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New Tools, New Targets Novel Approaches for Identifying and Characterizing
Epigenetic Modifications Webinar
25 June 2014 [0:00:00] Slide 2 Sean Sanders: Hello everyone and a very warm welcome to the Science/AAAS webinar,
New Tools, New Targets: Novel Approaches for Identifying and Characterizing Epigenetic Modifications. My name is Sean Sanders and I'm Editor for Custom Publishing at Science.
Epigenetic enzymes are currently among the most important
pharmaceutical targets due to their involvement in numerous key cellular processes and the etiology of many devastating diseases including cancer, neurodegenerative disease, and developmental disorders. Efforts are underway in many laboratories to identify and validate new so‐called reader, writer, and eraser enzymes, as well as their histone substrates in order to generate new therapeutics.
While various molecular biology techniques were initially used to identify
and validate epigenetic enzymes and their substrates, novel approaches are now available to perform pharmacological characterization of histone‐modifying enzymes. During this webinar, our experts will talk about methods to characterize histone‐modifying enzymes and present their own research data obtained using some novel experimental approaches.
It's my pleasure to introduce those speakers to you today and they are
Mr. Bill Janzen from the University of North Carolina at Chapel Hill and Dr. Yan‐Ling Zhang from the Broad Institute of MIT and Harvard in Cambridge, Massachusetts ‐‐ a very warm welcome to you both.
Slide 1 Before we get started, I have some information for our audience. Note
that you can resize or hide any of the windows in your viewing console.
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The widgets at the bottom of the console control what you see. Click on these to see speaker bios, additional information about technologies related to today's discussion, or to download a PDF of the slides.
Each of our guests will give a short presentation followed by a Q&A
session, during which they will address questions submitted by our live online viewers, so if you're joining us live, start thinking about some questions now and submit them at any time by typing them into the box on the bottom left of your viewing console and clicking the "submit" button.
If you don't see this box, click the red Q&A widget at the bottom of the
screen. Please remember to keep your questions short and concise as that will give them the best chance of being put to our panel.
You can also log in to your Facebook, Twitter, or LinkedIn accounts during
the webinar to post updates or send tweets about the event. Just click the relevant widgets at the bottom of the screen. For tweets, you can add the hashtag #ScienceWebinar. Finally, thank you to PerkinElmer for sponsoring today's webinar. Now, I'd like to introduce our first speaker, Mr. Bill Janzen.
Slide 3 Mr. Janzen worked in both industry and academia during his nearly 20
years in the drug discovery arena. Since 2008, he has been a professor of the practice and Director of Assay Development and Compound Profiling at the Center for Integrative Chemical Biology and Drug Discovery at the University of North Carolina at Chapel Hill.
A very warm welcome to you, Mr. Janzen. Thanks so much for being here. Dr. William Janzen: Thank you, Sean, and hello everyone. I'd like to thank Sean and the
organizers for the opportunity to speak to you today. I want to tell you a little bit about what we've done at the University of North Carolina in the area of Epigenetic Probe Discovery, but first, I'd like to talk a little bit about epigenetics.
Slide 4 The literal translation of epigenetics obviously means "above genetics".
Epigenetics is really a form of genetic control which is driven by factors other than DNA sequence. Epigenetic changes primarily switch genes on
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or off and determine chromatin structure, and this in turn can then control which proteins are transcribed.
Here, the term "heritable" refers to a stable cellular or organism‐specific
event, but there is some evidence that we can see intergenerational epigenetic changes as well.
Slide 5 Epigenetics has a number of important areas in biology. The most
commonly discussed one is in cell differentiation. The precursor cells or embryonic stem cells obviously have to differentiate into a variety of different cell and tissue types as an organism is formed all from the same genetic code, and this is an epigenetically‐driven process.
Epigenetics is also heavily involved in gene silencing. Genes can be active
genes, which are being transcribed; poised genes, genes which are on DNA or which have promoters ready to transcribe them from DNA; or silenced. This is driven through both changes in methylation states of DNA and changes in the histone tails in chromatin nucleosomes.
Slide 6 Epigenetics in biology can be demonstrated through X chromosome
silencing. In X chromosome silencing, it's very important because female mammals have two X chromosomes that one of these genes be silenced. This is necessary so that they won't have twice the number of X chromosome gene expression events and misregulation of this process can lead to many diseases, for example, autoimmune thyroid disorders and primary billiary cirrhosis.
[0:05:16] Slide 7 It's also important in biology and is shown through the effects of
maternal care. It's been shown that maternal grooming of offspring has been shown to persistently alter histone acetylation and transcription factor levels primarily NGFIA and binding to the GR promoter, which lead to differences in later stress response from the offspring.
Slide 8
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Perhaps one of the best demonstrations of epigenetic effects is shown in the "Dutch famine" effect from humans. Nearly six decades after the "Dutch famine" or "Hongerwinter" of 1943 through 1947, Lumey and colleagues isolated DNA from Hongerwinter individuals and found below average methylation of the insulin‐like growth factor 2 gene (IGF2), which codes for growth hormones critical to gestation.
