Most vendors of epigenetic modification proteins and reagents have enhanced and broadened their offerings in recent years. Of particular note is the increasing range of proteins and binding assays that now support research on the Bromodomains and other reader proteins.
Opinion on the status of epigenetic targets is relatively evenly split between maybe a major new category for successful new drug research, but a lot more work is needed and certain to be a major new category for successful drug research within three to five years.
Survey feedback on the status of epigenetic targets similarly ranged from high enthusiasm to moderate scepticism. The continued development of epigenetic target-based assays can be expected to progress along the lines of kinase research, ie working towards complete coverage of all epigenetic targets types from binding domains to enzymatic marks to facilitate screening, profiling and the validation of hits. Commitment to epigenetic target research should grow if ongoing clinical trials are successful.
Epigenetics is the study of heritable changes in gene expression caused by non-genetic mechanisms without alterations in gene structure or DNA sequence. Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation and chromatin remodelling are implicated in a number of cancer, immunological and neurodegenerative conditions.
Several years ago in DDW (1) we reported on the potential increasing importance of epigenetic modification research to some disease areas, particularly in cancer. We also detailed growing vendor interest in developing new tools and assays to support drug screening and sequencing against DNA methylation and histone modifications. Since that time it is probably fair to say that epigenetics has not quite taken off to the extent that some might have predicted and several companies that started off as enthusiastic exponents of epigenetic modification research have scaled back efforts in the area.
The reality is epigenetic research is proving a tough and time-consuming exercise compared to other areas in the field of oncology and drug discovery. One of the main constraints is the underlying biology of many epigenetic targets is still poorly understood, such that it is hard to make a major breakthrough in drug discovery unless biological knowledge significantly improves and the epigenetic proteins that are targetable for disease prevention are fully validated.
Progress has been further hampered in some areas by the lack of effective tools (particularly antibodies and validated tool compounds) to investigate these complex signalling and modification events in a variety of contexts. It seems that a major thrust by the pharmaceutical industry may also be needed to grow and progress this area and some companies may be holding off pending successful clinical outcomes (eg DNAdemethylating agents, pan-deacetylase inhibitors and inhibitors disrupting interactions of the BET family Bromodomains are all currently in clinical trials for various cancer indications) before making major investments into epigenetic modification research.
In March 2015, HTStec undertook a market survey on epigenetic proteins and reagents (2) mainly among research labs in pharma, biotech and academia. The main objective of this survey was to understand the current and future potential of epigenetic target research. In this article highlights from this market survey are reported and the findings are discussed together with vendor updates on the assays, kits, proteins, reagents and tools supporting epigenetic modification research and drug discovery.
Epigenetic proteins of greatest interest
The single category of epigenetic proteins of most interest to survey respondents was methyltransferases (30% selecting). This was followed by HDAC (23%); other (14%); reader domains (11%); DNMTs (8%); demethylases (7%); PARPs (4%); and then IDH (2%). The median number of different epigenetic proteins under investigation in survey respondents’ labs today (2015) was two to three proteins (Figure 1).
Disease areas targeting epigenetic modification research
The majority (72%) of survey respondents were targeting, using or planning to use epigenetic modification research within the oncology therapeutic area (TA). This was followed by the inflammatory disease/autoimmune (28% targeting); CNS/neurodegeneration/ pain (22% targeting); metabolic disease/diabetes (16% targeting); cardiovascular (12% targeting); anti-infectives/anti-viral (11% targeting) and other (9% targeting) (Figure 2).
Assay reagent preference Survey respondents’ reagent preference for epigenetic modification research or internal drug discovery was use commercial assay kits (65% preferring). The remaining (35%) preferred to develop their own assays using available proteins and reagents (Figure 3).
Main source of epigenetic proteins
The main sources/origins of epigenetic proteins used today (2015) were commercial sources (64% using), followed by in-house (15% using), academic institutes (13% using), collaborators (8% using) and then others (1% using) (Figure 4).
Preferred biochemical assay format
Survey respondents preferred biochemical assay format for internal work using commercial assay kits for epigenetic modification assays was ELISA. This was followed by chromatin immunoprecipitation (ChIP-Seq); and then fluorimetric/fluorescent intensity; HTRF (TR-FRET); and AlphaScreen/ AlphaLISA. The least preferred biochemical assay format was LabChip separation (Figure 5).
