LOPAC Library Research Articles

Abstract LB-129: Small molecular library screening to find methyl binding domain 2 protein inhibitors

Abstract DNA 5’ cytosine methylation at CpG dinucleotides, particularly at gene promoters and regulatory elements, can mediate epigenetic silencing of key tumor suppressor and caretaker genes in human cancers. This is largely mediated through the activity of methyl binding domain (MBD) “reader” proteins that bind methylated DNA and recruit chromatin repression complexes. Of the MBD family of proteins, MBD2 is a particularly attractive therapeutic target since targeted genetic disruption of this protein is well tolerated and can greatly attenuate tumor formation in cancer prone mice and growth of cancerous cells lines. Therefore development of pharmacological approaches for targeting MBD2 may have utility in cancer therapeutics. In this work we describe a time resolved fluorescence energy transfer (TR-FRET) based assay to find small molecule inhibitors of MBD2. First we optimized conditions to establish an assay that was amenable for high throughput screening (HTS) in 384 well plates, obtaining a Z’ factor of 0.58 (HTS target >0.5). To validate our assay we applied it to the 1280 compound Sigma® LOPAC library. Among 10 primary hits, four (Mitoxantrone, idarubicin, aurintricarboxylic acid, and NF449) showed dose responsive inhibition of MBD2 binding to methylated DNA. However, a counter screen showed these 4 compounds also inhibited interaction of the transcription factor SP-1 to DNA, suggesting their mode of action was not specific to MBD2. This pilot screen provided proof principle that our assay format could identify MBD2 inhibitors and that a counter screen strategy could evaluate the specificity of identified hit compounds. In collaboration with the Scripps Institute in Florida, we further miniaturized, refined, and deployed the MBD2 TR-FRET assay to screen the NCI small molecule library of 370,276 compounds. The primary screening effort identified 1,149 active compounds (able to disrupt MBD2-DNA interaction) at 4.4µM. In the secondary screen each of these were run in triplicate at 4.4µM, with 271 (26.2%) of them validating as active hits. Next, a TR-FRET assay to evaluate inhibition of binding of the ubiquitin-like with PHD and ring finger domains 1 (UHRF1) protein to methylated DNA was utilized to evaluate specificity of the 271 active compounds that passed secondary screening. This yielded 48 small molecules that appeared to specifically inhibit the MBD2-DNA binding interaction. These 48 were subjected to the TR-FRET assay in a dose response from ∼10nM to 50µM on both MBD2 and UHRF1, yielding 18 active compounds which specifically inhibit MBD2-DNA interactions in a dose responsive manner with IC50's between 1µM and 17µM. Work is currently underway to test these compounds for biological activity. Acknowledgments/Funding- The authors would like to thank Peter Hodder, Virneliz Fernandez-Vega and Tom Bannister at the Scripps Institute-Florida for their efforts with this project. Funding provided by Department of Defense award W81XWH-11-1-0618 (to NW), NIH/NCI grants CA70196 and CA58236 (to WGN and SY), and the Prostate Cancer Foundation. Citation Format: Nicolas A. Wyhs, Hugh Giovinazzo, David Walker, Srinivasan Yegnasubramanian, William G. Nelson. Small molecular library screening to find methyl binding domain 2 protein inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2014-LB-129

Abstract 2709: Identification of Aurintricarboxylic Acid (ATA) as an inhibitor of TWEAK-Fn14 signaling in glioblastoma cells

Abstract The long-term survival of patients with glioblastoma (GB) is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. In order to discover a small molecule inhibitors that disrupt Fn14-TWEAK signaling axis and subsequently glioma cell invasion, we developed a cell-based drug screening assay using HEK293 cells that overexpresses Fn14 and harbors NF-κB driven Firefly luciferase protein expression. In preliminary drug screening assay using LOPAC1280 library of 1280 pharmacologically active compound, Aurintricarboxylic Acid (ATA) showed suppression of NF-κB driven Firefly Luciferase activity downstreatm of TWEAK-Fn14 signaling. Conversely, ATA did not show any suppression in NF-κB driven Firefly Luciferase activity downstream of TNFα-TNFR1 signaling, suggesting that ATA is a specific inhibitor of TWEAK-Fn14-NF-κB signaling. In vitro, we demonstrated that ATA suppresses TWEAK induced chemotactic migration of glioma cells (T98G and A172) and display no general cytotoxicity. In summary, this work reports a new small molecule inhibitor of TWEAK-Fn14 signaling that could be useful in enhancing the therapeutic targeting of this deadly disease. Citation Format: Harshil D. Dhruv, Ethan Holiday, Donald Chow, Holly Yin, Michael E. Berens, Nhan L. Tran. Identification of Aurintricarboxylic Acid (ATA) as an inhibitor of TWEAK-Fn14 signaling in glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2709. doi:10.1158/1538-7445.AM2014-2709

Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2

Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)–based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z′ factor = 0.58) emerged as a superior screening strategy compared with FP (Z′ factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET–based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.