Exposure to famine during gestation resulted in a number of
abnormalities including reduced stature, and interestingly, it was found that this reduced stature was passed on generationally although the effect is lessened, so the research is ongoing to see how many generations this would then be carried.
It also resulted in a number of other changes including increases in
impaired glucose tolerance, obesity, coronary heart disease, atherogenic lipid profiles, schizophrenia, and a number of other effects. On the same vein, a number of Swedish research scientists recently showed that paternal access to food can influence the health of the children.
Deaths related to diabetes decreased for children when excess food is
available to the father during a critical period of development, but if food is plentiful during that period for the paternal grandfather, the risk is actually increased. So obviously, this is a very complex process.
Slide 9 Perhaps the most visual evidence of epigenetic changes is in coloration in
felines. On Valentine's Day 2002, the cloning of the first cat was announced by researchers at the College of Veterinary Medicine at Texas A&M. Copy Cat or CC was developed in partnership with a biotechnology company called Genetic Savings & Clone. It was immediately apparent that CC did not physically resemble Rainbow, her DNA donor mother.
Now, this is actually another great example of X inactivation.
Tortoiseshell and calico cats are always female, and black and orange alleles of fur coloration gene reside on the X chromosome. Because one allele is inactivated in every cell and the process is random, the coat will exhibit patches of both colors and the pattern cannot be predicted.
This obviously also is not very good for Genetic Savings & Clone. It pretty
much spoiled their business model and I think they went out of business at that time.
Slide 10
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But our interest today is the chemical biology regulation of chromatin
and how we can use chemical biology to understand chromatin regulation.
Chromatin, as we all know, involve the DNA helix wrapped tightly around
nucleosomes. Nucleosomes also have histone tails, which can be modified. This is where the term "writers, readers, and erasers" has come in. On the histone tails, we can have a number of different marks, acetylation marks, phosphorylation, ubiquitination, or methylation.
The writers or readers of this code are demonstrated here. For
acetylation, these are the histone acetyltransferases, the bromodomains, and the histone deacetylases. For methylation, the PKMT, the protein methyltransferases, the Royal family binders, and the demethylases, which also remove that methyl mark. For phosphorylation, these obviously are kinases and phosphatases.
Slide 11 Here at the University of North Carolina in the Center for Integrative
Chemical Biology and Drug Discovery ‐‐ and I apologize for the long name ‐‐ we have a broad mission to interact with faculty members to bring either drug or probe discovery projects through the pipeline.
We have targets coming from two potential sources, target proposals
from UNC faculty or responsive collaborations, but what I'm going to talk about today are the center‐initiated projects in chemical biology which focus around the epigenetics or more perspective science.
We are well‐equipped to prosecute these projects with a good group of
biologists, biochemists, 18 medicinal chemists and three computational chemists, and a library of over 300,000 small molecules, the goal of this being the development of small molecule probes for epigenetics.
[0:10:03] Slide 12 So one of the obvious questions is, "Why chemical probes?" We have
many molecular biology techniques which can knock down proteins or change their function, but chemical probes offer a number of advantages, the temporal resolution of being able to add a chemical compound and see a response rather than being forced to create a cell which is missing
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that response; mechanistic flexibility to alter targets separate of the functions of the protein; ease of delivery, once a compound is shown to be cell permeable or has a potential oral activity, it can be delivered easily to a biological system; and then of course the application to later drug discovery.
As we develop these probes, we're always trying to ensure that they have
drugable characteristics so that if a therapeutic intervention target is identified, we can move forward into lead discovery.
Slide 13 The second question that arises from that is, "What is a quality chemical
probe?" Obviously, chemical probes need to be very selective. In fact, more effort needs to be spent on selectivity and the understanding of the mechanism of a probe than of a drug.
Drugs can be highly nonselective and still be very, very potent and very
good therapeutic measures, but in order to use a probe to understand a biological system, we need to understand its selectivity very carefully, so they need to be profiled both in vitro and hopefully in vivo. Their mechanism of action needs to be understood.
We need to understand what is the identity of the active species, and
finally, proven utility as a probe. There needs to be cellular activity data to address at least one of the hypotheses of the role of the molecular target.
Most importantly, they need to be available. A probe is really not very
much used to the broader scientific community unless it's available with no restrictions on its use, so everything we're doing in the epigenetic area is intellectual property free and published, and the probes are freely available through commercial suppliers.
Slide 14 So coming back again to our picture of the epigenetic code and histone
tails, we made the decision early on to focus on methylation as an area where we felt we could have an impact, so our focus has been chemical probes for the Royal family binders to methyl tails on histone and the PKMT family.