What will most influence initiation of epigenetic research
Survey respondents ranked in-house research results as the information that has influenced or will most influence the decision to initiate large scale research using epigenetic proteins. This was closely followed by publications/reviews and successful epigenetic drugs in clinic. Ranked least influential was acquiring an epigenetic lead compound or epigenetic company (Figure 6).
Biggest obstacles to epigenetic targets gaining industry-wide acceptance
Survey respondents ranked biology not adequately understood as the biggest obstacle (limitation) to epigenetic targets gaining industry-wide acceptance. This was followed by targets are inherently difficult to drug; we just need more time – it is a new category; and need better tools and more assay kits available, which were all equally ranked. No clinical successes yet – it is a ‘me-too’ industry was ranked least limiting (Figure 7).
Latest developments in epigenetic drug screening
The following vendor snapshots provide additional details and describe some of the latest developments in assays, kits, associated reagents and tools used for the screening and investigation of epigenetic target enzymes and proteins:
Abcam (www.abcam.com/epigenetics) offers a wide range of scientific tools to help researchers working in epigenetics drug discovery to better understand human health and disease. The range includes kits and assays for histone modification, DNA methylation, sample preparation, ChIP studies, cell-based studies and also inhibitors of epigenetics- related proteins.
Some of Abcam’s newest and most innovative products in epigenetics include highly-specific monoclonal antibodies using RabMAb® technology and multiplex miRNA biomarker profiling assays using the company’s new Firefly™ particle technology. RabMAb primary antibodies are an ideal tool for detecting epigenetic marks on modified histones and DNA as the rabbit immune system has a much better response to small epitopes, offering high affinity and specificity to the modified target.
These high quality antibodies are able to detect subtle changes in epitopes such as post-translational modifications and activations. miRNAs have been shown to be involved in both DNA methylation and histone modifications.
Abcam’s new range of multiplex assays for high throughput miRNA biomarker discovery and validation use the company’s proprietary Firefly particle technology and can be read us ing standard flow cytometers. The platform enables rapid, highly sensitive detection of up to 68 miRNAs per well, working from either purified RNA or directly from biofluids such as serum, plasma and exosomes, without RNA purification.
The assays offer a convenient workflow for larger sample numbers when profiling miRNA compared to alternative methods such as qPCR, microarrays and sequencing. Offering bespoke assay development services using both proprietary technologies, Abcam is collaborating with several global pharma, biotech and research institutes to develop innovative tools to help them to discover more in their epigenetics research (Figure 8).
To best mimic cellular biology in an in vitro assay, Active Motif (www.activemotif.com) offers preassembled Recombinant Nucleosomes to use as substrates for performing drug discovery screens, analysing enzyme kinetics or studying histone modifications. In critical substrate selection, nucleosomes offer the advantage of providing a more biologically relevant histone conformation when compared to histone proteins alone or synthetic peptides (Figure 9).
Each of Active Motif’s Recombinant Nucleosomes is comprised of octamers of unmodified core histone proteins (H2A, H2B, H3 and H4) bound by DNA. Nucleosomes are available to study both canonical Histone H3.1 and Histone H3.3 variants. Additionally, a choice of unlabelled or biotinlabelled nucleosomes are provided for flexibility in experimental design. Active Motif has also partnered with Luminex®, the leader in multiplex technology, to develop the first ever multiplex epigenetic assay for screening changes in histone posttranslational modifications (PTMs).
The Histone H3 PTM Multiplex Assay, designed for use with Luminex instruments, is a three-hour assay that enables the simultaneous interrogation of multiple PTMs in a single well. The Histone H3 PTM Multiplex Kit’s ability to multiplex analysis of up to 13 different histone modifications from nanogram sample amounts in a 96-well platebased format renders a significant advantage over traditional approaches for analysis of epigenetic modulation by now providing a method that enables not only high-throughput, but also highcontent screening of specific and off-target effects.
Time-consuming Western blots can now be replaced in early lead generation or target validation programmes to assess the effects of drug treatments and disease states on histone modifications (Figure 10).
