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Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and requires new therapeutic strategies to improve clinical outcomes. EOC metastasizes in the abdominal cavity through dissemination in the peritoneal fluid and ascites, efficiently adapt to the nutrient-deprived microenvironment, and resist current chemotherapeutic agents. Accumulating evidence suggests that mitochondrial oxidative phosphorylation is critical for the adaptation of EOC cells to this otherwise hostile microenvironment. Although chemical mitochondrial uncouplers can impair mitochondrial functions and thereby target multiple, essential pathways for cancer cell proliferation, traditional mitochondria uncouplers often cause toxicity that precludes their clinical application. In this study, we demonstrated that a mitochondrial uncoupler, specifically 2,5-dichloro-N-(4-nitronaphthalen-1-yl)benzenesulfonamide, hereinafter named Y3, was an antineoplastic agent in ovarian cancer models. Y3 treatment activated AMP-activated protein kinase and resulted in the activation of endoplasmic reticulum stress sensors as well as growth inhibition and apoptosis in ovarian cancer cells in vitro. Y3 was well tolerated in vivo and effectively suppressed tumor progression in three mouse models of EOC, and Y3 also induced immunogenic cell death of cancer cells that involved the release of damage-associated molecular patterns and the activation of antitumor adaptive immune responses. These findings suggest that mitochondrial uncouplers hold promise in developing new anticancer therapies that delay tumor progression and protect patients with ovarian cancer against relapse.

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.

Cancer cells undergo significant “metabolic remodeling” to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer cells, ATP is produced by mitochondrial oxidative phosphorylation and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in colorectal cancer cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit colorectal cancer cell growth in vitro and in vivo. This study suggests that an electron transport chain inhibitor (i.e., DBI-1) and a glucose transport inhibitor, (i.e., BAY-876) are potentially effective combination for colorectal cancer treatment.

Supplementary Data from Diaminobutoxy-substituted Isoflavonoid (DBI-1) Enhances the Therapeutic Efficacy of GLUT1 Inhibitor BAY-876 by Modulating Metabolic Pathways in Colon Cancer Cells

Supplementary Information

The deregulation in the Wnt/β-catenin signaling pathway is associated with many human cancers, particularly colorectal cancer (CRC) and, therefore, represents a promising target for drug development. We have screened over 300 semisynthetic and natural compounds using a Wnt reporter assay and identified a family of novel chalcone derivatives (CXs) that inhibited Wnt signaling and CRC cell proliferation. Among them, we selected CX258 for further in vitro and in vivo study to investigate the molecular mechanisms. We found that CX258 significantly inhibited β-catenin expression and nuclear translocation, inducing cell cycle arrest at the G2/M phase in CRC cells. Additionally, CX258 reduced the expression of DNA Topoisomerase II alpha (TOP2A) in CRC cells. Moreover, knocking down TOP2A by siRNAs inhibited the Wnt/β-catenin signaling pathway, a finding suggesting that CX258 inhibited Wnt/β-catenin signaling and CRC cell proliferation at least partially by modulating TOP2A. Further studies showed that CDK1 that interacts with TOP2A was significantly reduced after TOP2A knockdown. We demonstrated that CX258 significantly inhibited DLD-1 CRC cell xenografts in SCID mice. In summary, we identified CX258 as a promising candidate for colorectal cancer treatment by targeting the TOP2A/Wnt/β-catenin signaling pathway.

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.

Cancer cells undergo significant “metabolic remodeling” to provide sufficient ATP to maintain cell survival and to promote rapid growth. In colorectal cancer cells, ATP is produced by mitochondrial oxidative phosphorylation and by substantially elevated cytoplasmic glucose fermentation (i.e., the Warburg effect). Glucose transporter 1 (GLUT1) expression is significantly increased in colorectal cancer cells, and GLUT1 inhibitors block glucose uptake and hence glycolysis crucial for cancer cell growth. In addition to ATP, these metabolic pathways also provide macromolecule building blocks and signaling molecules required for tumor growth. In this study, we identify a diaminobutoxy-substituted isoflavonoid (DBI-1) that inhibits mitochondrial complex I and deprives rapidly growing cancer cells of energy needed for growth. DBI-1 and the GLUT1 inhibitor, BAY-876, synergistically inhibit colorectal cancer cell growth in vitro and in vivo. This study suggests that an electron transport chain inhibitor (i.e., DBI-1) and a glucose transport inhibitor, (i.e., BAY-876) are potentially effective combination for colorectal cancer treatment.