Slide 15
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I'm going to talk mostly about our program in methyl‐lysine reader families. There are six primary families that have been identified, the Chromo domains, Tudors, MBTs, PHDs, PWWPs, and WD40 domains. The MBT domains are where we began this research. All these domains have very specific binding to methylation states of the histone tail. For example, MBT domains will bind either a mono or dimethyl, but will not bond tri or unmethylated.
Slide 16 So we began this work by developing an AlphaScreen Assay Platform. For
those of you not familiar with AlphaScreen, it involves a donor and acceptor bead. When the donor bead is excited by specific wavelengths of light, a singlet oxygen is released. That singlet oxygen has a transition distance in an aqueous environment of approximately 200 nanometers. So if an acceptor bead has been brought within that distance through a binding event, it will then emit light.
The way we have built our program is that we create methyl‐lysine
reader proteins which have specific binding tags that are both used during purification and used for coupling to the bead. Shown here is a 6×His tag with a nickel bead so that the bead can then bind directly to that protein. When the protein binds an acceptor motif on a peptide, that peptide then has a biotinylation tag which can be bound to a streptavidin donor bead, thus bringing these within the close proximity.
We found this to be very effective especially since many of these binder
proteins have low affinities and the AlphaScreen platform allows us to work with lower levels of protein than fluorescence polarization and some of the other techniques that could be used.
Slide 17 This work was first published in 2010 by Tim Wigel who was then a
postdoc in my laboratory, and we have continued to expand this platform.
Slide 18 The work I'm showing you here is primarily that of Tim, Victoria
Korboukh, and Brandi Baughman. Brandi is currently a postdoc in the lab. Victoria is also a former postdoc who's going on to an industry position, but this is now being expanded to create a panel which we can use in a chemical biology approach.
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[0:15:10] We have five MBT domains, five Tudor domains, arguably eight PHD
domains, although as you can see from this, six of those eight are different derivatives of a single protein, UHRF1, where we have a very heavy research focus at this point, and one Chromo domain.
The way our program works is that all compounds that are synthesized or
that are of interest in the reader program are screened through all members of this panel usually in an IC50 format so that we gather potency.
So we do primary screen in an AlphaScreen and then those are then
confirmed both in secondary assays to ensure specificity, and then through ITC fluorescence polarization or SPR plasmon resonance reads to show that the compound actually does bind directly to the protein. We then follow that with cell‐based assays to potentially move forward to in vivo assessment.
For our counterscreen, the peptides are shown in the blue box on the
lower left side of your screen. We do have multiple labels that we can use and can bind both GST and His‐tags for our proteins, but one of the areas we have to be very careful about is to ensure that the compounds that we're finding are in fact affecting binding and are not affecting the release of singlet oxygen or quenching of the light from the acceptor bead.
To do this, we use peptides. The top two are shown in the box where we
have a direct biotin and either His or GST tag so that we can directly couple the two beads and show that the compound is not having an effect on that interaction. We also have an unmethylated peptide so we can show specificity for the methylation mark and unlabeled peptides that we can use as competition agents.
Slide 19 This program has been running for a number of years. Over the last five
years, we've collected approximately 800,000 wells of data. As you can see on the top graph shown here, these show the number of compounds read per day.
The very large spike in the beginning of this graph shows where Tim
Wigel at one point over a three‐day period ran our 100,000 compound
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diversity library and to give us initial starting point data for this program, but you can see that we've had average runs of approximately 4000 wells a day through the life of the program.
You can also see the breakdown by the different proteins in the lower
side of this. Clearly, the L3MBTL1 program has been one of the most prolific, but also MBTD1 and the other MBT programs. Again, this is data that was primarily generated by Brandi, Victoria, and Tim.
Another important factor is the fact that we do run things primarily in
potency curves, so this data does represent 51,000 potency curves that we have available for data mining. From this, we've been able to do quite a bit of probe discovery.
Slide 20 Our primary work has, as I mentioned, been in the MBT domains and
we've been able to publish several structure‐activity relationship papers and small molecule ligands of methyl‐lysine binding domains. Shown here are two programs primarily from Martin Herold, also a former postdoc in the lab.
Slide 21 And also a much deeper understanding of the computational nature of
binding to L3MBTL1, but probably the biggest success has been in the generation of actual probes.
Slide 22 In 2013, Lindsey Ingerman James published this article in Nature
Chemical Biology showing the probe UNC1215 as a potent selective antagonist of L3MBTL3.
This was a very interesting program because this began as a compound
designed to bind tandem Tudor domains by taking a motif that had been shown to bind L3MBTL1 and a number of the other MBT domains. The thought was if we created essentially a dimer or doubling of this compound, it would be able to bind tandem Tudor domains and have greater efficacy.
What we found instead is that it was very, very potent against L3MBTL3
and from that, Lindsey has now been able to show that L3MBTL3 actually
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binds as a dimer to this molecule, so essentially it binds two molecules of L3MBTL3 in order to inactivate.