Epigenetic regulation has been implicated in diverse diseases including cancer, diabetes and inflammation and drug discovery efforts have focused on high throughput screening for histone methyltransferase (HMT) inhibitors. Universal assays that detect the S-adenosylhomocysteine (SAH) product of S-adenosylmethionine (SAM)-dependent methylation reactions allow screening and profiling of diverse HMTs with a single assay method.
The Transcreener® EPIGEN Methyltransferase Assay from BellBrook Labs (www.bellbrooklabs.com) detects enzymes that produce SAH, including HMTs and DNA methyltransferases, in an HTSready mix-and-read format (3). The EPIGEN Assay combines the validated Transcreener AMP2/GMP2 Assay for fluorescent immunodetection of AMP with coupling enzymes that convert SAH to AMP. This sensitive assay allows the use of physiological SAM concentrations and provides excellent signal at low substrate conversion, with a Z’ ≥ 0.7 under normal reaction conditions.
The assay relies on a ratiometric fluorescence polarisation readout with a far red tracer to minimise compound interference. To eliminate assay development and streamline inhibitor selectivity studies, BellBrook partnered with Reaction Biology to develop the Transzyme Methyltransferase Assay kits as a complete solution for HMT assays (4).
The Transzyme kits bundle everything needed for target HMT detection: the Transcreener EPIGEN Methyltransferase Assay detection reagents and optimised protocol, purified enzymes from Reaction Biology, including DNMT1, DOT1L, EZH2, G9a, MLL4, PRMT1, PRMT3, SET7/9, SET8, SUV39H1, GLP, G9a-GLP, PRMT4 (CARM1), or PRMT8, and substrates. Pre-calibrated enzymes and substrates eliminate the need for assay development and produce outstanding assay windows under initial velocity conditions (Figure 11).
BioVision (www.biovision.com) offers a broad range of products for study of all three major activities involved in epigenetic control – erasers, readers and writers. It offers a number of DNA methyltransferases, histone demethylases, deacetylases (HDACs) and acetyltransferases (HATs) that control eukaryotic gene transcription. Its products enable scientists to analyse their activity and study effects of inhibitors with easy to use kits for activity and inhibitor screening in HTP format. They also offer histones, sirtuins, Bromodomain and Tudor domain proteins and generic protein modifying enzymes.
It is well established that HDAC isoform distribution varies by type of tissue source and cell type as well as by intracellular localisation. BioVision offers unique immuno-precipitation (IP) and activity kits that enable scientists to isolate specific HDAC isoform complexes from their own samples. Scientists can follow up their studies of HDAC’s activity with screen for inhibitors of HDAC activity with its comprehensive inhibitor screening assay kits.
As HDACs regulate the expression of different genes, their localisation, post-translational modification and activity can be linked with specific gene products. For example, BioVision’s HDAC3 IP & Activity Assay Kit (K344-25) provides an antibody-based method to specifically isolate the HDAC3 complex from cells and/or tissues and to measure HDAC3 activity with high sensitivity.
Scientists can follow up with the HDAC3 Inhibitor Screening Kit (K363-100) for compound effect study on enzyme activity. BioVision also provides a number of inhibitor screening kits and a broad range of biochemicals, inhibitors, blocking peptides and antibodies for all three major groups of control proteins (Figure 12).
BPS Bioscience (www.bpsbioscience.com) has been supplying researchers with the highest quality epigenetic enzymes, assay kits and screening services for almost a decade. Leading with the first active EZH2 complex, BPS was the first company to provide a full suite of HDAC and SIRT enzymes and assays as well as the first to offer dozens of unique DNA and histone methyltransferases. BPS has continued to drive epigenetic lead discovery, expanding its portfolio to include more than 150 assay kits for screening inhibitors of deacetylases, methyltransferases, demethylases, bromodomains and more.
In addition to an unmatched portfolio of recombinant enzymes, assay kits, antibodies and inhibitors, BPS also provides rapid and reliable highthroughput screening and profiling services for epigenetic targets. BPS’s screening services are fully customisable and include one of the largest commercially available panels of epigenetic reader/ writer/eraser proteins for drug discovery. In particular, BPS offers a customisable panel of up to 17 unique histone demethylases, and an exclusive panel of 13 different PARP isozymes, including tankyrases.
