Library Of Pharmacologically Active Compounds Research Articles

A high-throughput cell-based screening method for Zika virus protease inhibitor discovery

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

B-364 Inhibition of Hypoxia-Inducible Factor-1a (HIF-1 a) Provides a Therapeutic Approach for Hepatocellular Carcinoma (HCC)

Abstract Background The most frequent type of primary liver cancer in adults is hepatocellular carcinoma (HCC) which is the third-highest cause of cancer-related death worldwide. Globally, people die from liver cancer each year, with HCC accounting for around 90% of those deaths, making it a serious public health concern. Cirrhosis, hepatitis B, hepatitis C, and non-alcoholic fatty liver disease are all high-risk factors for HCC development. In addition, HCC is one of the most frequent solid tumors found in humans. Due to insufficient blood supply, these solid tumors usually are devoid of oxygen and nutrients. A reduction in oxygen concentration results in the formation of a hypoxic environment that downregulates the metabolic process and is fatal to cancer cells. To circumvent this barrier to growth, cancer cells produce hypoxia-inducible factor-1 (HIF-1), which alters their metabolic pathways and allows them to survive. HIF-1alpha (HIF-1α), a cytoplasmic subunit of HIF-1 can be overexpressed in hypoxic conditions. HCC cells cannot proliferate under hypoxia without a high level of HIF-1α expression, making it as a potential cancer treatment target. Using the LOPAC (Library of Pharmacologically Active Compounds) drug library, we have identified multiple drug molecules that inhibit the expression of HIF-1α. Methods We constructed specific SK-HEP-1 cells, a human liver cancer cell line, expressing the luciferase reporter gene under the promoter of HIF-1α. By using the cells, we screened the (LOPAC) library. Other bioanalytical methods were used to confirm and analyze identified candidates. Results We successfully identified several compounds that are able to inhibit HIF-1α expression by using luciferase activity assays and Western blot analysis (WB). Further investigation was conducted to determine the molecular mechanism and function for one of the drug candidates, termed as UZ-1, in inhibiting HIF-1α in HCC by using the MTT assay and WB. UZ-1, an antirheumatic agent, showed a strong inhibitory effect on the expression of HIF-1α in SK-HEP-1 cells. In addition, it suppressed the signaling pathways associated with HIF-1α, leading to the apoptosis of HCC cells. Conclusion The UZ-1 molecule inhibits HIF-1α expression, as a result, and displays anticancer function in HCC cells through inducing apoptosis. Our result suggests that UZ-1 may be a potential drug candidate for the treatment of HCC.

High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation.

Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not β-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception.

IDF21-0319 Novel compounds found to regulate VEGF-A alternative splicing in diabetic podocytes

Background: Alternative splicing (AS) gives rise to multiple proteins from the same gene, by skipping or including exons or parts of them, or by keeping introns as coding sequences. The resulting AS isoforms may function differently from each other. Vascular endothelial growth factor A (VEGF-A) is an angiogenic protein. The alternatively spliced variant, VEGF-A165b, is formed when a distal 3’ splice site in exon 8 is selected. The anti-angiogenic and anti-permeability VEGF-A165b has reno-protective properties and has been shown to rescue kidney function in mouse models of diabetic nephropathy. Aim: The aim of this study was to investigate three novel compounds that regulate VEGF-A AS in podocytes exposed to a diabetic environment. Method: Two compounds trovafloxacin and 10058-F4 (5-[(4-Ethylphenyl) methylene]-2-thioxo-4-thiazolidinone), were identified to switch VEGF-A splicing in a previous screen performed by our lab using a library of pharmacologically active compounds (LOPAC). The third compound, delphinidin, was found to be key compound regulating VEGF-A AS in a natural blueberry and sea buckthorn extract (DIAVIT). Human podocytes were exposed to a diabetic environment (glucose soup [GS]: 25 mM glucose, 1 ng/ml TNF-α, 1 ng/ml IL-6, and 100 nM insulin), in comparison to a normal glucose (5.5 mM glucose) and an osmotic (5.5 mM glucose % 2B 19.5 mM mannitol) control, for 48 hours. RNA and protein were extracted for RT-PCR and Western blotting analysis of splice isoforms. Results: Changes in the VEGF-A165a/VEGF-A165b splicing ratio as well as the activation and expression of splice factors known to regulate this event will be compared between the diabetic environment and controls, in addition to the effects of each compound. Trovafloxacin (10 mM), 10058-F4 (10 mM), and delphinidin (10 mg/ml) significantly increased the anti-angiogenic VEGF-A165b relative to pro-angiogenic VEGF-A165a in podocytes exposed to a normal glucose environment at either the mRNA or protein level. Furthermore, 10058-F4 and delphinidin were also found to increase the VEGF-A165b/VEGF-A165a ratio in podocytes exposed to a diabetic environment. Regarding 10058-F4, pilot data suggests that this compound may influence the expression of Clk-1, a kinase known to regulate VEGF-A splice site selection, in diabetic podocytes. On the other hand, delphinidin was found to increase the phosphorylation of SRSF6, a splice factor known to promote VEGF-A165b splice site selection, in diabetic podocytes. 10058-F4 was found to downregulate SRSF1, which may be through c-myc inhibition. Discussion: We have identified three novel compounds that regulate VEGF-A AS to promote the expression of the reno-protective VEGF-A165b isoform in podocytes. This study will further investigate the mechanism of action of these three compounds regarding VEGF-A splicing regulation in diabetic podocytes. The final goal of this project is to identify whether these novel AS regulatory compounds can be used to develop new therapeutic strategies in diabetic nephropathy.