[0:20:08] Slide 23 The other area that we've worked in the epigenetics area has been in the
area of histone methyltransferases. This work was published earlier in Chemistry & Biology in 2010, also by Tim Wigel. This is an effort that has been led by Jian Jin, one of my faculty colleagues at the University of North Carolina. I just want to quickly touch on a couple of his successes.
Slide 24 He has been able to publish multiple probes that selectively inhibit G9a
and GLP. This has been work that's been in collaboration with the Structural Genomics Consortium. Rather than read all the names on this very large publication, I do want to just point out Masoud Vedadi, who is the first author here, and also obviously Jian Jin, our director Stephen Frye, Peter Brown, a number of people who contributed to this, as you can see.
The first publication was on UNC638 as a selective probe of G9a, but that
was later then optimized to UNC642, which was improved PK properties and was an in vivo probe.
Slide 25 Jian has also been able to develop UNC1999 and orally bioavailable probe
of the methyltransferase EZH2 and EZH1, again, in collaboration with the SGC. This is work from Kyle Konze, Anqi Ma, and a number of other chemists and our collaborators at the Structural Genomics Consortium.
Slide 26 With that, I'd like to end. I'd like to thank the entire group at UNC Center
for Chemical Biology, particularly Stephen Frye who is not shown in this picture because he is our team photographer and was taking the picture.
I'd like to thank the University of Lineberger University Cancer Research
Fund for funding, the Eshelman School of Pharmacy, as well as National Institutes of Health for funding this work. I will conclude at this point and allow us to move on to our next speaker.
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Sean Sanders: Great! Thank you so much, Mr. Janzen, excellent presentation. As you
mentioned, we are going to move on to our second speaker. Slide 27 Just a quick reminder to those watching live, you can still submit your
questions at any time. Just type them into the question box on the left of your screen and click "submit".
Our second speaker today is Dr. Yan‐Ling Zhang. Dr. Zhang is currently
the director of In Vitro Pharmacology in the Therapeutics Platform at the Broad Institute of MIT and Harvard.
Slide 28 In this role, she focuses on the development of biologically‐relevant, high
throughput biochemical and phenotypic assays to enable high throughput screening for a variety of drug targets for psychiatric disorders, and is currently also interested in developing a high throughput calcium dynamics assay to monitor synaptic networks.
A very warm welcome to you, Dr. Zhang. Dr. Yan‐Ling Zhang: Thank you, Sean, for the introduction. Hello, everyone! Here, I'll present
our case study in developing isoform specific HDAC inhibitors specifically focusing on kinetic characterization of HDAC inhibition with microfluidic mobility shift assay.
Slide 29 HDAC catalyzes the removal of the acetyl group from lysine residues in
histone tails and also some non‐histone proteins. It plays a critical role in chromatin structure. Upon deacetylation, positive charge of the histones interact with the negative charge of DNA forming highly compacted chromatin structure, shutting down the gene transcription, so histone acetylation plays an important role in the regulation of gene expression.
Slide 30 Zinc‐dependent HDACs are classified in three classes depending on the
sequence homology to its yeast counterpart and localization. Class I HDAC contains HDAC1, 2, 3, and 8. They are most located in the nucleus.
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The crystal structure of HDAC2, 3, and 8 are available. Their catalytic domains are highly conserved.
Slide 31 Because of the importance of HDACs in cell cycling, differentiation, and
apoptosis, many efforts have been put across industries and academic labs in the development of small‐molecule HDAC inhibitors.
[0:24:58] Here, I listed four classes of known HDAC inhibitors, hydroxamic acid,
which is represented by SAHA as the approved drug marketed under the name Zolinza for the treatment of cutaneous T‐cell lymphoma. It inhibits Class I and some Class II HDACs with nanomolar to micromolar potency.
Cyclic peptide is another class of HDAC inhibitors. Their representative
compound is FK‐228, which is also a recently FDA‐approved drug for cutaneous T‐cell lymphoma indication. It's a nanomolar, a Class I HDAC inhibitor.
Some short‐chain fatty acids such as Butyrate also show inhibiting activity
in the HDACS as a millimolar range of potency. Finally, benzamides like MS‐275 and CI‐994 will target HDAC1, 2, 3 inhibitors. Some of them are in clinical trials for treatment of various cancers.
By looking at the target column in this table, you'll find that none of these
known inhibitors are isoform‐selective. In the past few years, we have been interested in developing isoform‐selective inhibitors to understand individual HDAC function.
Slide 32 We're particularly interested in HDAC2 because of its function in the
other diseases. Dr. Li‐Huei Tsai's lab at MIT has shown that neuro‐specific overexpression
of HDAC2 to the mice reduced dendritic spine density, synapse numbers, synapse plasticity, and memory formation. So conversely, HDAC2 deficiency resulted in increased synapse numbers and memory facilitation, so similar to chronic HDAC inhibitor treatment.
