TOOLS FOR DRUG DISCOVERY

Transcription

1 TOOLS FOR DRUG DISCOVERY targeting epigenetics and transcription Epigenetics Gene Regulation mirna

2 DRUG DISCOVERY TOOLBOX assays, reagents and services to accelerate your research Target Identification Target Validation Drug Discovery Preclinical Testing Drug discovery programs that focus on epigenetic targets have attracted much attention and investment in recent years. This is driven by the potential to develop novel therapeutic strategies that can reverse transcriptional and epigenetic abnormalities. Active Motif is the industry leader in providing innovative technologies for epigenetics and gene regulation research. This brochure features assays, reagents and services that can easily be integrated into your drug discovery workflow to help you streamline and advance the process. Target-relevant Antibodies Assay-ready Proteins Histone Substrates Multiplex Assays for Modifications Genome-wide Services Screening Tools mirna Functional Assays Pathway Analysis Global Response Kits For a complete list of available products, please visit us at 2

3 ANTIBODIES for discovery and analysis of druggable targets Active Motif is more than just an antibody supplier. We identify, manufacture and rigorously test each antibody in our vast collection to ensure that we offer only the highest quality product. We monitor every step from immunogen design and specificity screening to application validation. This thorough process ensures our antibodies meet the high standards for consistency and quality you require to achieve the goals of your drug discovery and development efforts. Rigorous application testing Assay-compatible formulations Large lot sizes Monoclonals to histone PTMs ANTIBODIES FOR RELEVANT DISEASE AREAS Oncology Neurobiology Immunology Metabolic disorders Inflammation Cardiovascular disease OVER 600 KEY DNA METHYLATION, HISTONE AND TRANSCRIPTION FACTOR TARGETS EPIGENETICS H2A, H2B, H3 and H4 mods DNA methylation Bromodomain proteins Methyltransferases Demethylases Acetyltransferases Deacetylases TRANSCRIPTION FACTORS Hormone receptors NFkB family members STAT family members IRF family members AP-1 family members p53 c-myc Product Catalog No. BRD4 antibody (pab) DOT1L antibody (pab) EED antibody (mab) EZH2 antibody (mab) HDAC1 antibody (mab) HDAC2 antibody (mab) Histone H3K4me3 antibody (mab) Histone H3K27me3 antibody (mab) Histone H3K36me2 antibody (mab) Histone H3K79me2 antibody (pab) LSD1 / KDM1A antibody (pab) MMSET / WHSC1 antibody (mab) Smyd3 antibody (pab) For more information and a complete list of available antibodies, please visit us at sales@activemotif.com 3

4 RECOMBINANT PROTEINS purified assay-ready proteins There is no need to waste your valuable time and resources producing proteins for assay development. Active Motif s comprehensive portfolio of high-quality recombinant proteins, enzymes and domains are ideal for use in your specific assays. With over 300 purified assay-ready recombinant proteins including transcription factors, histones, DNA and histone modifying enzymes and bromodomains you are sure to find what you need to develop more efficient assays for your drug discovery and development programs. PROTEINS FOR BIOCHEMICAL ASSAYS Enzyme activity assays Inhibitor screens Binding studies TR-FRET AlphaLISA OFFERING THE LARGEST COLLECTION OF RECOMBINANT BROMODOMAINS Select from over 40 bromodomains Designed specifically for use in assays and screens BRD4 Ac Ac Transcription I-BET Ac Ac BRD4 I-BET COMPREHENSIVE PORTFOLIO OF READY-TO-USE RECOMBINANT PROTEINS EPIGENETICS Methyltransferases Demethylases Acetyltransferases Deacetylases Chromatin modifiers Product Bromodomains TRANSCRIPTION FACTORS Hormone receptors NFkB JAK/STAT PPAR p53 c-myc Catalog No. BRD4 ( ) protein CARM1 protein, active ER protein EZH2 protein complex, active LSD1 / KDM1A protein, active NFkB p65 protein p53 protein p300 protein PRMT5 complex, active Set2 protein, active SIRT1 protein, active Smyd3 protein For a complete list of available purified recombinant proteins, please visit us at 4

