyrido[3',2':4,5]thieno[2,3-e]pyridazin-6-ones 2 that inhibited the intermediary filament protein, vimentin, at low micromolar concentrations. This inhibition promoted the secretion of Prostate Apoptosis Response-4 protein (Par-4), which selectively triggered apoptosis in prostate cancer cells such as CWR22Rv1, LNCaP-derivative C4-2B, PC-3 and its aggressive analog, PC-3 MM2.

Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled ( E , E )-1-[ 3 H]-2,5-bis(4′-hydroxy-3′-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils.

Adenosine monophosphate-activated kinase (AMPK) plays a central role in regulating energy homeostasis in eukaryotic cells. AMPK also regulates lipid synthesis by inhibiting acetyl-CoA carboxylase (ACC) and regulates mTOR signaling by activating TSC2. Due to its important roles in cell metabolism, AMPK is an attractive target for metabolic diseases, such as type II diabetes and obesity. AMPK activators, such as metformin, that are used for diabetes treatment are also effective anticancer agents. However, the efficacies of many known AMPK activators are relatively low. For example, metformin activates AMPK at millimolar levels. In this study, we identified a novel family of AMPK activators, namely fluorinated N,N’-diarylureas, that activate AMPK at 1–3µM concentrations. These novel agents strongly inhibit the proliferation of colon cancer cells. We studied the potential mechanisms of these agents, performed a structure-activity relationship (SAR) study and identified several fluorinated N,N’- diarylureas as potent AMPK activators.

Mycosin protease-1 (MycP1) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP1 variants from Mycobacterium thermoresistible (MycP1mth), Mycobacterium smegmatis (MycP1msm) and M. tuberculosis (MycP1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)2 (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO2(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC50 values 121.6±25.3 μM for MycP1mth, 93.2±37.3 μM for MycP1msm and 37.9±5.2 μM for MycP1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP1 catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs.

In an effort to screen natural compounds and their derivatives as novel agents for cancer treatment, we developed two semisynthetic nature compounds, A14 and A52, which have potencies in an in vitro cell proliferation studies against LS174T and PC-3 cells in the mid-nanomolar range. A14 inhibited more than 90% of in vitro cancer cell proliferation around 300 nM and also inhibited in vivo PC-3 prostate cancer xenografts in nude mice. Cell morphology studies and comparisons with known antineoplastic agents suggested that A14 disrupted microtubule organization. In vitro tubulin polymerization assay suggested that A14 inhibited this process. Based on molecular docking, A14 fits very well in the colchicine-binding site at the interface between the alpha-tubulin and beta-tubulin. Further analyses suggest that A14 sensitivity may correlate with CDK/cyclin activity. A14 inhibited PC-3 prostate cancer xenografts in nude mice. In addition to solid tumors, A14 and its analog A52 strongly inhibited a panel of human leukemia cancer cell lines. Furthermore, these compounds inhibited Myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model. Although many tubulin inhibitors were available, there is no compound that targeting the colchicine-binding site has been approved by FDA for cancer treatment. This new family of tubulin polymerization inhibitors could be further developed for the treatment of both solid and liquid tumors. Citation Format: Yanqi Xie, Liliia Kril, Wen Zhang, Mykhaylo Frasinyuk, Svitlana Bondarenko, Elizabeth Hausman, Zachary Martin, Przemyslaw Wyrebek, Tianxin Yu, Changguo Zhan, Vitaliy Sviripa, Jessica Blackburn, David Watt, Chunming Liu. Novel tubulin polymerization inhibitors repress tumor xenografts in nude mice and leukemia in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2674.

Purpose: The aim of this paper is to raise awareness within built environment and associated disciplines regarding the potential for initiating and triggering events associated with chemical sensitivity as a result of chemical treatments used for the control or eradication of fungal infections and/or insect infestations in buildings. Design: Literature review and interviews with affected individuals. Materials and methods: The information has been obtained by literature search and review of published sources from built environment and associated disciplines; also from personal communications with those affected by chemical treatments and treatment residues in buildings. Results: Chemical treatments used for the control or eradication of fungal infections and/or insect infestations in buildings and the residues of such treatments do not pose a proven hazard to the general population. There is, however, an apparent potential risk from such treatments and from the presence of treatment residues in treated bu...