Discovery and characterization of potent And-1 inhibitors for cancer treatment.

Acidic nucleoplasmic DNA‐binding protein 1 (And‐1), an important factor for deoxyribonucleic acid (DNA) replication and repair, is overexpressed in many types of cancer but not in normal tissues. Although multiple independent studies have elucidated And‐1 as a promising target gene for cancer therapy, an And‐1 inhibitor has yet to be identified. Using an And‐1 luciferase reporter assay to screen the Library of Pharmacologically Active Compounds (LOPAC) in a high throughput screening (HTS) platform, and then further screen the compound analog collection, we identified two potent And‐1 inhibitors, bazedoxifene acetate (BZA) and an uncharacterized compound [(E)‐5‐(3,4‐dichlorostyryl)benzo[c][1,2]oxaborol‐1(3H)‐ol] (CH3), which specifically inhibit And‐1 by promoting its degradation. Specifically, through direct interaction with And‐1 WD40 domain, CH3 interrupts the polymerization of And‐1. Depolymerization of And‐1 promotes its interaction with E3 ligase Cullin 4B (CUL4B), resulting in its ubiquitination and subsequent degradation. Furthermore, CH3 suppresses the growth of a broad range of cancers. Moreover, And‐1 inhibitors re‐sensitize platinum‐resistant ovarian cancer cells to platinum drugs in vitro and in vivo. Since BZA is an FDA approved drug, we expect a clinical trial of BZA‐mediated cancer therapy in the near future. Taken together, our findings suggest that targeting And‐1 by its inhibitors is a potential broad‐spectrum anti‐cancer chemotherapy regimen.

What Makes a Compound a Sodium Channel Inhibitor

Voltage-gated sodium channels has been found to be the most promiscuous group of protein drug targets. To develop drugs for this uniquely indiscriminate binding site is a challenge that requires an unconventional approach. A practicable strategy would be to focus on mechanism of action, not affinity. One of the most significant questions regarding mechanism is to distinguish nonspecific and specific binding. We expect that those compounds which bind specifically to the Phe residue of the local anesthetic binding site (F1579 in Nav1.4, F1760 in Nav1.5) will act predominantly by modulation, and therefore will have a preferable therapeutic profile.In this study we identified chemical properties which determine effective binding involving this residue. We used the data from a study (Zheng et al., 2014), where both wild type and binding site mutant channels were screened using the Library of Pharmacologically Active Compounds (LOPAC). We have found that those compounds which are highly potent, but only in the presence of the Phe residue, occupy a remarkably small fraction of the chemical space filled up with compounds of the LOPAC.These are strongly lipophilic compounds, with 2-3 aromatic rings, low polar surface area, containing a positively charged nitrogen, which however is able to deprotonate at physiological pH. Quantitatively, these compound have molecular weight in the range of 250-450, aromatic atom count: 12-18, logP: 3-6, pKa: 7-10, polar surface area: 4-70 Å2. The chemical space delineated by these criteria contains 8 % of all compounds, of which 56 % were potent (IC50 less than 5 µM) and Phe-sensitive (larger than 5x difference in IC50) sodium channel inhibitors. Of the remaining 92 % of all compounds, located outside this restricted section of the chemical space, only 2.8 % were both potent and Phe-sensitive.