5 FULL-LENGTH MODIFIED HISTONES biologically relevant assay substrates Active Motif offers an exclusive collection of 60 full-length unmodified and modified recombinant histones, including site- and degree-specific modifications such as methylation, acetylation and phosphorylation. Because of our patented Expressed Protein Ligation (EPL) and Methylated Lysine Analog (MLA) synthesis technologies, Active Motif is the only company to offer full-length modified histones. Your in vitro biochemical assay can better identify and validate enzyme inhibitors when the system mimics actual cellular biology. To help you make the correct choice of substrate, Active Motif offers over 60 different recombinant modified H3 and H4 histones. Use as stand-alone substrates or assemble along with recombinant H2A and H2B to generate nucleosome and oligonucleosomes. By choosing the most biologically relevant substrate, you can ensure the most accurate result. WHICH HISTONE SUBSTRATE IS RIGHT FOR YOUR ASSAY? THE LARGEST COLLECTION OF RECOMBINANT HISTONES AVAILABLE Over 60 recombinant histones Site- and degree-specific modifications Patented EPL & MLA synthesis technologies Biotinylated histones available for FRET Product Catalog No. Histone H3 biotinylated Histone H2A Histone H2B Histone H Histone H3K4me Histone H3K4me Histone H3K4me Histone H3K9me Histone H3K9me Histone H3K9me Histone H3K9ac Histone H3K14ac Histone H3K18ac Histone H3K27ac Histone H3K27me Histone H3K36me Histone H3K79me Histone H4K20me Chromatin Assembly Kit Nucleosome Assembly Control DNA Histone Purification Kit Histone Purification Mini Kit Choosing the correct histone substrate for assay development is key to achieving the best results. Opinion leaders in epigenetics recognize the power of reconstituting recombinant chromatin for creating biologically relevant substrates. For a complete list of available recombinant histones, please visit us at sales@activemotif.com 5

6 HISTONE MODIFICATION PROFILING Luminex Histone H3 PTM Multiplex Assays Partnering with Luminex, the multiplexing leader, Active Motif has developed the first multiplex epigenetic assay for studying histone post-translational modifications (PTMs). The Histone H3 PTM Multiplex Assay is designed for use with Luminex instruments, enabling high-throughput processing of nanogram-sized samples to simultaneously interrogate multiple PTMs in a single reaction. Time-consuming Westerns blots can now be replaced in early target validation programs that correlate global histone modification enrichment or depletion with disease. The Histone H3 PTM Multiplex Assays provide a rapid, highly sensitive, high-throughput method for profiling changes in histone PTMs in a singleplex or multiplex format. You will get more data from less input, in less time and at a lower cost. Streptavidin-phycoerythrin Biotinylated Histone H3 Ab Histone containing PTM PTM Ab-conjugated bead H3 Ac H3 SA-PE SA-PE Schematic of the Histone H3 PTM Multiplex Assay. The assay works as a solution-based sandwich ELISA to evaluate histone H3 PTM levels. Biotin ASSAY FEATURES Enabling the only kit available to perform multiplexed histone analysis Efficient use less input and less time to assay multiple histone PTMs in a single reaction Sensitive requires only nanogram amounts of crude acid extracts or purified histones Accurate detect PTM differences between samples Biotin P Me3 H3 Biotin SA-PE Median Fluorescent Intensity (MFI) Median Fluorescent Intensity (MFI) H3K9ac H3 Total MW (kda) plex Histone H3 PTM Assay H3K9me2 H3K9me1 2 g/ml H3 (total) H3S10ph H3K4me3 H3K9me3 4 g/ml HeLa Acid Extract /- colcemid treatment (8 g/lane) Histone H3 Normalized Detection of H3S10ph Untreated Colcemid treated 2 g/ml H3K27me3 H3K27me2 H3 pan-acetyl H3S10ph 4 g/ml HeLa Acid Extract (ng) 1 g 500 ng 250 ng 125 ng Blank Screen multiple histone PTMs in a single well. HeLa acid extract was tested at varying concentrations using 10 antibody-conjugated bead sets in multiplex. Comparison of H3S10ph data from a Western blot or the Histone H3 PTM Assay. Total histone H3 and H3S10ph levels from untreated or colcemid-treated HeLa acid extract were compared using Western blot (top image) or a 2-plex Histone H3 PTM Assay (bottom image). To learn more about our Luminex assays, visit us at 6