Two metabolites of the tranquilizer azaperone were extracted from alkalinized horse urine after treatment with beta-glucuronidase/sulfatase from limpets (Patella vulgata). The metabolites were identified by a combination of independent chemical synthesis and GC/MS and 1H NMR analysis. The metabolites were identified as 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanol, designated as 5'-hydroxy-azaperol, and 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanone, designated as 5'-hydroxyazaperone. A TLC screening test was developed for detecting both metabolites in basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The screening test was used to detect azaperone metabolites in extracts of horse urine collected for 24 h after intravenous administration of azaperone. The administration of azaperone to horses was confirmed by GC/MS identification of 5'-hydroxyazaperone and 5'-hydroxyazaperol from basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The extracted metabolites were treated with bis(trimethylsilyl)acetamide to produce trimethylsilyl (TMS) ether derivatives, and mass spectra and retention times were compared to those of the synthesized metabolites treated in the same manner.

In oils and Precambrian- to Miocene-age source rocks from varying depositional environments, we have conclusively identified several novel 3-alkyl sterane and triaromatic steroid series, including (1) 3β-n-pentyl steranes, (2) 3β-isopentyl steranes, (3) 3β-n-hexyl steranes, (4) 3β-n-heptyl steranes, (5) 3,4-dimethyl steranes, (6) 3β-butyl,4-methyl steranes, (7) triaromatic 3-n-pentyl steroids, and (8) triaromatic 3-isopentyl steroids. We have also tentatively identified additional homologs with 3-alkyl substituents as large as C11. The relative abundances of these compounds vary substantially between samples, as indicated by (1) the ratio of 3β-n-pentyl steranes to 3β-isopentyl steranes and (2) the ratio of 3-n-pentyl triaromatic steroids to 3-isopentyl triaromatic steroids. These data suggest possible utility for these parameters as tools for oil-source rock correlations and reconstruction of depositional environments. Although no 3-alkyl steroid natural products are currently known, several lines of evidence suggest that 3β-alkyl steroids result from bacterial side-chain additions to diagenetic Δ2-sterenes.

Colorectal (CRC) cancer is the second leading cause of cancer deaths in the US; treatment of metastatic CRCs is limited due to drug resistance and eventual relapse. Cancer stem cells and PI3K mutation (i.e., pik3ca mutation) have been implicated in the relapse and lack of treatment response for many cancers including CRCs; therefore, it is critical to develop a therapeutic strategy that eliminates both the fast-growing cancer cells and the more resistant cancer stem cells. The purpose of the present study was to evaluate the anticancer activity of recently-described, novel AMPK activators, fluorinated N, N-diarylureas (FNDs), against CRC metastatic cell lines, pik3ca mutant cell lines, and CRC stem cells. METHODS. i) First, to assess AMPK activation, metastatic CRC cell lines (HT29 and KM20 cells), HCT116 pik3ca wild-type (WT) and mutant (MUT) cell lines, and CRC stem cell lines (from Celprogen) were treated with varying concentrations of 8 FNDs (4a, 4b, 4h, 4j, 4k, 4z, 4aa, 4bb). AMPK activation and activation of the upstream regulator, LKB1, was assessed by western blot. ii) Next, we determined the effect of the FNDs and, for comparison, metformin (an AMPK activator) on induction of cell cycle suppression and induction of apoptosis as assessed by cyclin D1 expression and PARP cleavage, respectively; β-actin antibody was used as a loading control. RESULTS. i) Treatment with the FNDs resulted in AMPK activation in HT29 and KM20 cells and CRC stem cells at concentrations as low as 10 μM with a peak activation at 12 h. We observed LKB1-independent AMPK kinase activation. Decreased LKB1 phosphorylation after FND treatment was dose dependent (i.e., decreased by ∼50% at 10 μM and undetectable at 50 μM). ii) All 8 FNDs (at a 25 μM dosage) completely suppressed cyclin D1 expression in HT29 and KM20 cells; a similar cyclin D1 suppression was noted with metformin at a 500x higher dosage (i.e., 10mM). Five of the 8 FNDs (4b, 4j, 4z, 4aa, 4bb) increased PARP cleavage in HT29 and KM20 cells. We used these 5 FNDs (at a dosage of 50 μM each) to treat the CRC stem cell lines; cyclin D1 suppression was noted for all 5 FNDs but strong induction of apoptosis was only identified using the 4b FND. Finally, we treated HCT116 pik3ca WT and MUT cell lines with 4b FND and found that cyclin D1 expression was completely suppressed at 40 μM and induction of apoptosis was noted at a dosage of 20 μM. CONCLUSIONS. We demonstrate cell cycle suppression and apoptosis in CRC cells and stem cells using the recently-developed FND compounds with the 4b compound as the most effective. These compounds appear to have considerable promise as a targeted cancer stem cell-specific agents and offer a potentially novel strategy for the treatment of CRC metastases. Citation Format: Piotr Rychahou, Dasha Kenlan, Vitaliy M. Sviripa, David S. Watt, B. Mark Evers. Fluorinated N, N-diarylureas treatment as effective strategy to target colorectal cancer stem 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 1222.

