Structure-Guided Design of DOT1L Methyltransferase Inhibitors By a Novel, Label Free Assay Platform

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4811-4811
Author(s):  
Joanna S. Yi ◽  
Alex Federation ◽  
Jun Qi ◽  
Sirano Dhe-Paganon ◽  
Michael Hadler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Target Genes ◽  
Histone 3 ◽  
Biochemical Assays ◽  
High Doses ◽  
H3k79 Methylation

Abstract Cooperation between several epigenetic modulators defines MLL-rearranged leukemia as an epigenomic-driven cancer. Wild type MLL catalyzes trimethylation of lysine 4 on histone 3 from the methyl donor S-adenosylmethionine (SAM) at homeobox and other genes important for hematopoiesis, promoting their expression during development. However, in MLL-rearrangements, its methyltransferase domain is ubiquitously lost and replaced with >70 known fusion partners. Many of these fusion partners recruit DOT1L, the only known SAM-dependent lysine methyltransferase responsible for the methylation of lysine 79 of histone 3 (H3K79)—a mark associated with most actively transcribed genes. Therefore, the recruitment of DOT1L by MLL fusion partners to MLL-target genes leads to aberrant H3K79 hypermethylation at these loci, resulting in inappropriate gene expression and leukemogenesis. DOT1L as a therapeutic target in MLL has been genetically validated by several groups, leading to the development of SAM-competitive small molecule inhibitors of DOT1L. These inhibitors exhibit excellent biochemical activity and selectivity, yet have delayed cellular activity and needing relatively high doses, with viability effects requiring 7-10 days and EC50s for H3K79 methylation depletion of 1-3 μM in cell lines. In animal studies, this translates to a modest survival benefit while requiring high doses through continuous osmotic subcutaneous infusion. Further optimization of DOT1L inhibitors is therefore needed. To date, development of DOT1L inhibitors has been slow, perhaps related to inadequacy of discovery chemistry assay technologies. All biochemical assays are radioactivity-based and are not miniaturizeable; low-throughput and delayed cellular effects of DOT1L inhibition all hamper the discovery of improved inhibitors. Therefore a pressing need towards improved DOT1L inhibitor discovery is a robust, accessible, and rapid profiling platform. Toward this goal, we synthesized both FITC- and biotin-tagged DOT1L probe ligands. We confirmed by structural studies that binding of the probes were similar to our previously published inhibitor, depleted H3K79 methylation, and had antiproliferative effects in MLL-rearranged cell lines. We then utilized the probes to devise two non-radioactive, orthogonal biochemical assays to competitively profile putative inhibitors: one employing bead-based, proxmity fluorescence technology and the second using fluorescence polarization technology. These assays are robust and adaptable to high-throughput screening. We also designed a miniaturizable high-content imaging, immunofluorescence-based assay to assess the effect of DOT1L inhibitors on H3K79 methylation, reporting cellular IC50s after just four days of treatment. These three assays were validated against three known DOT1L inhibitors of different potencies, accurately differentiating between the compounds. Together, these orthogonal assays define an accessible platform capability to discover and optimize DOT1L inhibitors. Our platform rank-ordered a library of SAM derivatives that we synthesized, indicating that large substituents off the SAM base does not affect DOT1L binding. We also explored other features of the SAM core structure, identifying several chlorinated probes that had increased cellular potency (IC50 values ~10nM) relative to the initial compounds published, without losing specificity for DOT1L. The inhibitory effect on MLL-target gene expression correlated to the H3K79me2 decrease reported in high content assay, validating that our high-content assay accurately reports on downstream biology seen later in treatment. And as expected, the high-content potencies of our chlorinated DOT1L probes also correlated to increased anti-proliferative effect in MLL cells. Overall, we utilized chemistry, biology, and chemical biology tools to develop this profiling platform capability for more rapid discovery and optimization of small molecule DOT1L inhibitors. These assays can additionally be used to screen for non-SAM competitive inhibitors in high-throughput fashion. Furthermore, the DOT1L inhibitors and probes synthesized here (available as open-source tools) are useful in deeper mechanistic studies of the DOT1L complex and its role in MLL. Disclosures Armstrong: Epizyme: Consultancy.