We started the lead optimization for HDAC inhibitors with the benzamide
class because of their unique inhibition kinetics, so we hoped by
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optimizing both potency and inhibition kinetics that we gain excellent selectivity.
Inhibition kinetics measurement requires a continuous enzymatic assay.
Traditional or commonly used HDAC assay is a trypsin‐coupled endpoint assay. The acetyl‐lysine containing peptide substrate is conjugated with coumarin. So upon deacetylation by HDAC, the coumarin will be hydrolyzed by trypsin, releasing coumarin and generating a fluorescence readout.
In the presence of trypsin, we found that some HDAC enzymes like
HDAC2 were not stable and would also be cleaved by trypsin, so this trypsin‐coupled assay is not optimal for continuously monitoring HDAC activity.
Slide 33 One method came up to our mind that will continue HDAC enzymatic
kinetic assay. It's the Caliper microfluidics LabChip technology. This technology is a capillary electrophoresis‐based separation technology. It has been widely used in inhibitor screening and MOA studies in kinase and protease fields.
The assay setup is quite straightforward, so we just incubated HDAC with
acetylated peptide substrate. The substrate is fluorescent‐labeled. Because of the charge difference between the acetylated substrate and deacetylated product, they will be separated by capillary electrophoresis in microfluidic chips and the substrate conversion can be continuously monitored and quantified by fluorescence peak from both product and the substrate.
Slide 34 What we call first generation HDAC Caliper substrates from PerkinElmer
are listed here. We could derive it either from Histone 3, 4, or p53 peptide. They are good substrates for HDAC3 and HDAC6. You can see the conversion here for HDAC3. However, very little activity was observed using this substrate for HDAC1,2 and at 200 nanomolar you barely see any conversion.
So working with our mechanism team and also application scientists from
PerkinElmer, we were able to synthesize the normal HDAC Caliper substrates, which show very good activity against all HDACs.
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[0:30:06] Slide 35 So here in the left panel is the comparison of in‐house substrate with first
generation of PerkinElmer Caliper substrate in HDAC1 activity. The right panel is the comparison of these substrates for HDAC2, 3
activity. You can see that our in‐house Caliper substrate is at least fortyfold more active than first generation, commercial available Caliper substrate, so HDAC1, 2. Both substrates are quite efficient for HDAC3.
Slide 36 So with these new substrates in hand, we have developed our SAR assay
using Caliper technology to support the lead optimization for our HDAC program. This is the screen view of those response data from SAR assay, so including those curves ‐‐ and I see 15 ‐‐ so it's quite a robust assay.
Slide 37 We also developed that Caliper assay using in‐house substrate for HDAC1
to 9 for selective profiling. All the substrate conversion reaches 20% to 40% in the presence of subnanomolar or low nanomolar HDAC enzymes.
Slide 38 Here, the selective profile for known HDAC inhibitors using Caliper assay,
you can see LBH and SAHA are hydroxamate‐based HDAC inhibitors with broad inhibition spectrum, and the CI‐994 and the Merck60 are benzamide‐based inhibitors with better cell activity against HDAC1,2 or HDAC1,2,3.
Slide 39 We also profiled in‐house HDAC inhibitors. Here are field profiling
examples on our in‐house HDAC1,2 selective inhibitors, HDAC3 specific inhibitors, as well as HDAC6,8 selective inhibitors. Most of benzamide as well as HDAC selective inhibitors are time‐dependent inhibitors.
Slide 40 Next, we would like to understand the inhibition kinetics. There are two
common mechanisms shown here with slow binding kinetics analysis.
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Mechanism one indicates a slow binding step, being really a limiting step of inhibiting kinetics. In this case, K‐observed, which is derived from the progression curve is proportional to inhibitory concentration and then the slope is exactly on the low substrate concentration.
In mechanism two, the slow inhibiting kinetics is mainly from slow
conformational change of enzyme inhibitor complex after inhibitor banding. So in this case, K‐observed reaches to hyperbolic saturation in the presence of a high inhibitor concentration.
Slide 41 In both cases, a Koff can be derived from a dilution experiment. Here, it
shows you a design of dilution experiment. We incubated the enzymes with a high concentration of inhibitor for one to two hours depending on their binding kinetics, then diluted it a hundredfold. The enzyme activity was monitored in continuous Caliper assay to follow the inhibitor's dissociation and calculated the off rate.
Slide 42 Here are a few examples on how to measure inhibiting kinetics of
parameters. We use HDAC2 and some known inhibitors as examples. First, we measured the inhibition kinetics for SAHA. SAHA is a fast on/fast off reversible HDAC2 inhibitor.
Here is the progression curve for HDAC2 catalyzed enzyme reaction
generated from Caliper assay. X‐X is time and Y‐X is substrate conversion. The off line with the largest slope is a time course of HDAC2 activity in the absence of the SAHA. The other lines are the time course of HDAC2 activity in the presence of SAHA at a different concentration. At one micromolar of SAHA concentration, HDAC2 completely lost activity, suggesting that SAHA is a tighter nanomolar range inhibitor.