Regulatory T cells (Tregs) are essential to maintain self-tolerance and immune homeostasis but, as components of the tumor microenvironment (TME), are also a major barrier to effective cancer immunosurveillance and immunotherapy. FH535 and its derivative Y3 are two N-aryl-benzene-sulfonamides (NABs) that inhibit HCC cell proliferation and tumor progression. However, the impact of NABs on the immune cells in the TME is not yet known. Analyses of explanted livers from patients with hepatocellular carcinoma (HCC) showed that high levels of tumor-infiltrating Tregs were associated with poor tumor differentiation. These results lead us to investigate the immunomodulatory effects of NABs in regulatory and effector T cells. Exposure of primary human Tregs to NABs induced a rapid but temporary increase of cell expansion, a gradual disruption of suppressor activity, and concomitant bioenergetics and autophagic flux dysregulations. In contrast to Tregs, no gross effects were observed in effector T cells. Addition of Rapamycin prevented the functional decay of Tregs and restored their metabolic profile, suggesting that NAB effects require the integrity of the mTOR pathway. This study revealed the immunomodulatory properties of NABs with a preferential impact on Treg activity and provided novel insights into the anti-tumor potential of sulfonamides.

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.

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis( N -amidinohydrazones) and N -(amidino)- N′ -aryl-bishydrazones for their antibacterial and antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi. We investigated their potential to develop resistance against both bacteria and fungi by a multi-step resistance-selection method, explored their potential to induce the production of reactive oxygen species, and assessed their toxicity. In summary, we found that these compounds exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains with minimum inhibitory concentration (MIC) values ranging from 500 μM against bacteria and 1.0 to >31.3 μg/mL against fungi; and in most cases, they exhibited either superior or similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also observed minimal emergence of drug resistance to these newly synthesized compounds by bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the human Ether-a-go-go -related gene (hERG) channel with acceptable IC 50 values ranging from 1.12 to 3.29 μM. Overall, these studies show that bis( N -amidinohydrazones) and N -(amidino)- N ′-aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial and antifungal agents.

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

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target β-catenin in this pathway. We now report that several “Wnt inhibitors” that allegedly target β-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known “Wnt inhibitor”, FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M) was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

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.

Summary Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (β-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity., Graphical Abstract, Highlights • A class of carboxamide-substituted benzhydryl amine (CBA) Wnt inhibitors • A biological active, biotinylated CBA to identify KDM3A as a direct target • CBA-1 interacted with the Mn2+ ion in the JmjC domains of KDM3A/3B • CBA-1 inhibited Wnt signaling in colon cancer cells and in zebrafish models, Biological Sciences; Biochemistry; Biochemical Mechanism

Advanced prostate tumors usually metastasize to the lung, bone, and other vital tissues and are resistant to conventional therapy. Prostate apoptosis response-4 protein (Par-4) is a tumor suppressor that causes apoptosis in therapy-resistant prostate cancer cells by binding specifically to a receptor, Glucose-regulated protein-78 (GRP78), found only on the surface of cancer cells. 3-Arylquinolines or "arylquins" induce normal cells to release Par-4 from the intermediate filament protein, vimentin and promote Par-4 secretion that targets cancer cells in a paracrine manner. A structure-activity study identified arylquins that promote Par-4 secretion, and an evaluation of arylquin binding to the hERG potassium ion channel using a [(3)H]-dofetilide binding assay permitted the identification of structural features that separated this undesired activity from the desired Par-4 secretory activity. A binding study that relied on the natural fluorescence of arylquins and that used the purified rod domain of vimentin (residues 99-411) suggested that the mechanism behind Par-4 release involved arylquin binding to multiple sites in the rod domain.

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 metabolism of a promising antineoplastic agent, trans-2,6-difluoro-4′-(N,N-dimethylamino)stilbene (DFS), was studied in mouse, rat, and human liver microsomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the multiple reaction monitoring-information-dependent acquisition-enhanced product ion scan (MRM-IDA-EPI) method. Ten putative metabolites were identified and the structures of four metabolites were confirmed using authentic standards. Since trans-2,6-difluoro-4′-(N-methylamino)stilbene (DMDFS, M1) was present in all species as metabolite and displayed in vitro growth inhibition superior to DFS, its pharmacokinetic profiles were examined in Sprague–Dawley rats using DFS as a comparator. A reliable LC-MS/MS multiple reaction monitoring (MRM) method was subsequently developed and validated for the simultaneous quantification of both DFS and DMDFS in rat plasma for this purpose. Upon intravenous administration (4 mg/kg), DFS had a moderate clearance (Cl = 62.7 ± 23.2 mL/min/kg), terminal elimination half-life (t 1/2 λZ = 299 ± 73 min), and mean transit time (MTT = 123 ± 14 min) with demethylation metabolism accounting for about 10 % of its total clearance. DMDFS possessed an intravenous pharmacokinetic profile similar to DFS. During oral dosing (10 mg/kg) where both DFS and DMDFS were absorbed rapidly, the oral bioavailability of DFS was approximately 2-fold greater than that of DMDFS (DFS: F = 42.1 ± 12.8 %; DMDFS: F = 18.7 ± 3.9 %). Interestingly, the DMDFS exposure after oral dosing of DFS (10 mg/kg) was comparable to that after oral administration of DMDFS (10 mg/kg) alone. As DFS displayed potent anticancer activities and excellent pharmacokinetic profiles, it appears to be a favorable candidate for further pharmaceutical development.