High-Throughput Screening Assays for Cancer Immunotherapy Targets: Ectonucleotidases CD39 and CD73.

Production of adenosine in the extracellular tumor microenvironment elicits strong immunosuppression and is associated with tumor progression. Thus, targeting adenosine-generating ectonucleotidases is a potential strategy to stimulate and prolong antitumor immunity. Because the reaction products of ectonucleotidases differ by a single phosphate group, selective detection in an assay format that is compatible with high-throughput screening (HTS) has been elusive. We report the development of biochemical assays capable of measuring the activity of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; also known as CD39) and ecto-5'-nucleotidase (CD73). Both assays leverage the Transcreener HTS Assay platform, which facilitates selective immunodetection of nucleotides with homogenous fluorescent readouts, fluorescence polarization or time-resolved fluorescence energy transfer. The Transcreener AMP2 Assay was used to measure CD39 activity, allowing detection of adenosine monophosphate (AMP) production (Z' > 0.6) with subnanomolar amounts of CD39, allowing IC50 determination for tool compounds, consistent with previously reported values. To detect the production of adenosine by CD73, the Transcreener ADP2 Assay was coupled with adenosine kinase (AK); conversion of adenosine to AMP and adenosine diphosphate (ADP) by AK allows detection with ADP2 antibody. The Transcreener AMP2 Assay was used to screen a 1280 Library of Pharmacologically Active Compounds (LOPAC) library and a 1600-compound subset of a ChemBridge diversity library for CD39 inhibitors, allowing the identification of nine and eight candidate compounds from each library, respectively. The Transcreener ADP2 Assay was used to screen 1600 compounds from the ChemBridge diversity library for CD73 inhibitors and identified 14 potential candidates. HTS-compatible assays for ectonucleotidase activity may allow identification of purinergic signaling pathway inhibitors important for tumor-specific immune responses during tumor pathogenesis.

A high-throughput screen of pharmacologically active compounds for inhibitors of UHRF1 reveals epigenetic activity of anthracycline derivative chemotherapeutic drugs.

DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z’ factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.

Detection of nanocarrier potentiation on drug induced phospholipidosis in cultured cells and primary hepatocyte spheroids by high content imaging and analysis

Considerable effort has been made to develop nanocarriers for controlled drug delivery over the last decade, while it remains unclear how the strength of adverse drug effect will be altered when a drug is loaded on the nanocarrier. Drug-induced phospholipidosis (DIP) is characterized with excessive accumulation of phospholipids in cells and is common for cationic amphiphilic drugs (CAD). Previously, we have reported that PEGylated graphene oxide (PEG-GO) loaded with several CAD can potentiate DIP. In current study, we extended our study on newly identified phospholipidosis (PLD) inducers that had been identified from the Library of Pharmacologically Active Compounds (LOPAC), to investigate if PEO-GO loaded with these CAD can alter DIP. Twenty-two CAD were respectively loaded on PEG-GO and incubated with RAW264.7, a macrophage cell line. The results showed that when a CAD was loaded on PEG-GO, its strength of PLD induction can be enhanced, unchanged or attenuated. PEG-GO loaded with Ifenprodil exhibited the highest PEG-GO potentiation effect compared to Ifenprodil treatment alone in RAW264.7 cells, and this effect was confirmed in human hepatocellular carcinoma HepG2, another cell line model for PLD induction. Primary hepatocyte culture and spheroids mimicking in vivo conditions were used to further validate nanocarrier potentiation on DIP by Ifenprodil. Stronger phospholipid accumulation was found in PEG-GO/Ifenprodil treated hepatocytes or spheroids than Ifenprodil treatment alone. Therefore, evidences were provided by us that nanocarriers may increase the adverse drug effects and guidance by regulatory agencies need to be drafted for the safe use of nanotechnology in drug delivery.

Combined chemical genetics and data-driven bioinformatics approach identifies receptor tyrosine kinase inhibitors as host-directed antimicrobials

Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.