[0:35:06] Upon dilution, the HDAC2 activity was completely recovered just like
DMSO control suggesting this inhibition is reversible. The off rate is also very fast. It reached up the limit of our detection.
Slide 43 We don't characterize the inhibition kinetics for benzamide inhibitor such
as CI‐994. CI‐994 clearly shows time‐dependent inhibition, so we can see
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through the progression curve together K‐observed ‐‐ K‐observed is proportional to CI‐994 concentration indicating a slow binding of inhibitor to HDAC2. The Kon is at 0.016 per minute or per micromolar. Upon dilution, the activity gradually comes back with the koff 0.0036 per minute, corresponding a half‐life of 190 minutes.
Slide 44 So similarly, Merck60 is also a slow tight‐binding inhibitor with even
slower off rate. Its half‐life was estimated to be three days, but I just want to mention that although Merck60 has very slow off rate, it does not form a covalent bond based on mass spec experiment.
Slide 45 So using these technologies, we have characterized the inhibition kinetics
for all of these compounds. Here is the summary table on the inhibiting kinetics and the binding affinity data we obtained to support our SAR. As you can see, CI‐994 is a nanomolar inhibitor for HDAC1, 2, 3 with slow‐binding kinetics. Their half lives are 74, 190, and 160 minutes respectively.
Our in‐house compounds, Compound 4 and Compound 5, also have slow‐
binding kinetics towards HDAC1, 2, 3. However, these two compounds have much slower on‐rate in HDAC3, which makes the HDAC1, 2 selective.
Slide 46 Comparing with CI‐994, Compound 6 has much faster off rate in HDAC1
inhibition and HDAC1 and 2 binding. It maintains long half‐life for HDAC3, about 29 minutes. We can make HDAC3 selective inhibitor.
Slide 47 So in summary, high efficiency HDAC Caliper substrates have been
synthesized and characterized for HDAC1‐9, including HDAC11. Here, I didn't show the data for HDAC11, but the substrates are also good for HDAC11. All these substrates and assay kits are now available in PerkinElmer. So by optimizing both potency and inhibiting kinetics data using Caliper assay, we're able to get isoform‐selective inhibitors.
Both our HDAC1, 2 and HDAC3 selective inhibitors have great PK/PD
properties and a few animal efficacy, which we will publish the data soon.
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Slide 48 This work is a team effort and a collaboration between several groups
from Broad, also MGH and MIT. I also want to thank the application scientists from PerkinElmer. Thank you.
Sean Sanders: Thank you so much, Dr. Zhang. Many thanks to both of our speakers for
the wonderful presentations and we're going to move right along to the questions submitted by our online viewers.
Slide 49 A quick reminder to those watching us live that you can still submit your
questions. Just type them into the text box and click the "submit" button. And again, if you don't see that box, click the red Q&A icon and it should appear.
The first question I'm going to put to you, Mr. Janzen, from a therapeutic
standpoint, would modulating the binding of RedA proteins to histone tails be more efficient or specific than utilizing histone methyltransferases or HDAC inhibitors?
[0:40:03] Dr. William Janzen: That's a very good question. The difficulty there is that it's always difficult
as you move into a new protein class to understand how specificity is going to translate into therapeutic efficacy. Clearly, as we're developing highly specific probes, these are some of the questions that we want to answer. And if the therapeutic events you're trying to interrupt does involve specific binding to a histone tail by an epigenetic complex, it's going to be more specific for that because if we inhibit the methyltransferases, those are going to have substrates which are non‐histone based.
I will say that for many of the probes that we're working on or actually for
most of the probes we're working on, we have not seen overt physiological effects as they've been administered. They do tend to be a bit more subtle. I think again our approach is to develop highly specific probes that we can make available to researchers so that we can answer exactly that question.
Sean Sanders: Dr. Zhang, a question that came in for you, is the microfluidic technology
that you're using applicable to targets other than HDACs?
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Dr. Yan‐Ling Zhang: Yes. Theoretically, an enzyme catalyzes that reaction. The substrate and
the product have different charges. You can separate it using this technology. This technology had been widely used in kinase field as well as protease field. Recently, it has been used in methyltransferase as well.
Sean Sanders: Great! Coming back to you, Mr. Janzen, maybe a broader question, how
could one specifically target drugs to a chosen gene in order to change its epigenetic status? Do you have any thoughts on that?
Dr. William Janzen: Chosen gene to change its epigenetic status, that's ‐‐ I think the answer
I'm going to give here is not going to satisfy whoever submitted it because I'm going to begin by saying I don't know. You could hypothesize some methods whereby you could target a specific gene if you understood clearly enough the expression patterns around that gene.