P98 Tetracyclin antibiotics as inhibitors of H2S synthesis

Hydrogen sulfide (H2S) has emerged as a gaseous signaling molecule with important roles in neuromodulation, blood pressure regulation, angiogenesis, cardioprotection, cellular energetics and apoptosis. H2S is produced endogenously by three enzymes in the sulfur metabolic network, namely cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Although several compounds that inhibit CSE are available, no selective inhibitors for CBS or 3MST currently exist. In the present study we screened the LOPAC library for inhibitors of H2S-producing enzymes. The methylene blue assay was used to measure H2S production from recombinant enzymes. HCT116 and HT29 cells were used for MTT-based proliferation assays. Screening of the LOPAC library yielded several hit compounds that blocked H2S-synthesizing enzymes to varying extents. Among those, doxycyclin and demeclocycline were found to inhibit 3MST by approximately 50% at 100 uM, with doxycyclin being a more potent 3MST inhibitor than demeclocycline in the in vitro activity assay. Tetracyclin, meclocyclin, oxycyclin, methacyclin, tigecyclin and minocyclin did not affect 3MST activity when used up to 300 uM. From the above-mentioned tetracyclines, tigecyclin at 100 uM caused only a slight inhibition of both CBS and CSE; tigecyclin additionally inhibited CSE. In a cell-based assay, demeclocyclin, but not doxycyclin, inhibited the growth of HCT116 and HT29, two colon cancer cell lines in which CBS is highly expressed and its product, H2S supports cell proliferation. We conclude that based on its selectivity towards 3MST, demeclocyclin could serve as a lead compound that after undergoing optimization could yield future selective 3MST inhibitors.

Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology

Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z' factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

Detection of Phospholipidosis Induction: A Cell-Based Assay in High-Throughput and High-Content Format

Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multilamellar bodies viewed under an electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. To validate this assay for high-throughput screening (HTS), the LOPAC library of 1280 compounds was screened using a quantitative HTS platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers, including NAN-190, ebastine, GR127935, and cis-(Z)-flupentixol, were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.

Development of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection

Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators.

A quantitative high throughput assay for identifying gametocytocidal compounds

Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z′-factor for this assay were 3.2-fold and 0.68, respectively. The IC50 value of epoxomicin, the positive control compound, was 1.42±0.09nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the AlamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1280 known compounds, revealed two selective gametocytocidal compounds having 54- and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line.

Etoposide increases equilibrative nucleoside transporter 1 activity and fluorothymidine uptake: Screening of 60 cytotoxic agents

Equilibrative nucleoside transporter 1 (ENT1) is a major regulator for the uptake of [(18)F]fluorothymidine ([(18)F]FLT), a promising positron emission tomography tracer for treatment monitoring. Various antimetabolites such as 5-fluorouracil often increase ENT1 activity and [(18)F]FLT uptake (flare) in spite of cell death. However, it has not yet been defined which agents induce [(18)F]FLT flare and what is the role of [(18)F]FLT flare in cell viability. Sixty cytotoxic agents from the LOPAC1280 library were screened for ENT1 activity in HeLa cells which predominantly express ENT1, and topoisomerase inhibitors (i.e., aurintricarboxylic acid, idarubicin, camptothecin and etoposide) were identified as potent inducers of ENT1 activity. The changes in ENT1 activity were closely correlated with [(3)H]FLT uptake (Spearman's correlation coefficient, r=0.66; P<0.01). Etoposide significantly increased ENT1 activity and [(3)H]FLT uptake accompanying cell cycle arrest at S/G2/M phase and the increase in TK1 expression and activity in both ENT1-low expressing HT29 and ENT1-high expressing MDA-MB-231 cells. The inhibition of ENT1 activity by dipyridamol or S-(p-nitrobenzyl)-6-thioinosine repressed the etoposide-induced cell death in HeLa cells, whereas it induced no changes in the other cell lines. In conclusion, etoposide is identified as a potent inducer for ENT1 activity and [(3)H]FLT uptake. The role of ENT1 activity by etoposide was cell-type dependent, which requests caution for the application of ENT1-mediated [(18)F]FLT flare for treatment monitoring.