There is significant interest in developing small molecule inhibitors of histone methyltransferases (HMTs) as drug candidates for certain disease pathologies, including cancer. The available HMT screening assays (mass spectrometry, antibodybased activity and radiometric binding/activity) present challenges in developing small molecule inhibitors of HMTs because they are expensive, labour- and time-intensive, frequently suffer from cross-reactivity and/or generate radioactive waste.
Cayman Chemical (www.caymanchem.com) offers SAM-Screener™, a novel fluorescent small molecule probe that binds specifically to the S-adenosylmethionine (SAM) binding site of several HMTs. Using this patented technology we have developed homogeneous, one-step fluorescence polarisation binding assays to probe the SAMbinding site of various HMTs. These assays are non-kinetic, substrate-independent and scalable to 1536-well format. Currently, they are available as high-throughput screening assay kits for SET7/9, GLP and MLL1.
Each kit includes recombinant enzyme, SAM-Binding Site Probe, assay buffer, a positive control compound and a 384-well plate. Custom assay development to adapt this assay to several other HMTs is also available through Cayman’s Epigenetic Screening and Profiling Service. This dedicated high-throughput screening laboratory can utilise this assay methodology to screen a chemical library against specific HMT targets. This novel fluorescence polarisation binding technology complements Cayman Chemical’s extensive range of products for epigenetic research, including additional assay kits, proteins, antibodies and biochemicals (Figure 13).
The development of epigenetic target-based assays has somehow followed a similar path to other biochemical enzymatic assays such as kinases. The need for extremely specific assays using carefully selected antibodies, or the optimisation of more universal approaches measuring a co-product of the enzymatic reaction, ie ADP for kinases, SAH for methyltransferases, show a lot of similarities.
As illustrated in Figure 14, the epigenetic ‘targetome’ may be broadly covered by the Cisbio Bioassays (www.cisbio.com) HTRF assay platform, which has rapidly expanded from histone modification (HMT, HDM, HDAC), to other important targets such bromo-, chromo-, tudor and MBT domain binding assays.
As cell-based approaches are more challenging to set up for each single target due to the high specificity requirements or material extraction, the assessment of biomarkers such IL6 or c-myc can represent a very smart alternative under a cell-based mode, and have been successfully used as a readout to BRD4 binding to histone, for instance.
Concurrently, Schulze et al (5) have recently published an original HTRF cell-based assay to investigate ligandinduced BRD4 protein stabilisation which they successfully correlated to a thermal shift assay, validating thereby the concept of domain compound binding for HTS, which the reference biophysical method hardly allows because of throughput limitations. Altogether, these different approaches prove once more the versatility of HTRF technology to address a broad range of targets and now for epigenetic ones.
EMD Millipore (www.emdmillipore.com/epigenetics) has supported epigenetics researcher for more than a decade. Its comprehensive epigenetics portfolio includes kits, recombinant histone proteins, antibodies and assays for all areas of epigenetics including DNA methylation, chromatin remodelling, histone modification and ncRNA biology. Recently, EMD Millipore has launched the AbSurance™ Pro Histone Peptide Microarray.
This microarray is ideal for histone protein-protein interaction analysis or histone antibody specificity screening using scanning densitometry or chemiluminescent detection. This expertly designed glass slide array contains peptides corresponding to unmodified and post-translationally modified sequences of all four core histone subunits (H2A, H2B, H3, H4) plus select variants. Designed to mimic post-translational modifications found in naturally occurring histones, multiple ~20mer peptides are spotted as unmodified, singly modified as well as multiply modified sequences with up to six modifications per peptide totalling 270 unique peptide sequences (Figure 15).
Enzo Life Sciences (www.enzolifesciences.com) provides a comprehensive tool set for epigenetic modifications including DNA modifications (5-mC and 5-hmC methylation) and histone post-translational modifications (demethylation, acetylation, ubiquitinylation, phosphorylation, SUMOylation, methylation and deacetylation). Enzo Life Sciences is a pioneer in non-radioactive HDAC and Sirtuin assays for drug screening. Enzo revolutionised the industry by introducing the first non-radioactive assay for screening epigenetic regulators.
For more than a decade, the FLUOR DE LYS® deacetylase assay platform has freed researchers from cumbersome protocols for screening a broad range of HDAC and Sirtuin activity. These assays are founded upon an industry-leading portfolio of active enzymes and high-purity peptide substrates to deliver the sensitivity needed when dissecting epigenetic pathways.