Where this can be done, some other work that we're doing with a
collaborator ‐‐ Ian Davis here at UNC ‐‐ is to look at accessibility of chromatin. I think one way you could look at targeting a specific gene is if that gene is known to be silenced or expressed by specific changes in the chromatin and we can find small molecules that then change that accessibility to the gene.
Sean Sanders: Dr. Zhang, any thoughts on that? Dr. Yan‐Ling Zhang: Yeah. I would think it depends on the target. If the epigenetic
modification is easy to target, then it'll be easier to find a small molecule to manipulate it that way. If the gene itself has a function which you can manipulate by a small molecule, I would think the gene directly, the targeted gene, will be a good approach as well.
Sean Sanders: Excellent. Dr. Zhang, let me stay with you and come back to the
techniques that you were using. This viewer asks if it would be possible to identify non‐competitive inhibitors or modulators, for example, allosteric modulators of HDACs using the microfluidic technology.
Dr. Yan‐Ling Zhang: Yes, I think so because the technology itself can monitor that mechanism
or study the mechanism; and if it's a competitive inhibitor, it will clearly show it competes with the substrate. If it's allosteric, you will see it won't compete with the substrate, and I think this technology can distinguish them.
Sean Sanders: Mr. Janzen, back to you. Have you done any work on chemical probes for
bromodomains?
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Dr. William Janzen: That work is being done by another group at the Structural Genomics
Consortium, so the work is essentially divided between the collaborators. Other than accessing some of their probes to use in our panels, we haven't done any direct discovery of bromodomains.
Sean Sanders: Do you have any places you can point the viewer to where they might
find some information from your colleagues? Dr. William Janzen: I would go to the Structural Genomics Consortium website and that's the
best starting point that will link in to all of the collaborators. [0:45:04] Sean Sanders: Perfect! Dr. Zhang, if available, could you mention where the information
about the development of quantitative SAR was published? Dr. Yan‐Ling Zhang: Yeah. I don't have the literature off the top of my head, but there are
some papers there including not just potency, but as well as all the kinetics as a comment to SAR to support the lead optimization.
Sean Sanders: Excellent! I think this one was also for you. The Caliper assay that you
mentioned, can they be used to study the kinetics of chemical compounds targeted to other histone modifying enzymes and have you tried this?
Dr. Yan‐Ling Zhang: We haven't tried that yet, but again like I said, there are some
publications that use this technology to do the methyltransferase. Also, some people use this one to test the p53 DNA binding as well.
Sean Sanders: Excellent! Mr. Janzen, back to you, do you have any idea of the current
status of measuring histone modifications using small numbers of cells, maybe with the ultimate goal of single cell sensitivity?
Dr. William Janzen: Well, histone changes, it's very difficult to pin that down to a specific
answer. The current status of that as far as I know is that people are working on moving this down into smaller cells. We are currently working on a platform, which is a derivation of the FAIRE platform, which uses formaldehyde‐assisted cross‐linking to look at the status of regulatory elements, trying to miniaturize that down to smaller number of cells, which may then also be available for ChIP assays.
As far as I know, people have been able to get down to small numbers of
cells, but not single cells. If there is a PCR readout for the change, we are
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hoping we can actually move these down into a single cell environment in the near future.
Sean Sanders: Excellent! Actually, let me stay with you. I'm going to come to Dr. Zhang
for this as well, but this viewer says, "It seems that the predisposition of certain enzymes is to act upon their respective histone residues within the proximity of a specific region of DNA such as a promoter or enhancer region. Is it known what confers this kind of targeted action when it seems that histones appear essentially unrecognizable from each other?" Do you have any thoughts on that one?
Dr. William Janzen: It seems that the histone regulators also are involved in targeting. One of
the things that's being seen in epigenetics is that the enzymes that modify histone tails or in some cases, even DNA methylation, don't work in isolation. They tend to come in as a complex.
So a reader domain may be involved in binding to a histone tail, but may
bring with it a large complex, which then modifies adjacent tails, adjacent marks on the tail, or may in fact open and close chromatin and allow transcription of regions.
We don't really know whether the presence of a secondary protein on
the chromatin is involved in targeting that as well, but it's highly likely that the presence of a transcription element could then recruit in binding elements that make epigenetic changes.
Sean Sanders: Dr. Zhang? Dr. Yan‐Ling Zhang: Yeah, I agree. As for HDAC, we already know a lot of HDACs actually in
vivo form complex forms. The complex form's substrate specificity could be different from a single HDAC enzyme in vitro, so those might involve the interaction and recognition with a different reading of a histone in the cell.
Sean Sanders: Excellent! Dr. Zhang, let me stay with you for another question on the
application of this technology that you're using. Can it be used to monitor changes to substrates other than peptide substrates such as nucleosomes or individual histones, if you have any experience in that area?
Dr. Yan‐Ling Zhang: I would think that it is possible. The only limitation for this technology is
that your substrate cannot be too big. [0:50:05]
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It's pretty much limited to within 20 amino acids. You can do DNA and peptide as well, which is derived from either protein or transcription factors, all kinds of in vivo proteins.