7 MEASURE GLOBAL CELLULAR RESPONSE tools to detect cellular changes in disease models Small molecule treatment of cell models can significantly alter gene expression through transcription factor and epigenetic modulation. Likewise, changes in regulatory pathways can distinguish diseased tissue from normal. Active Motif offers simple-to-use assays to accurately measure such global changes. Our epigenetic screening assays are robust, reliable and amenable to high-throughput processing, enabling you to quickly and easily profile cellular changes in the activity of a variety of targets. MEASURE GLOBAL CELLULAR CHANGES TRANSCRIPTION FACTOR BINDING TransAM Transcription Factor Assays Assays for 40 different transcription factors, including: c-myc NFkB IRF-3 HIF-1 STAT3 ER GR p53 Nrf2 and more... EPIGENETIC ENZYME ACTIVITY Histone Acetyltransferase (HAT) Assays Histone Deacetylase (HDAC) Assays Histone Demethylase (HDM) Assays HISTONE MODIFICATIONS Histone ELISAs for: Total Histone H3K4me1 H3K4me2 H3K4me3 H3K9me2 H3K9me3 H3K27me1 H3K27me3 H3K9ac H3K14ac H3S10ph H3S28ph To learn more about our assays and see a complete listing, please visit us at sales@activemotif.com 7

8 CELL-BASED PATHWAY ANALYSIS identify biological responses to compound treatment In understanding a compound s mechanism of action it is crucial to identify activation of both intended and unintended cellular pathways. LightSwitch Pathway Screening Assays help you characterize cellular responses to compound treatment and understand off-target effects. Our unique collection of validated promoter reporters measure gene expression changes associated with a number of disease-related biological pathways. Our standard LightSwitch Pathway Screening Assays are available as stable cell lines or transfection-ready plasmids. Custom pathway sets are also available. Analyze 15 pathways in an HTS environment Identify compound-mediated pathway activation Determine the MOA and off-target effects of your lead compounds Available as a product or a service Cell Treatments TCDD R1881 PMA Estradiol DMSO Prednisone Heat shock DFO 1% O 2 TNF-a IFNa IFNg Nutlin Lovastatin Synthecol Controls Tox/Ahr Androgen CREB Estrogen Glucocorticoid Heat shock Hypoxia NFkB STAT p53 Representative Reporter Constructs Pathway Profiling Panel 48 promoters and controls Pathway activity readout for: HIF-1 NF B CREB HSF1 p53 STAT SREBP ER AR GR AhR Hypoxia Inflammation cyclic AMP Heat shock DNA damage, apoptosis Interferon Cholesterol biosynthesis Estrogen Androgen Glucocorticoid Toxicity LightSwitch Pathway Screening Services can profile pathway responses to treatments. The heat map above shows the inducible activity of 29 different reporter constructs that represent 10 different pathways in response to 15 different treatments. To learn more about our LightSwitch Pathway Screening Assays and Services, please visit us at 8