scholarly journals Development of a Cell-Based Reporter Assay for Screening of Inhibitors of Hypoxia-Inducible Factor 2-Induced Gene Expression

2006 ◽  
Vol 11 (6) ◽  
pp. 678-687 ◽  
Author(s):  
Girma M. Woldemichael ◽  
James R. Vasselli ◽  
Roberta S. Gardella ◽  
Tawnya C. Mckee ◽  
W. Marston Linehan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Clear Cell ◽  
Hypoxia Inducible Factor ◽  
Luciferase Reporter ◽  
Reporter Cell

Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

scholarly journals Screening for Small-Molecule Modulators of Long Noncoding RNA-Protein Interactions Using AlphaScreen

2015 ◽  
Vol 20 (9) ◽  
pp. 1132-1141 ◽  
Author(s):  
Roya Pedram Fatemi ◽  
Sultan Salah-Uddin ◽  
Farzaneh Modarresi ◽  
Nathalie Khoury ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Noncoding Rna ◽  
Target Genes ◽  
Regulatory Function ◽  
Downstream Target ◽  
Small Molecule Modulators ◽  
In Cells

Long non–protein coding RNAs (lncRNAs) are an important class of molecules that help orchestrate key cellular events. Although their functional roles in cells are not well understood, thousands of lncRNAs and a number of possible mechanisms by which they act have been reported. LncRNAs can exert their regulatory function in cells by interacting with epigenetic enzymes. In this study, we developed a tool to study lncRNA-protein interactions for high-throughput screening of small-molecule modulators using AlphaScreen technology. We tested the interaction of two lncRNAs: brain-derived neurotrophic factor antisense ( BDNF-AS) and Hox transcript antisense RNA ( HOTAIR), with Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase against a phytochemical library, to look for small-molecule inhibitors that can alter the expression of downstream target genes. We identified ellipticine, a compound that up-regulates BDNF transcription. Our study shows the feasibility of using high-throughput screening to identify modulators of lncRNA-protein interactions and paves the road for targeting lncRNAs that are dysregulated in human disorders using small-molecule therapies.

Intersecting Chemical Genomic and Genetic Screens Identifies Glycogen Synthase Kinase-3α (GSK-3α) as a Modulator of Differentiation In Acute Myeloid Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1000-1000
Author(s):  
Versha Banerji ◽  
Kenneth N. Ross ◽  
Loretta S. Li ◽  
Stacey M. Frumm ◽  
Anna C. Schinzel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Small Molecule ◽  
High Throughput ◽  
Glycogen Synthase ◽  
Morphological Changes ◽  
Gene Expression Signature ◽  
Gsk 3Β ◽  
Shrna Screen

Abstract Abstract 1000 The treatment of acute myeloid leukemia (AML) poses a vexing challenge despite an improved understanding of its molecular pathogenesis. A two-hit theory has been proposed for the pathogenesis of AML where the first hit imparts a proliferation defect and the second a block in differentiation. Drug discovery efforts for AML, however, have largely focused on the proliferation defect. Using the intersection of chemical biology and high-throughput genetic screening we sought to identify new AML differentiation targets by measuring the induction of a complex gene expression signature of myeloid maturation. We performed two independent small molecule library screens and a high-throughput shRNA screen for perturbations that induce differentiation in AML cells. We measured this differentiation signature using the previously described gene expression-based high-throughput screening (GE-HTS) approach in which gene expression signatures serve as surrogates for different biological states. Glycogen Synthase Kinase-3 (GSK-3) emerged as a target at the intersection of these three screens. GSK-3 is a multifunctional serine threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation and proliferation, with an emerging role in human leukemia. We demonstrate that the GSK-3 inhibitors scoring in the primary screens indeed induce the differentiation signature with a dose-response in AML cell lines. In order to further validate GSK-3 as a target, we extended testing to lithium chloride and SB216763, two commonly used GSK-3 inhibitors not in the original screens. Both of these molecules induced AML differentiation as measured by gene expression and morphological changes in multiple AML cell lines and in primary patient blasts in vitro. GSK-3 is expressed as two highly homologous but non-redundant isoforms, GSK-3α and GSK-3β, with small molecule inhibitors non-selectively reported to target both. In order to further validate the findings of the small molecule library screens, we performed a high-throughput shRNA screen targeting the human kinome for shRNAs that induce differentiation. Multiple hairpins against GSK-3α scored. In secondary testing of four AML cell lines, we found that genetic loss of GSK-3α induced differentiation as measured by induction of the complex gene expression signature, alterations in genome-wide expression, and morphological changes associated with maturation. Moreover, colony formation in methylcellulose was impeded. These effects could be rescued with a GSK-3α cDNA immune to the effects of the shRNA, further supporting the on-target activity of the hairpin. In contrast, GSK-3β-directed hairpins induced minimal differentiation. We next extended testing to an in vivo U937 orthotopic model of AML. While pan-GSK-3 inhibition with lithium chloride treatment did not demonstrate efficacy in this model, inhibition of GSK-3α with shRNA attenuated development of disease compared to an shRNA control. We hypothesize that the rise in β-catenin with pan-inhibition of GSK-3 may attenuate the response to lithium in vivo. In contrast, isolated knockdown of GSK-3α does not induce β-catenin. While much of the prior cancer literature has focused on the role of GSK-3β in human malignancy, these studies suggest a role for GSK-3α in AML differentiation and support a role for GSK-3α-directed targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Next-Generation NAMPT Inhibitors For ALL Identified By Sequential High-Throughput Phenotypic Chemical and Functional Genomic Screens