Sean Sanders: Excellent! Mr. Janzen, back to you, can you describe the paradigm for
your tertiary cell‐based screens, specifically the platform and end points? Dr. William Janzen: Each one is different because in each case, we're usually measuring
access to a different chemical target. For example, for the reader domains, what we have tended to do is to look first at things like FRAP experiments for GFP‐labeled reader proteins, but then also the chemists have been very creative and have created click chemistry modifications of the compounds so that we can actually photoaffinity tag or tag these with fluorescent moieties after they're in the cell so that we can allow them to bind and then track.
There's publication recently on what's been called Chem ChIP of being
able to use these tagged chemical compounds within the cell to then pull down and analyze the complexes that are active within the cell, so each one is different. There's not a standard panel of phenotypic assays that we look at.
Obviously, we do the basic assays, looking at cytotoxicity across a number
of cells. And if the function of a protein is known, then we will look at that specific readout, but in most cases in the readers, the function is largely poorly understood and we just want to show that we're having an intracellular effect.
Sean Sanders: Excellent! Dr. Zhang, towards the end of your talk, some of the HDAC
inhibitors you discussed had very slow binding kinetics. What might be the clinical impact of this if it were to be applied in a clinical setting?
Dr. Yan‐Ling Zhang: Yeah. Slow kinetic binding can mean longer residence times, which can
give you better efficacy and also can affect the dose as well. Sean Sanders: Excellent! I have another question for you, Dr. Zhang. How is it thought
that simple short‐chain fatty acids inhibit HDACs? Do you have any thoughts on the mechanisms?
Dr. Yan‐Ling Zhang: Those inhibitors vary. The potencies is not as great as others, are
minimolar. So in terms of in vivo efficacy, people actually don't know exactly whether that's because of inhibition of HDACs or some other related targets.
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Sean Sanders: Great! A pretty broad question here, this is probably from somebody who's starting off in the field. They're saying for someone wanting to enter the field of epigenetics research, where is a good place to start and what areas should they be looking at studying? Maybe Mr. Janzen, I'll ask you that one first.
Dr. William Janzen: Without knowing a little bit about their background, that's very difficult
to predict again. I would say the place to start is by learning as much about epigenetics as possible, seeing where your current research background fits well into that. We obviously chose methylation because it was an area that was poorly understood and we felt that we could have a real impact there by generating these probes.
It also depends very much on whether the individual entering the field is
interested in academic pursuit, drug discovery, or more of getting into the chemical biology side. The HDACs are very heavily researched. I think quite a bit is known about that. Probably the area that is most interesting now is trying to understand how the CpG islands actually affect transcription, so there's quite a bit. It's such a huge area that it's hard to isolate this down and say where one should enter it.
[0:55:04] There are so many opportunities to have an impact right now that I think
almost any area you choose, you will be able to find something interesting to do there and something that will have a large impact on the field.
Sean Sanders: Dr. Zhang, your thoughts? Dr. Yan‐Ling Zhang: Yeah, I agree. It depends on their interest and their background. You can
either focus on individual targets or enzymes in epigenetics, or you can look from the epigenetic effects on either developmental or disease‐focused. Either way will be interesting.
Sean Sanders: All right, so we're coming to the end of the webinar, but I have just one
more question I wanted to put to you. Well, maybe we'll start with Dr. Zhang this time. Where do you see the technology moving in the next, say, two to five years and what would you like to see that would enable your research and enable you to dig deeper into this subject area?
Dr. Yan‐Ling Zhang: Specifically for HDAC, I would think a big challenge here is currently we
do the in vitro to purify the system. It will be great if we can get the
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complex form of what exists in cell in vivo. I think mass spectroscopy would be pretty helpful there.
Sean Sanders: Great! Dr. Janzen, any last thoughts? Dr. William Janzen: Yeah. I think the field is actually moving in the directions we would like to
see it go. One of the biggest challenges we've had has been being able to work with isolated nucleosomes. There are a couple of small companies now that are starting up and are beginning to produce recombinant nucleosomes that work in the ‐‐ it look like they would work at least in the AlphaScreen format. I think that's going to be a huge help to us all. The antibodies are getting better.
One of the other challenges we've had has been the specificity of
methylation, so I think we're gaining the tools that we need. The final thing we need, I think, is what was asked by a question earlier if we can get down to single cell level of changes.
Sean Sanders: Excellent! Well, we are unfortunately out of time for this webinar, so on
behalf of myself and our viewing audience, I want to thank our speakers very much for being with us today, Mr. Bill Janzen from the University of North Carolina at Chapel Hill and Dr. Yan‐Ling Zhang from the Broad Institute of MIT and Harvard.
Please go to the URL now at the bottom of your slide view to learn more
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sponsorship of today's educational seminar. Goodbye. [0:58:25] End of Audio