We have raised antibodies and developed one-step enzyme-linked immunosorbent assays (ELISA) for the diuretics ethacrynic acid and bumetanide as part of a panel of pre- and post-race tests for high potency drugs in racing horses. These ELISA tests are rapid (completed within one hour), sensitive, and can be read by eye. The ELISA detects ethacrynic acid at a drug concentration for half-maximal inhibition (I-50) of about 2.5 ng/mL for the parent drug. After dosing horses intravenously with 5 mg ethacrynic acid per horse, the parent drug or its metabolites are detectable in urine for at least 8 hours. The bumetanide ELISA has an I-50 for the parent drug of about 2.0 ng/mL and will detect bumetanide or its metabolites for about 8 hours in urine after intravenous administration of a 1.7-mg dose per horse. Both antibodies are relatively specific for each drug and do not cross-react with other commonly used diuretics or other acidic compounds often found in post-race equine urine samples. Ethacrynic acid and bumetanide are potent diuretics suspected of being illegally substituted for furosemide in certain racing jurisdictions. Development of these rapid, sensitive, and simple tests for these agents will allow more effective pre- and post-race control of the use of these agents in racing horses. Both tests have recently uncovered several "positives" for these medications in a midwestern racing jurisdiction.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC), or β-catenin gene. These mutations stabilize β-catenin and constitutively activate Wnt/β-catenin target genes, such as c-Myc and cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations less than 10 μM. To understand the structure--activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20 mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of β-catenin, probably at the transcriptional level.

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.

Two synthetic strategies permitted the synthesis of various metabolites of detomidine (1) and medetomidine (4), potent α-2 adrenoceptor agonists that undergo rapid oxidative metabolism at the aromatic methyl group distal to the imidazole ring. In the detomidine series, the addition of a Grignard reagent prepared from 2-((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-bromotoluene (13) to imidazole-4(5)-carboxaldehyde (7) provided 2-(((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-(1′-hydroxy-1′-(5′-imidazolyl)methyl)tolulene (14). In a subsequent reduction, it was possible to differentiate between the secondary benzylic hydroxyl group and the primary benzylic hydroxyl group protected as a 3,4-dimethoxybenzyl ether. Removal of the protecting group provided 3-(hydroxymethyl)detomidine (3-HD)(2) and an oxidation furnished 3-carboxydetomidine (3-CD)(3). However, in the medetomidine series, a similar hydrogenolysis of 2-(((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-(1′-hydroxy-1′-methyl-1′-(5′-imidazolyl)methyl)toluene (17) failed, and an alternate, longer route involving dehydration and reduction was necessary to secure 3-(hydroxymethyl)medetomidine (3-HM) (5) and following an oxidation, 3-carboxymedetomidine (3-CM) (6). Finally, an expeditious route to 3-CM (6) involved the addition of the Grignard reagent prepared from 2-(3-bromo-2-methylphenyl)-4,4-dimethyl-2-oxazoline (22) to 4-acetyl-1H-imidazole and the hydrogenolysis and hydrolysis of 2-(1-(4,4-dimethyl-2-oxazolyl))-6-(1′-oxo-1′-(5′-imidazolyl)methyl)toluene (23).

Efficient routes for the preparation of selected C-23 and C-24 diastereomers of the C 30 biological markers 4α-methyl-5α-stigmastane (1) and 5α-dinosterane (2) involved the alkylation of 20-(iodomethyl)-4α-methyl-5α-pregnane with either saturated or α,β-unsaturated esters. The alkylation of (20S)-20-(iodomethyl)-4α-methyl-5α-pregnane with methyl (3R)-3-ethyl-4-methylpentanoate furnished methyl (20R,23ζ,24S)-4α-methyl-5α-stigmastane-23-carboxylate, and a subsequent decarbomethoxylation provided (20R,24R)-1. The alkylation of (20S)-20-(iodomethyl)-4α-methyl-5α-pregnane with methyl (3S)-3,4-dimethylpentanoate led to methyl (20R,23ζ,24R)-4α,24-dimethyl-5α-cholestane-23-carboxylate, and reduction of this mixture provided principally (20R,23S,24R)-5α-dinosteran-29-ol. The further reduction of the mesylate of this isomer secured (20R,23S,24R)-5α-dinosterane