Abstract PR15: Rho-associated kinase 1 inhibition is synthetically lethal with von Hippel-Lindau deficiency in clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (CC-RCC) is the most lethal of all genitourinary cancers. The functional loss of the von Hippel-Lindau (VHL) gene occurs in 90% of CC-RCC, driving cancer progression. The objective of this study was to identify chemical compounds that are synthetically lethal with VHL deficiency in CC-RCC. An annotated chemical library, the library of pharmacologically active compounds (LOPAC), was screened in parallel on VHL-deficient RCC4 cells and RCC4VHL cells with re-introduced VHL. The ROCK inhibitor, Y-27632, was identified and validated for selective targeting of VHL-deficient CC-RCC in multiple genetic backgrounds by clonogenic assays. Downregulation of ROCK1 by small interfering RNA (siRNA) selectively reduced the colony forming ability of VHL-deficient CC-RCC, thus mimicking the effect of Y-27632 treatment, whereas downregulation of ROCK2 had no effect. In addition, three other ROCK inhibitors, RKI 1447, GSK 429286, AT13148 selectively targeted VHL-deficient CC-RCC. Importantly, we found that mammalian target of rapamycin (mTOR) inhibitor Everolimus synergized with ROCK inhibitor RKI 1447 in targeting VHL-deficient cells. Accordingly, AT13148, an AGC kinase inhibitor blocking S6 kinase (S6K, downstream target of mTOR) in addition to ROCK1&2, showed the most strong synthetic lethality effect out of all ROCK inhibitors tested. CC-RCC treatment with ROCK inhibitors is cytotoxic and cytostatic based on bromodeoxyuridine (BrdU) assay, propidium iodide (PI) staining and growth curves, and blocks cell migration based on transwell assay. On the one hand, knockdown of hypoxia-inducible factor (HIF) β in the VHL-deficient CC-RCC had a protective effect against Y-27632 treatment, mimicking VHL reintroduction. On the other hand, CC-RCCVHL cells were sensitized to Y-27632 treatment in hypoxia (2% O2). These results suggest that synthetic lethality between ROCK inhibition and VHL deficiency is dependent on HIF activation. Moreover, HIF1α or HIF2α overexpression in CC-RCCVHL cells is sufficient to sensitize them to ROCK inhibition. Finally, Y-27632 treatment inhibited growth of subcutaneous 786-OT1 CC-RCC tumors in mice. Thus, ROCK inhibitors represent potential therapeutics for VHL-deficient CC-RCC. Citation Format: Jordan M. Thompson, Quy H. Nguyen, Manpreet Singh, Matthew W. Pavesic, Irina Nesterenko, Luke J. Nelson, Alicia Liao, Olga V. Razorenova. Rho-associated kinase 1 inhibition is synthetically lethal with von Hippel-Lindau deficiency in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR15.

Development of Novel Cell Lines for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators

Estrogen-related receptor alpha (ERRα), the first orphan nuclear receptor discovered, is crucial for the control of cellular energy metabolism. ERRα and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), are required for rapid energy production in response to environmental challenges. They have been implicated in the etiology of metabolic disorders such as type 2 diabetes and metabolic syndrome. ERRα also plays a role in the pathogenesis of breast cancer. Identification of compounds that modulate ERRα signaling may elucidate environmental factors associated with these diseases. Therefore, we developed stable cell lines containing an intact ERRα signaling pathway, with and without the coactivator PGC-1α, to use as high-throughput screening tools to detect ERRα modulators. The lentiviral PGC-1α expression constructs and ERRα multiple hormone response element (MHRE) reporters were introduced into HEK293T cells that express endogenous ERRα. A cell line expressing the reporter alone was designated “ERR.” A second cell line expressing both reporter and PGC-1α was named “PGC/ERR.” Initial screenings of the Library of Pharmacologically Active Compounds (LOPAC) identified 33 ERR and 22 PGC/ERR agonists, and 54 ERR and 15 PGC/ERR antagonists. Several potent ERRα agonists were dietary plant compounds (e.g., genistein). In conclusion, these cell lines are suitable for high-throughput screens to identify environmental chemicals affecting metabolic pathways and breast cancer progression.

Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms.

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell-based assays in high-throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH-SY5Y). An enzyme-based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high-throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.

Rho-associated kinase 1 inhibition is synthetically lethal with von Hippel-Lindau deficiency in clear cell renal cell carcinoma.