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 171-171
Author(s):  
Michael C. Wei ◽  
Christina J. Matheny ◽  
Michael C. Bassik ◽  
Alicia J. Donnelly ◽  
Martin Kampmann ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chemical Genetics ◽  
Specific Cell ◽  
Functional Genomic ◽  
A Cell ◽  
Nanomolar Range

Abstract There is a critical need for new agents with novel therapeutic targets and improved safety profiles in high-risk acute lymphoblastic leukemia (ALL), which is a significant cause of morbidity and mortality in pediatric and adult populations. Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of their mechanistically relevant targets remains a major experimental challenge. We applied a chemical genetics approach involving sequential unbiased high-throughput chemical and ultra-complex, genome-scale shRNA screens to address this challenge and identify novel agents in ALL. A cell-based phenotypic high-throughput chemical screen of 115,000 compounds identified 640 compounds that inhibited growth of one or both ALL cell lines with high-risk Mixed Lineage Leukemia (MLL) genetic abnormalities, but did not inhibit the growth of a cell line lacking MLL rearrangement. The most potent and selective 64 were tested on an expanded panel of eight human B-ALL cell lines to identify lead compound STF-118804. STF-118804 inhibited the growth of most B-ALL cell lines with high potency demonstrating IC50 values in the low nanomolar range. Leukemic samples from five pediatric ALL patients were also sensitive to STF-118804 in the low nanomolar range. STF-118804 displayed 5–10 fold more potency against most leukemias in comparison to cycling human (lineage-negative cord blood) and murine (c-kit+ bone marrow) progenitor cells, demonstrating a therapeutic index. STF-118804 displays distinctive cytotoxicity by inducing apoptosis without causing a phase-specific cell cycle arrest. To discover the molecular target of STF-118804, a functional genomic screen was performed to identify shRNAs that conferred sensitivity or resistance to STF-118804, utilizing an ultra-complex (∼25 shRNAs per gene) library targeting in total ∼9300 human genes and 1000s of negative control shRNAs. NAMPT was the most statistically significant gene to confer sensitivity to STF-118804, suggesting that STF-118804 functioned as a NAMPT inhibitor. NAMPT encodes nicotinamide phosphoribosyl transferase, a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in many biochemical processes. STF-118804 was confirmed as a novel class of NAMPT inhibitor through metabolic rescue, enzymatic, and genetic studies. STF-118804 displayed strong inhibitory activity in in vitro NAMPT enzymatic assays. Over-expression of wild-type or mutant NAMPT in cells indicated that STF-118804 cytotoxicity is a result of its ability to inhibit NAMPT, and that STF-118804 does not have significant off-target effects on cell viability. The potential efficacy of STF-118804 in vivo was assessed in an orthotopic xenograft model of ALL. Sublethally irradiated immunodeficient mice were transplanted with human ALL cells engineered to constitutively express firefly luciferase. Dosing of STF-118804 was initiated two weeks post-transplant when ALL cells had engrafted and bioluminescent signal was detectable. Mice treated with STF-118804 showed regression of leukemia by bioimaging and significantly extended survival. The leukemia initiating cell (LIC) frequency in STF-118804 treated mice was significantly lower (∼8 fold) than vehicle treated mice, showing that STF-118804 was effective in reducing LICs. In summary, tandem high-throughput screening identified a highly-specific, potent, and structurally novel small molecule inhibitor of NAMPT that is active in ALL. Tandem high throughput screening using chemical and ultra-complex shRNA libraries provides a rapid chemical genetics approach for seamless progression from small molecule lead identification to target discovery and validation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of a chemical modulator of EZH2-mediated silencing by cell-based high-throughput screening assay