Two synthetic strategies permitted the synthesis of various metabolites of detomidine (1) and medetomidine (4), potent α-2 adrenoceptor agonists that undergo rapid oxidative metabolism at the aromatic methyl group distal to the imidazole ring. In the detomidine series, the addition of a Grignard reagent prepared from 2-((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-bromotoluene (13) to imidazole-4(5)-carboxaldehyde (7) provided 2-(((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-(1′-hydroxy-1′-(5′-imidazolyl)methyl)tolulene (14). In a subsequent reduction, it was possible to differentiate between the secondary benzylic hydroxyl group and the primary benzylic hydroxyl group protected as a 3,4-dimethoxybenzyl ether. Removal of the protecting group provided 3-(hydroxymethyl)detomidine (3-HD)(2) and an oxidation furnished 3-carboxydetomidine (3-CD)(3). However, in the medetomidine series, a similar hydrogenolysis of 2-(((3′,4′-dimethoxyphenyl)methoxy)methyl)-6-(1′-hydroxy-1′-methyl-1′-(5′-imidazolyl)methyl)toluene (17) failed, and an alternate, longer route involving dehydration and reduction was necessary to secure 3-(hydroxymethyl)medetomidine (3-HM) (5) and following an oxidation, 3-carboxymedetomidine (3-CM) (6). Finally, an expeditious route to 3-CM (6) involved the addition of the Grignard reagent prepared from 2-(3-bromo-2-methylphenyl)-4,4-dimethyl-2-oxazoline (22) to 4-acetyl-1H-imidazole and the hydrogenolysis and hydrolysis of 2-(1-(4,4-dimethyl-2-oxazolyl))-6-(1′-oxo-1′-(5′-imidazolyl)methyl)toluene (23).

The compound (1S,3aR,4R,6S,8aS,9S)-1-tert-butoxy-9-chloro-2,3,3a,4,6,7,8,8a-octahydro-8a-methyl-4-methylthio-1H-3a,6-methanoazulen-5-one, C 17 H 27 ClO 2 S, was identified as the reaction product when the acid chloride formed from a trans-fused perhydroindane derivative underwent a Friedel-Crafts acylation of the internal olefin

Various photoactive phenothiazines were synthesized that possessed a 2-azido, 3-azido, 2-benzoyl, or 1,3,4-trifluoro-2-azido functionality in combination with various modifications of the N-alkyl side chain. These phenothiazines were evaluated for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase (PDE). All were active in inhibiting the action of calmodulin (CaM), but those possessing either a 3-azido and a 4-(4-methyl-1-piperazinyl)butyl side chain or a 2-benzoyl group and 3-(dimethylamino)propyl side chain proved to be most active (I50 = 14 +/- 3 microM and 7 +/- 1 microM, respectively) when compared to the known inhibitor, chlorpromazine (CPZ, I50 = 30 microM). Calmodulin was photolabeled with ca. 35% efficiency in a light- and calcium-dependent fashion using a radiolabeled analog, 3-azido-10-(4-(4-[14C]methyl-1-piperazinyl)butyl)phenothiazine, of one of these compounds. Competition studies using this radiolabeled analog and CPZ were consistent with binding to one or both of the hydrophobic binding pockets of CaM.

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Stereoselective routes for the preparation of C-23 and C-24 diastereomers of the C30 biological marker, 5α-dinosterane (1), involved the alkylation of (20S)-20-(iodomethyl)-4α-methyl-5α-pregnane (7) with either a saturated ester, methyl 3,4-dimethylpentanoate (9), followed by reduction to give principally the erythro-diastereomers or the alkylation of 7 with an α,β-unsaturated ester, methyl 3,4-dimethylpentenoate (12), followed by reduction to give principally the threo-diastereomers.

A novel, specific, and potent biological action of leukotriene C 4 (LTC 4 ) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the conferment of protection against subsequent γ-irradiation. Consequently, studies were conducted to determine whether LTC 4 -conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C 4 (LTC 4 ) were identified and characterized using intact V79 cells incubated at 4°C in the presence of serine-borate, during which time conversion of LTC 4 to LTD 4 or LTE 4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca 2+ , Mg 2+ , and Na 2+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leikotrienes C 4 , D 4 , or E 4 , or by the leokotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 ± 0.63 nM, approximately 9.9 × 10 5 sites/cell] and low-affinity [Kd = 350 ± 211 nM, approximately 2.7 × 10 6 sites/cell] binding sites. The observed binding characteristics of LTC 4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC 4 , binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.