Clear Cell Renal Cell Carcinoma (CC-RCC) is the most lethal of all genitourinary cancers. The functional loss of the von Hippel-Lindau (VHL) gene occurs in 90% of CC-RCC, driving cancer progression. The objective of this study was to identify chemical compounds that are synthetically lethal with VHL deficiency in CC-RCC. An annotated chemical library, the Library of Pharmacologically Active Compounds (LOPAC), was screened in parallel on VHL-deficient RCC4 cells and RCC4VHL cells with re-introduced VHL cDNA. The ROCK inhibitor, Y-27632, was identified and validated for selective targeting of VHL-deficient CC-RCC in multiple genetic backgrounds by clonogenic assays. Downregulation of ROCK1 by siRNA selectively reduced the colony forming ability of VHL-deficient CC-RCC, thus mimicking the effect of Y-27632 treatment, whereas downregulation of ROCK2 had no effect. In addition, two other ROCK inhibitors, RKI 1447 and GSK 429286, selectively targeted VHL-deficient CC-RCC. CC-RCC treatment with ROCK inhibitors is cytotoxic and cytostatic based on BrdU assay, Propidium Iodide (PI) staining, and growth curves; and blocks cell migration based on transwell assay. Importantly, knockdown of Hypoxia Inducible Factor (HIF) β in the VHL-deficient CC-RCC had a protective effect against Y-27632 treatment, mimicking VHL reintroduction. On the other hand, CC-RCCVHL cells were sensitized to Y-27632 treatment in hypoxia (2% O2). These results suggest that synthetic lethality between ROCK inhibition and VHL deficiency is dependent on HIF activation. Moreover, HIF1α or HIF2α overexpression in CC-RCCVHL cells is sufficient to sensitize them to ROCK inhibition. Finally, Y-27632 treatment inhibited growth of subcutaneous 786-OT1 CC-RCC tumors in mice. Thus, ROCK inhibitors represent potential therapeutics for VHL-deficient CC-RCC.

FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay: New Internally Quenched Fluorogenic Substrates for High-Throughput Screening

In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.

A cell-based quantitative high-throughput image screening identified novel autophagy modulators

Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.

Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

Abstract 3924: A homogenous bioluminescent system for measuring GTPase, GAP and GEF activities

Abstract GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay (GTPase/GAP/GEF-Glo) system to analyze these proteins in a simple, convenient “add-mix-read” format. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP and the process of nucleotide exchange. Manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and detection of the ATP using luciferin/luciferase combination. We show that the assay is sensitive, and robust when analyzing for analyzing intrinsic GTPase- activity, GAP-stimulated GTPase- activity, GAP-activity and GEF- activity. The system can also be used to analyze these proteins expressed in cells as a fusion protein performing the assay in a pulldown -format. Furthermore, there was minimal false hits when tested for compound interference using the library of pharmacologically active compounds (LOPAC) and its robustness as indicated by high Z’-factor of 0.93 and CV of 2.2%. Citation Format: Subhanjan Mondal, Kevin Hsiao, Said A. Goueli. A homogenous bioluminescent system for measuring GTPase, GAP and GEF activities. [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 3924. doi:10.1158/1538-7445.AM2015-3924

Discovery of an inhibitor of Z-alpha1 antitrypsin polymerization.

Polymerization of the Z variant alpha-1-antitrypsin (Z-α1AT) results in the most common and severe form of α1AT deficiency (α1ATD), a debilitating genetic disorder whose clinical manifestations range from asymptomatic to fatal liver and/or lung disease. As the altered conformation of Z-α1AT and its attendant aggregation are responsible for pathogenesis, the polymerization process per se has become a major target for the development of therapeutics. Based on the ability of Z-α1AT to aggregate by recruiting the reactive center loop (RCL) of another Z-α1AT into its s4A cavity, we developed a high-throughput screening assay that uses a modified 6-mer peptide mimicking the RCL to screen for inhibitors of Z-α1AT polymer growth. A subset of compounds from the Library of Pharmacologically Active Compounds (LOPAC) with molecular weights ranging from 300 to 700 Da, was used to evaluate the assay’s capabilities. The inhibitor S-(4-nitrobenzyl)-6-thioguanosine was identified as a lead compound and its ability to prevent Z-α1AT polymerization confirmed by secondary assays. To further investigate the binding location of S-(4-nitrobenzyl)-6-thioguanosine, an in silico strategy was pursued and the intermediate α1AT M* state modeled to allow molecular docking simulations and explore various potential binding sites. Docking results predict that S-(4-nitrobenzyl)-6-thioguanosine can bind at the s4A cavity and at the edge of β-sheet A. The former binding site would directly block RCL insertion whereas the latter site would prevent β-sheet A from expanding between s3A/s5A, and thus indirectly impede RCL insertion. Altogether, our investigations have revealed a novel compound that inhibits the formation of Z-α1AT polymers, as well as in vitro and in silico strategies for identifying and characterizing additional blocking molecules of Z-α1AT polymerization.

Identification of signalling cascades involved in red blood cell shrinkage and vesiculation

Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.