The aminomethylation of hydroxylated isoflavones with 2-aminoethanol, 3-amino-1-propanol, 4-amino-1-butanol, and 5-amino-1-pentanol in the presence of excess formaldehyde led principally to 9-(2-hydroalkyl)-9,10-dihydro-4H,8H-chromeno[8,7-e][1,3]-oxazin-4-ones 4 and/or the tautomeric 7-hydroxy-8-(1,3-oxazepan-3-ylmethyl)-4H-chromen-4-ones 5. The ratio of these tautomers was dependent on solvent polarity, electronic effects of aryl substituents in the isoflavone and the structure of the amino alcohol. NMR studies confirmed the interconversion of tautomeric forms.

Two factors contribute to the inefficiency associated with screening pharmaceutical library collections as a means of identifying new drugs: [1] the limited success of virtual screening (VS) methods in identifying new scaffolds; [2] the limited accuracy of computational methods in predicting off-target effects. We recently introduced a 3D shape-based similarity algorithm of the SABRE program, which encodes a consensus molecular shape pattern of a set of active ligands into a 4D fingerprint descriptor. Here, we report a mathematical model for shape similarity comparisons and ligand database filtering using this 4D fingerprint method and benchmarked the scoring function HWK (Hamza–Wei–Korotkov), using the 81 targets of the DEKOIS database. Subsequently, we applied our combined 4D fingerprint and HWK scoring function VS approach in scaffold-hopping and drug repurposing using the National Cancer Institute (NCI) and Food and Drug Administration (FDA) databases, and we identified new inhibitors with different scaffolds of MycP1 protease from the mycobacterial ESX-1 secretion system. Experimental evaluation of nine compounds from the NCI database and three from the FDA database displayed IC50 values ranging from 70 to 100 μM against MycP1 and possessed high structural diversity, which provides departure points for further structure–activity relationship (SAR) optimization. In addition, this study demonstrates that the combination of our 4D fingerprint algorithm and the HWK scoring function may provide a means for identifying repurposed drugs for the treatment of infectious diseases and may be used in the drug-target profile strategy.

Inhibition of the catalytic subunit of the heterodimeric methionine S-adenosyl transferase-2 (MAT2A) with fluorinated N,N-dialkylaminostilbenes (FIDAS agents) offers a potential avenue for the treatment of liver and colorectal cancers where upregulation of this enzyme occurs. A study of structure-activity relationships led to the identification of the most active compounds as those with (1) either a 2,6-difluorostyryl or 2-chloro-6-fluorostyryl subunit, (2) either an N-methylamino or N,N-dimethylamino group attached in a para orientation relative to the 2,6-dihalostyryl subunit, and (3) either an N-methylaniline or a 2-(N,N-dimethylamino)pyridine ring. These modifications led to FIDAS agents that were active in the low nanomolar range, that formed water-soluble hydrochloride salts, and that possessed the desired property of not inhibiting the human hERG potassium ion channel at concentrations at which the FIDAS agents inhibit MAT2A. The active FIDAS agents may inhibit cancer cells through alterations of methylation reactions essential for cancer cell survival and growth.

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.

Structure-activity relationships (SAR) in 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535) were examined as part of a program to identify agents that inhibit the Wnt/β-catenin signaling pathway that is frequently upregulated in hepatocellular carcinoma (HCC). FH535 was reported as an inhibitor of both β-catenin in the Wnt signaling pathway and the peroxisome proliferator-activated receptor (PPAR). A β-catenin/T-cell factor (TCF)/Lymphoid-enhancer factor (LEF)-dependent assay (i.e., luciferase-based TOPFlash assay) as well as a [(3)H]-thymidine incorporation assay were used to explore SAR modifications of FH535. Although replacing the 2,5-dichlorophenylsulfonyl substituent in FH535 with a 2,6-dihalogenation pattern generally produced more biologically active analogs than FH535, other SAR modifications led only to FH535 analogs with comparable or slightly improved activity in these two assays. The absence of a clear SAR pattern in activity suggested a multiplicity of target effectors for N-aryl benzenesulfonamides.

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.

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. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. 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 cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.