Enzo’s high-quality chemiluminescent, fluorescent and colorimetric assays, which are amenable to automated HTS platforms, deliver more high-quality hits and are backed by a broad panel of characterised inhibitors and PTMspecific antibodies for ubiquitinylation, sumoylation, methylation, acetylation and phosphorylation. In support of screening efforts, Enzo offers a SCREEN-WELL® Epigenetics compound library, a collection of 43 biochemicals with defined activity against epigenetic regulating enzymes, each of which can be supplied individually upon request (Figure 16).
For years, histone methylation of lysine residues has been widely studied and is considered a well-established epigenetic mechanism. However, there has been a significant increase in attention towards arginine-based histone methylation and its related protein arginine methyltransferases (PRMTs), serving as possible new epigenetic markers. Increased understanding of arginine methylation could potentially lead to the development of a powerful PRMT inhibitor and subsequently cancer diagnostic tools, but efficient validation of the functional purposes of such arginine methylation patterns necessitates useful and robust reagents.
Epigentek (www.epigentek.com/arginine), a comprehensive provider of epigenetic reagents and services, recently launched an extensive line of both symmetric and asymmetric histone arginine methylation antibodies, including the intriguing patterns of H4R3 and H3R2. Specifically catering to the rising demand by biopharmaceutical organisations for arginine methylation- associated drug discovery projects, Epigentek’s arginine methylation antibodies are thoroughly validated for a multitude of applications including Western blot, immunofluorescence, immunohistochemistry, ELISA and, in particular, ChIP and ChIP-seq. These antibodies can be found on Epigentek’s website, and are covered under a sixmonth application guarantee (Figure 17).
PerkinElmer (www.perkinelmer.com) offers a comprehensive suite of radiometric and non-radiometric solutions to characterise epigenetics writer and eraser enzymes, as well as reader proteins acting on DNA and Histones. These solutions were developed to help scientists performing fundamental research or drug discovery activities on a multitude of new biological targets often intractable. Traditional Michaelis-Menten enzyme kinetics analyses can be performed with tritiated co-factors (eg 3H-SAM, 3H-AcCoA) or using the LabChip EZ-reader microfluidics system.
Straightforward biochemical characterisation of epigenetics writer and eraser enzymes with histone tail peptides is achieved using homogeneous immunoassays such as LANCE (TR-FRET) and AlphaLISA. The latter technology is also applicable to more biologically relevant paradigms involving full length histones, recombinant nucleosomes and cellular models (recombinant, primary or stem cells). Due to its ability to work with large protein complexes, AlphaLISA is also used to study the interaction of Bromodomains with several acetylated H4 variants (Figure 18).
Histone deacetylase (HDAC) enzymes remain important drug targets for a number of human infirmities. However, the full promise of clinical utility for existing HDAC inhibitors (HDACi) is hampered by significant dose-limiting toxicities. These off-target effects have led to a renewed focus on basic HDAC biology and the development of isoenzyme-selective HDACi, which could better target the pertinent HDAC while reducing or eliminating unintended toxicity.
The suite of selective HDAC-Glo™ Assays offered by Promega (www.promega.com) represent a new set of tools to both identify and characterise the selectivity profiles of novel HDAC inhibitors. These assays use optimised substrates, with extended peptide sequences and novel chemical modifications, to impart their isoform/isotype selectivity. HDAC activity is measured by making a homogeneous addition of the selective HDAC-Glo™ reagent to assay wells containing a HDAC source and test compound, then detecting luminescence, which is proportional to HDAC activity.
In addition to being isoenzyme selective, these novel substrates are cell permeable, allowing use with live cells in addition to lytic modes. Whether using purified enzymes or cell-based screening systems, all three HDAC-Glo™ Assays produce broad linear responses (two to three orders of magnitude) with large dynamic ranges. This improved ‘assay window’ allows for convenient miniaturisation for standard screening or qHTS applications.
Furthermore, because of the steady-state ‘glow’ of the luminescence it is also possible to initiate a HDAC-Glo™ reaction and introduce the test HDACi to measure both the kinetics of cell permeability and rate of target engagement in real time (Figure 19).