9 FUNCTIONAL ASSAYS FOR mirnas measure mirna function and validate mirna targets MicroRNAs (mirnas) are involved in many areas of disease biology, and are actively being tested in the clinic as drug targets and as therapeutic molecules themselves. The LightSwitch 3 UTR Reporter Assay provides an HTS system that is ideally suited for screening the effects of small molecules and chemical modifications on mirnas. The LightSwitch 3 UTR Reporter GoClone Collection includes over 12,000 ready-to-transfect human 3 UTR reporter vectors and 1000 synthetic mirna target reporters to study mirna-3 UTR interactions, validate mirna targets, and measure RNA stability, translation efficiency and the functional impact of mirnas on a gene-by-gene basis. Measure the effects of small molecules or chemical modifications on mirna activity Validate downstream mirna targets Identify all mirnas that regulate a gene of interest mirna screening services also available Understanding the biological role of a mirna often requires identification of downstream targets. The LightSwitch 3 UTR reporter system is a validated assay that can rapidly identify hundreds of mirna target genes in a single experiment. Log2 Ratio (mirna/control) RIMS1 G6PC3 GNPDA2 PPIA1 ANKRD13C No Insert Interaction of mir-122 with 3 UTR targets. 3 UTR targets of mir-122 were cloned into the LightSwitch 3 UTR vector and co-transfected with either mir-122 mimic or a non-targeting control mirna in K-562 cells. The graph shows the four strongest responders along with two non-responding controls. PRODUCTS FOR mirna FUNCTIONAL SCREENS 12,000 pre-cloned human 3 UTR reporters Synthetic target reporters containing sequences fully complementary to 1000 human mirnas mirna mimics and inhibitors Validated 3 UTR controls Complete screening service to validate 3 UTR targets for any mirna For more complete information on our mirna targets, mimics, inhibitors and services, please visit sales@activemotif.com 9

10 GENOME-WIDE SOLUTIONS characterize inhibitors of epigenetic targets Targeting epigenetic modifying enzymes for drug development has great potential for advancing research in the fields of cancer, inflammation, development and neurobiology. The in-depth characterization of lead compounds in these areas requires a genome-wide mechanistic perspective of how these compounds alter epigenetic profiles. Active Motif s Epigenetic Services team specializes in genome-wide compound characterization and offers this assay as an end-to-end ChIP-Seq service to accelerate your drug discovery and development program. { Vehicle HAT ChIP-Seq Inhibitor { Vehicle Acetylated Histone ChIP-Seq Inhibitor 80,000 33,100,000 ZBTB80S 33,120,000 Reduced Acetyltransferase Occupancy Reduced Histone Acetylation RBBP4 33,140,000 SYNC 33,160,000 33,180,000 33,200,000 KIAA ,220,000 ChIP-Seq in small molecule inhibitor-treated cells. Antibodies that target a specific HAT and its corresponding histone modification were used in ChIP-Seq. Arrows indicate site-specific reductions in HAT occupancy and its corresponding histone modification. RELATED SERVICES Getting the most out of genome-wide data requires overlaying multiple types of data sets. In addition to ChIP-Seq, Active Motif also offers several other end-to-end whole-genome services that can be combined to meet your specific research needs. RNA Pol II ChIP-Seq measure transcription rates RNA-Seq determine steady-state mrna levels MeDIP-Seq profile genome-wide DNA methylation VALIDATED ASSAYS FOR OVER 200 TARGETS BRD4 H3K9ac EZH2 H3K27me3 LSD1 H3K4me2 To view the full range of services and for more information, please visit us at

11 REPORTER ASSAYS FOR DRUG SCREENING accelerate reporter assay screens with the LightSwitch System The LightSwitch Luciferase Assay System dramatically reduces the time investment needed to identify the most responsive promoter to develop as a new HTS reporter assay. Over 18,000 pre-cloned human promoter reporter vectors are available to choose from for immediate incorporation into plate-based screens. 18,000 pre-cloned human promoter reporters for screening expression changes of any human gene 100 transcription factor response element reporters for screening transcription factor activity Validated promoter reporters for measuring pathway activation are also available (see page 8) N S N N N O O Cl Dose response of LightSwitch SYT8 Promoter Reporter construct to estrogen compounds. HT1080 cells were co-transfected with a SYT8 Promoter GoClone (Product ID S714388) and an ER cdna expression plasmid, then treated with five different estrogen compounds for 24 hours before the luminescence was measured using the LightSwitch Luciferase Assay Kit. CUSTOM SERVICES AVAILABLE Stable cell line generation Custom assay development Custom cloning of novel regulatory elements (or TF response elements) For more complete information, please visit us at sales@activemotif.com 11

12 Enabling Epigenetics Research NORTH AMERICA Toll Free: Direct: Fax: JAPAN Direct: +81 (0) Fax: +81 (0) EUROPE GERMANY 0800/ UNITED KINGDOM 0800/ FRANCE 0800/ OTHER COUNTRIES, DIRECT +32 (0) Fax: +32 (0)