Abstract 1135: Bioluminescent methods to investigate cellular metabolic status: NAD(P)/NAD(P)H levels, redox potential, and hydrogen peroxide concentration

Abstract Cancer is a disease defined by uncontrolled cell growth where cellular energy metabolism pathways must evolve for tumors to survive and proliferate. NAD(P) and NAD(P)H play a major role in oxidative phosphorylation and their role in aerobic glycolysis is of great interest. Additionally, NAD(P)/NAD(P)H act as co-factors for enzymes involved in cancer pathogenesis through regulation of chromatin structure, DNA repair, and transcription (e.g. sirtuins and poly(ADP-ribose) polymerase). The study of how NAD(P)/NAD(P)H are generated and utilized during adaptive cancer cell energy metabolism would benefit from the development of a rapid, sensitive, and homogeneous assay to measure the level of these nucleotides. We developed a bioluminescent assay for measuring NAD(P)/NAD(P)H that can detect ≤ 0.1 µM NADH and has a 1000-fold dynamic range. The assay is well suited for high throughput screening (Z’ = 0.92, S/B = 95) and has been used to screen the LOPAC library. By coupling other reactions with NAD(P)/NAD(P)H measurement, we analyzed the levels of metabolites and the activity of enzymes, including isocitrate dehydrogenase and pyruvate dehydrogenase, in enzyme preparations and crude cell lysates. This method is applicable for measuring cellular NAD and NADH levels directly from cell culture without further sample handling. This assay is based on a novel proluciferin derivative that is processed in vitro through an enzymatic reaction. The released luciferin is detected in a coupled luciferin/luciferase reaction and the luminescent signal is correlated with the amount of NAD(P)/NAD(P)H present in the sample. As an extension of this approach, similar proluciferin chemistries were developed to detect the reducing potential of metabolically active cells. This robust luminescent assay can detect the reducing potential of less than 100 cells/well in 96-well format and distinguish small changes in cell number. Multiple cell lines were treated with cancer therapeutic compounds and the performance of the proluciferin approach was compared to commonly used colorimetric (e.g. MTT, MTS, and XTT) and fluorogenic (e.g. resazurin) methods. The proluciferin assay resulted in comparable pharmacological responses, significantly increased signal window, and superior sensitivity. Additionally, the proluciferin-based approach has been extended to other metabolic readouts. For example, novel proluciferin substrates were developed and applied to the detection of hydrogen peroxide in cells and in biological samples through a HRP-independent process. Bioluminescent assays provide greater sensitivity and dynamic range than most fluorescent or colorimetric assays, and are better suited for high throughput screening. Applying these assays to the study of cancer cell energy metabolism will provide a significant advantage over existing methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1135. doi:1538-7445.AM2012-1135

Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.

N-Acetylfarnesylcysteine Is a Novel Class of Peroxisome Proliferator-activated Receptor γ Ligand with Partial and Full Agonist Activity in Vitro and in Vivo

The thiazolidedione (TZD) class of drugs is clinically approved for the treatment of type 2 diabetes. The therapeutic actions of TZDs are mediated via activation of peroxisome proliferator-activated receptor γ (PPARγ). Despite their widespread use, concern exists regarding the safety of currently used TZDs. This has prompted the development of selective PPARγ modulators (SPPARMs), compounds that promote glucose homeostasis but with reduced side effects due to partial PPARγ agonism. However, this also results in partial agonism with respect to PPARγ target genes promoting glucose homeostasis. Using a gene expression-based screening approach we identified N-acetylfarnesylcysteine (AFC) as both a full and partial agonist depending on the PPARγ target gene (differential SPPARM). AFC activated PPARγ as effectively as rosiglitazone with regard to Adrp, Angptl4, and AdipoQ, but was a partial agonist of aP2, a PPARγ target gene associated with increased adiposity. Induction of adipogenesis by AFC was also attenuated compared with rosiglitazone. Reporter, ligand binding assays, and dynamic modeling demonstrate that AFC binds and activates PPARγ in a unique manner compared with other PPARγ ligands. Importantly, treatment of mice with AFC improved glucose tolerance similar to rosiglitazone, but AFC did not promote weight gain to the same extent. Finally, AFC had effects on adipose tissue remodeling similar to those of rosiglitazone and had enhanced antiinflammatory effects. In conclusion, we describe a new approach for the identification of differential SPPARMs and have identified AFC as a novel class of PPARγ ligand with both full and partial agonist activity in vitro and in vivo.