Reaction Biology Corporation (RBC) (www.reactionbiology.com) is a contract research organisation specialising in epigenetic drug profiling, screening and early drug discovery collaborations. RBC offers a large collection of epigenetic targets for biochemical and cell-based assays, including kinases, reader domains, methyltransferases, histone deacetylases, histone acetylases, demethylases, deubiquitinases and PARPs. Kinases, Methyltransferases, Histone Acetylases and PARPs are tested using RBC’s proprietary HotSpotSM radioisotope technology.
Since detection does not rely on antibodies, coupling enzymes or fluorescence, major causes of false positives, false negatives and compound interference are eliminated. While working towards complete coverage of the bromodomain family, RBC has developed various types of assays to facilitate screening, drug discovery and validation of hits, including AlphaScreen, Thermal Shift, HTRF, FP and ITC assays for more than 100 reader domains.
The BromoMELT thermal melt stability assay kit is also available, including 76 bromodomains in total. RBC’s cell-based epigenetic assays include deacetylation, methylation and protein-protein interaction assays. RBC also offers a variety of high quality epigenetic proteins including methyltransferases, reader domains and demethylases, among others. Each protein is checked for purity, in addition to being tested in biochemical assays before being sold as a product.
RBC’s newest epigenetic addition is an expanding line of epigenetic inhibitors. During early stage discovery and development for emerging epigenetic targets, it is critical to deeply understand the biology of a target, to develop powerful assay platforms and to utilise screening expertise to allow for rapid hit identification and hit to lead development. By embracing these pillars, RBC continues to be a leading epigenetic research provider within the drug discovery industry (Figure 20).
Table 1 attempts to summarise the offerings discussed in the above vendor snapshots. What is apparent is that most vendor’s actually have reasonable coverage in terms of the diversity of different tool types provided and only a few are confined to specific epigenetic target assays based on a single approach or technology.
Overall all vendors have enhanced and broadened their epigenetic modification offerings since our previous DDW article in 2011 (1). One aspect of note is the increasing range of proteins and binding assays that now support research on the Bromodomains and other reader proteins. This probably reflects the increased interest in these readers as a result of ongoing clinical trials for various cancer indications of inhibitors disrupting interactions of the BET family Bromodomains.
The jury appears to be still out on the future of epigenetic research with the professional opinion of survey respondents on the status of epigenetic targets relatively evenly split between: 51% maybe a major new category for successful new drug research, but a lot more work is needed; and 47% certain to be a major new category for successful drug research; with the remaining 2% expressing the view it was unlikely to ever be a major new category for successful drug research (Figure 21).
The median timeframe when epigenetic targets are expected by survey respondents to be a new category for successful drug research was three to five years. Some comments made by survey respondents on the status of epigenetic targets are presented in Table 2.
Again, comments range from high enthusiasm to moderate scepticism. In conclusion, the development of epigenetic target-based assays have advanced greatly over the past decade and can be expected to progress along the lines of kinase research, ie working towards complete coverage of all epigenetic targets types from binding domains to enzymatic marks to facilitate screening, profiling and the validation of hits. Commitment to epigenetic target research is expected to grow if ongoing clinical trials are successful. DDW
Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation; assay methodologies and reagent offerings) to drug discovery and the life sciences. Since its formation 13 years ago, HTStec has published 114 market reports on enabling technologies and Dr Comley has authored 53 review articles in Drug Discovery World. Please contact firstname.lastname@example.org for more information about HTStec reports.
1 Comley, J (2011). Epigenetics – an emerging target class for drug screening. DDW 14(2): 43-50.
2 Epigenetic Proteins & Reagents Trends 2015. Published by HTStec Limited, Godalming, UK, April 2015.
3 Klink, TA et al (2012). Development and validation of a generic fluorescent methyltransferase activity assay based on the transcreener AMP/GMP assay. J. Biomol. Screen 17(1): 59-70.
4 Kumar, M et al (2015). Biochemical Assay Development for Histone Methyltransferases Using a Transcreener-Based Assay for S-Adenosylhomocysteine. Assay Drug Dev Technol. 24 Feb 2015 [Epub ahead of print].
5 Schulze, J et al (2015). Cellbased protein stabilization assays for the detection of interactions between smallmolecule inhibitors and BRD4. J. Biomol. Screen. 20(2):180-9.