Colorectal (CRC) cancer is the second leading cause of cancer deaths in the United States in 2014. Patients often die due to metastasis of CRC rather than from the primary disease, and it is thought that cancer stem cells play a major role in the survival and metastasis of cancer cells after treatment. A new class of small molecules, fluorinated N,N-diarylureas (FNDs), have been formulated and shown to reduce viability of CRC cells through AMPK activation. The purpose of the present study was to evaluate whether FNDs inhibit growth and induce apoptosis of CRC metastatic cell lines and CRC stem cells in vitro and to identify an FND for development as a potential chemotherapeutic agent. METHODS. FNDs were identified in a high-throughput screen and selected for study based on their relative induction of AMPK activity. Compounds are arbitrarily designated as 4a, 4b, 4h, 4j, 4k, 4z, 4aa, 4bb. Metastatic CRC cell lines HT29, HTC116, and KM20, were purchased from American Type Culture Collection. Two CRC stem cell lines were purchased from Celprogen. Cell viability and IC50 concentrations were measured using a sulforhodamine B colorimetric (SRB) cytotoxicity assay. Western blots were analyzed with antibodies: PARP for apoptosis, cyclin D1 for cell cycle progression, pAMPK to confirm AMPK activation, and β-actin as a loading control. RESULTS. AMPK activation after FND treatment was confirmed by western blot in metastatic CRC and stem cells with a peak activation at 12 h. Furthermore, AMPK activation was noted at concentrations as low as 5 μM, compared to metformin (an AMPK activator drug for DMII) which is typically dosed at 5-20 mM for metastatic cell lines. CRC stem cells were more resistant to FND treatment than regular metastatic CRC cell lines, with an IC50 concentration of 50 μM for stem cells as compared to 10-25 μM for metastatic cell lines. Cyclin D1 expression was undetectable with FND drug treatments at IC50 concentrations in CRC stem cell lines. FND 4b stood out as the strongest inducer of apoptosis, but all of the FND compounds increased apoptosis compared to control. CONCLUSIONS. AMPK is a key regulator of energy metabolism in cells, and cancer cells effectively make use of this pathway for their unregulated proliferation. Activation of AMPK by all FND compounds successfully inhibited the cell cycle, inhibited subsequent cellular proliferation, and induced a remarkable level of apoptosis in select FNDs in CRC metastatic and stem cells. Additionally, these effects were observed at much lower doses than other AMPK activators such as metformin and AICAR. Therefore, FNDs have considerable promise in the treatment of metastatic colon cancer, especially by preventing progression and metastasis as a result of their inhibition of CRC stem cells. Citation Format: Dasha Kenlan, Piotr G. Rychahou, Vitaliy Sviripa, David Watt, B. Mark Evers. New potent AMPK activators against colorectal cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2015-4461

Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. In normal liver, methionine S-adenosyltransferase 1 (MAT1), but not MAT2A is expressed. MAT2A is induced in liver cancer. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells and liver cells in vitro and in vivo. Using a biotinylated FIDAS analog, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. FIDAS agents do not bind MAT1A. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC and liver cancer cell growth. FIDAS agents also inhibited specific histone methylation and repressed transcription of c-myc and other oncogenes. FIDAS agents delivered orally repressed CRC xenografts in athymic nude mice. Based on SAR and docking studies, new generations of FIDAS agents were synthesized and tested. An active in vivo metabolite of FIDAS was identified. PK/PD studies were performed. Our findings suggest that FIDAS analogs targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment. Citation Format: Wen Zhang, Vitaliy Sviripa, David Watt, Chunming Liu. Methionine S-adenosyltransferase 2A (MAT2A) inhibitors for cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2015-5388

The compound (1S,3aR,4R,6S,8aS,9S)-1-tert-butoxy-9-chloro-2,3,3a,4,6,7,8,8a-octahydro-8a-methyl-4-methylthio-1H-3a,6-methanoazulen-5-one, C 17 H 27 ClO 2 S, was identified as the reaction product when the acid chloride formed from a trans-fused perhydroindane derivative underwent a Friedel-Crafts acylation of the internal olefin