2019 ◽  
Vol 166 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Akihiro Murashima ◽  
Keiko Shinjo ◽  
Keisuke Katsushima ◽  
Tetsuo Onuki ◽  
Yasumitsu Kondoh ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Target Genes ◽  
Cancer Cell Lines ◽  
Methyltransferase Activity ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Catalytic Inhibitor

Abstract Dysregulation of enhancer of zeste homologue 2 (EZH2), a methyltransferase component of polycomb repressive complex 2, is found in many types of cancers especially those that are highly progressive and aggressive. Specific catalytic inhibitors of EZH2 have high anti-tumour activity, particularly in lymphomas with EZH2 activating mutations. However, the clinical benefits of EZH2 catalytic inhibitors in tumours overexpressing EZH2 are still limited. Here, we identified NPD13668, a novel modulator of EZH2-mediated gene silencing, from 329,049 small chemical compounds using a cell-based high-throughput screening assay. NPD13668 reactivated the expression of silenced H3K27me3 target genes together with depletion of the H3K27me3 modification. In addition, NPD13668 repressed the cell growth of prostate cancer cell lines (PC3 and LNCaP) and ovarian cancer cell lines (SKOV3 and NIH-OVCAR3). NPD13668 partially inhibited the methyltransferase activity of EZH2 in vitro. Genome-wide expression analysis revealed that after NPD13668 treatment, about half of the upregulated genes overlapped with genes upregulated after treatment with GSK126, well-known EZH2 catalytic inhibitor, indicating that NPD13668 is a potential modulator of EZH2 methyltransferase activity. Our data demonstrated that targeting the pharmacological inhibition of EZH2 activity by NPD13668 might be a novel cancer treatment.

scholarly journals High-throughput screening and genome-wide analyses of 44 anticancer drugs in the 1000 Genomes cell lines reveals an association of the NQO1 gene with the response of multiple anticancer drugs

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009732
Author(s):  
Farida S. Akhtari ◽  
Adrian J. Green ◽  
George W. Small ◽  
Tammy M. Havener ◽  
John S. House ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cancer Patients ◽  
Cell Lines ◽  
Anticancer Drugs ◽  
High Throughput ◽  
High Throughput Screening ◽  
Genome Wide Association ◽  
1000 Genomes ◽  
Genome Wide ◽  
Drug Treatments

Cancer patients exhibit a broad range of inter-individual variability in response and toxicity to widely used anticancer drugs, and genetic variation is a major contributor to this variability. To identify new genes that influence the response of 44 FDA-approved anticancer drug treatments widely used to treat various types of cancer, we conducted high-throughput screening and genome-wide association mapping using 680 lymphoblastoid cell lines from the 1000 Genomes Project. The drug treatments considered in this study represent nine drug classes widely used in the treatment of cancer in addition to the paclitaxel + epirubicin combination therapy commonly used for breast cancer patients. Our genome-wide association study (GWAS) found several significant and suggestive associations. We prioritized consistent associations for functional follow-up using gene-expression analyses. The NAD(P)H quinone dehydrogenase 1 (NQO1) gene was found to be associated with the dose-response of arsenic trioxide, erlotinib, trametinib, and a combination treatment of paclitaxel + epirubicin. NQO1 has previously been shown as a biomarker of epirubicin response, but our results reveal novel associations with these additional treatments. Baseline gene expression of NQO1 was positively correlated with response for 43 of the 44 treatments surveyed. By interrogating the functional mechanisms of this association, the results demonstrate differences in both baseline and drug-exposed induction.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

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