A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K D of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.

Inhibition of Morphine-Induced cAMP Overshoot: A Cell-Based Assay Model in a High-Throughput Format

Opiates are not only potent analgesics but also drugs of abuse mainly because they produce euphoria. Chronic use of opiates results in the development of tolerance and dependence. Dr Marshall Nirenberg's group at the National Institutes of Health (NIH) was the first to use a cellular model system of Neuroblastoma × Glioma hybrid cells (NG108-15) to study morphine addiction. They showed that opiates affect adenylyl cyclase (AC) by two opposing mechanisms mediated by the opiate receptor. Although the cellular mechanisms that cause addiction are not yet completely understood, the most observed correlative biochemical adaptation is the upregulation of AC. This model also provides the opportunity to look for compounds which could dissociate the acute effect of opiates from the delayed response, upregulation of AC, and thus lead to the discovery of non-addictive drugs. To identify small molecule compounds that can inhibit morphine-induced cAMP overshoot, we have validated and optimized a cell-based assay in a high throughput format that measures cellular cAMP production after morphine withdrawal. The assay performed well in the 1536-well plate format. The LOPAC library of 1,280 compounds was screened in this assay on a quantitative high-throughput screening (qHTS) platform. A group of compounds that can inhibit morphine-induced cAMP overshoot were identified. The most potent compounds are eight naloxone-related compounds, including levallorphan tartrate, naloxonazine dihydrochloride, naloxone hydrochloride, naltrexone hydrochloride, and naltriben methanesulfonate. The qHTS approach we used in this study will be useful in identifying novel inhibitors of morphine induced addiction from a larger scale screening.

Abstract 5342: Assessing EWS/FLI1 activity as a read-out for small-molecule drug screening in Ewing's sarcoma

Abstract Background: Ewing's sarcoma (ES) is the second most common bone malignancy in children and adolescents and accounts for approximately three percent of pediatric cancers. The hallmark of Ewing Sarcoma Family of Tumors (ESFT) is a chromosomal translocation involving the EWS gene on chromosome 22q12 with a member of the ETS transcription factor family. The derived EWS/FLI1 chimeric fusion protein, found in 85% of tumors, is a potent transcription factor, regulating malignant transformation and maintenance of the oncogenic phenotype by aberrant expression of its target genes. Hence, EWS/FLI1 represents an attractive therapeutic target whose transcriptional activity might be modulated by already existing small molecule inhibitors. Material and Methods: We screened the LOPAC1280 library (Sigma-Aldrich) consisting of 1280 different drug-like and well annotated compounds covering all major drug types. In addition we screened a smaller library composed of the latest targeted agents in the field of oncology, including most characterized kinase inhibitors. The effect of the compounds on EWS/FLI1 transcriptional activity was measured in three ES cell lines, using quantitative RT-PCR. Our read out was relative expression of three well-described EWS/FLI1 target genes: NR0B1, NKX2.2 and CAV1 and one new target gene, PHLDA1 (unpublished data) as well as the expression of the fusion protein itself. In parallel we measured proliferation using WST-1 assays. Results: We identified several well known chemotherapeutics such as camptothecin, doxorubicin, vincristine and etoposide as modulators of EWS/FLI1 target gene expression. The current usage of some of these agents in the treatment of Ewing's sarcoma validated our screening approach in an ideal and unbiased way. In addition we identified inhibitors targeting several signalling pathways, both known and unknown to play a role in sarcomas. The most prominent among them is the phosphoinositide-3-kinase (PI3K) pathway, playing a prominent role in many cancer types. Three inhibitors targeting this pathway revealed a significant modulation of EWS/FLI1 target genes and a strong inhibition of cell proliferation in all three ES cell lines. Conclusion: Screening of small molecule inhibitors was used to identify inhibitors of potential molecular pathways regulating the transcriptional activity of EWS/FLI1. Work continues to investigate these pathways in more detail on the molecular level. Genetic-loss of function experiments will be carried out to exclude unspecific off-target effects. Our findings will help to prioritize specific pathway inhibitors for more detailed investigations to accelerate the development of improved and novel therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2011-5342

Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors

The objective of this study was to measure the efficacy of Mycobacterium smegmatis as a surrogate in vitro model for the detection of compounds which are inhibitory to the growth of Mycobacterium tuberculosis. A chemical screen of the LOPAC library for anti-mycobacterial compounds was performed using M. smegmatis. Parallel screens were conducted with another tuberculosis model, Mycobacterium bovis BCG, and with M. tuberculosis under identical growth conditions and the inhibitors detected across the three species were compared. 50% of compounds that were detected as active against M.tuberculosis were not detected using M. smegmatis compared to 21% of compounds using M. bovis BCG. To examine whether these findings were unique to LOPAC, screens were performed with the NIH Diversity Set and Spectrum Collection. An even higher proportion of M. tuberculosis inhibitors were not detected from the NIH Diversity Set and Spectrum Collection using M. smegmatis compared to M. bovis BCG. These data reveal that a significant proportion of M. tuberculosis inhibitors are missed in library screening with M.smegmatis. The basis of the variation in the inhibitory profiles of M. smegmatis and M. tuberculosis has yet to be fully determined, however, our genomic comparisons indicate that approximately 30% of M.tuberculosis proteins lack conserved orthologues in M. smegmatis compared to 3% being absent in M.bovis BCG. In conclusion, although M. smegmatis offers some technical benefits such as a shorter generation time and negligible risk to laboratory workers, it is significantly less effective in the detection of anti-M. tuberculosis compounds relative to M. bovis BCG. This limitation needs to be taken into consideration when selecting an in vitro screening model for tuberculosis drug discovery.

Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange– and Mass Spectrometry–Based Strategy

Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Described here is use of an H/D exchange- and a matrix assisted laser desorption/ionization (MALDI) mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen 2 chemical libraries, including the 1280-compound LOPAC library and the 9600-compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which is demonstrated here to yield screening rates as fast as 6 s/ligand. The false-positive and false-negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false-positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered, including 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1, 4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., K(d) values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA-targeted diagnostic imaging or therapeutic agents for lung cancer.

Abstract A168: Quantitative high-throughput screening for inhibitors of human apurinic/apyrimidinic endonuclease APE1

Abstract The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of spontaneous hydrolytic, oxidative, and non-enzymatic alkylation base damage via the DNA base excision repair (BER) pathway. The increased activity of APE1, often observed in tumor cells, is thought to contribute to the development of resistance to various anticancer drugs; conversely, its down-regulation sensitizes tumor cells to DNA damaging agents via induction of apoptosis. Therefore, inhibiting APE1 within the BER pathway in cancer cells is an attractive strategy in the attempts to overcome chemotherapeutic resistance. Despite ongoing efforts, potent specific inhibitors of APE1 have yet to be discovered. We developed a fluorogenic substrate operating in the red-shifted fluorescence spectral region to configure a highly robust kinetic assay for use in 1536-well based automated high-throughput screening (HTS). The assay was used in a titration-based screen of the LOPAC1280 library to identify potential APE1 inhibitors which were further validated and profiled in a panel of assays including radiotracer-based incision assay, Thiazole Orange fluorophore displacement test for promiscuous DNA binders, and E. coli Endo IV counterscreen. Select compounds which passed the above validation steps were progressed to studies of increased biochemical complexity and were shown to inhibit abasic-site incision activity in whole cell protein extracts and to potentiate the genotoxic effect of methyl methanesulfonate (MMS), consistent with suppression of BER on a cellular level. Subsequently, a fully automated HTS was conducted against a collection of 241,291 diverse compounds tested as 7-concentration series at a 4 uL reaction volume in 1536-well plate format. To our knowledge, this represents the first large-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A168.

A High-Throughput Approach for Identification of Novel General Anesthetics

Anesthetic development has been a largely empirical process. Recently, we described a GABAergic mimetic model system for anesthetic binding, based on apoferritin and an environment-sensitive fluorescent probe. Here, a competition assay based on 1-aminoanthracene and apoferritin has been taken to a high throughput screening level, and validated using the LOPAC1280 library of drug-like compounds. A raw hit rate of ∼15% was reduced through the use of computational filters to yield an overall hit rate of ∼1%. These hits were validated using isothermal titration calorimetry. The success of this initial screen and computational triage provides feasibility to undergo a large scale campaign to discover novel general anesthetics.