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New Tools, New TargetsNovel Approaches for Identifying and Characterizing Epigenetic Modifications
Instructions for Viewers
Webinar Series
William P. JanzenUniversity of North CarolinaChapel Hill, NC
Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
June 25, 2014
Participating Experts
Yan-Ling Zhang, Ph.D.Broad Institute of MIT and HarvardCambridge, MA
Webinar Series
New Tools, New TargetsNovel Approaches for Identifying and Characterizing Epigenetic Modifications
Epigenetic Probe Discovery
William P Janzen
William Janzen, Center for Integrative
Chemical Biology& Drug Discovery
University of North Carolina Chapel Hill
http://www.pharmacy.unc.edu/labs/center-for-integrative-chemical-biology-and-drug-discovery
Center For Integrative Chemical Biology & Drug Discovery
EPIGENETICS – “above genetics”
• Definition: ‘‘An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.’’
Berger, S.L., Kouzarides, T., Shiekhattar, R. & Shilatifard, A. An operational definition of epigenetics. Genes Dev 23, 781-3 (2009).
Center For Integrative Chemical Biology & Drug Discovery
Epigenetics in biology
• Cell differentiation
Lunyak V V , Rosenfeld M G Hum. Mol. Genet. 2008;17:R28-R36
Center For Integrative Chemical Biology & Drug Discovery
Epigenetics in biology
• Cell differentiation
• X chromosome silencing
Wolf Reik & Annabelle LewisNature Reviews Genetics 6, 403-410 (May 2005)
Center For Integrative Chemical Biology & Drug Discovery
Epigenetics in biology
• Cell differentiation
• X chromosome silencing
• Effect of maternal care Maternal care alters cytosine methylation of GR promoter.Maternal care alters cytosine methylation of GR promoter.Maternal care alters cytosine methylation of GR promoter.Maternal care alters cytosine methylation of GR promoter.
Ian C G Weaver, et.el.,Nature Neuroscience 7, 847 - 854 (2004)
Center For Integrative Chemical Biology & Drug Discovery
Epigenetics in biology
• Cell differentiation
• X chromosome silencing
• Effect of maternal care
• ‘Dutch famine’ effect in humans
Difference in DNA methylation of CpGdinucleotides in siblings discordant for periconceptional exposure to famine.
Tobi E W et al. Hum. Mol. Genet. 2009;18:4046-4053
Center For Integrative Chemical Biology & Drug Discovery
Epigenetics in biology
• Cell differentiation
• X chromosome silencing
• Effect of maternal care
• ‘Dutch famine’ effect in humans
• Coloration in felines
Chemical Biology of Chromatin Regulation
PKMT PKDMRoyal
HAT HDACBromo
Histone code: e.g.H3K4Me3H3K9Me3
Frye, S. V.; Heightman, T.; Jin, J. Targeting Methyl Lysine. Annu.Rep. Med. Chem. 2010, 45, 329-343.
*Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature (2010).
*
Center For Integrative Chemical Biology & Drug Discovery
CICBDD Mission and Pipeline
Collaborative Center; mostly grant funded, not fee for service Portfolio of 18‐24 pre‐projects and projects running concurrently Extensive pharma experience among center leadership and staff, including contributions
to several FDA drugs (i.e., Avodart, Tykerb) and multiple clinical candidates
Target Proposalsfrom
UNC Faculty
“responsivecollaborations”
Target Proposalsfrom
UNC Faculty
“responsivecollaborations”
Small molecule ‘probes’
Target validation & Drug leads
Core Staff & Broader UNC FacultyCenter ‐InitiatedProjects inChemical Biology
“prospectivescience”
Center ‐InitiatedProjects inChemical Biology
“prospectivescience”
12 Biologists/Biochemists18 Chemists/3 Comp. Chem>300,000 Small Molecules
11
Center For Integrative Chemical Biology & Drug Discovery
Why chemical probes?
• Temporal resolution– rapid exposure and elimination of effects are possible– can readily pair with other stimuli
• Mechanistic flexibility– can potentially target separate functions of a protein, as opposed
to ablating them all
• Ease of delivery– Can be freely cell permeable, potential for oral activity
• Applicability to drug discovery– transition from target validation to therapeutic intervention is
more direct
Weiss, W.A., S.S. Taylor, and K.M. Shokat, Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat Chem Biol, 2007. 3(12): p. 739-44.
Center For Integrative Chemical Biology & Drug Discovery
What is a quality chemical probe?
• Molecular Profiling: Sufficient in vitro potency and selectivity data to confidentlyassociate its in vitro profile to its cellular or in vivo profile.
• Mechanism of Action: Activity in a cell-based or cell-free assay influences aphysiologic function of the target in a dose-dependent manner.
• Identity of the Active Species: Has sufficient chemical and physical property datato permit interpretations of results to be attributed to its intact structure or a wellcharacterized derivative.
• Proven Utility as a Probe: Cellular activity data available to confidently address atleast one hypothesis about the role of the molecular target in a cell’s response to itsenvironment.
• Availability: Is readily available to the academic community with no restrictions onuse.
– Frye, S. V. The art of the chemical probe. Nat Chem Biol 2010, 6, 159-161.
Center For Integrative Chemical Biology & Drug Discovery
Chemical Biology of Chromatin Regulation
PKMT PKDMRoyal
HAT HDACBromo
GOAL = Chemical Probes
Histone code: e.g.H3K4Me3H3K9Me3
Frye, S. V.; Heightman, T.; Jin, J. Targeting Methyl Lysine. Annu.Rep. Med. Chem. 2010, 45, 329-343.
*Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature (2010).
*
Center For Integrative Chemical Biology & Drug Discovery
34 Chromo Domains (Kme3) 37 Tudor Domains (Kme2 & 3) 9 MBT Domains (Kme1 & 2)
102 PHD Domains (Kme0‐3) 22 PWWP Domains (Kme3) 2 WD40 Domains (Kme2 & 3)
Methyl-lysine Reader Families
Center For Integrative Chemical Biology & Drug Discovery
AlphaScreen Assay Platform
Center For Integrative Chemical Biology & Drug Discovery
Wigle et.al, (2010) Screening for Inhibitors of Low Affinity Epigenetic Peptide-Protein Interactions: An AlphaScreen™-Based Assay for Antagonists of Methyl-Lysine Binding Proteins, Journal of Biomolecular Screening 15: (1) 62-71
Center For Integrative Chemical Biology & Drug Discovery
Chemical Biology ApproachMBT
1 L3MBTL12 L3MBTL23 L3MBTL34 MBTD15 SFMBT1
CHROMO1 CBX7
TUDOR1 UHRF1
(TTD)2 53BP13 PHF14 PHF195 PHF20
PHD1 JARID1A2 PHF233 UHRF1 (TTD-PHD)4 UHRF2 (TTD-PHD)5 UHRF1 (PHD)6 UHRF2 (PHD)7 UHRF1 (TTD*-PHD)8 UHRF1 (full-length)Counterscreen Peptides
Brandi BaughmanVictoria Korboukh Tim Wigle
Center For Integrative Chemical Biology & Drug Discovery
• ~800,000 wells of data collected over 5 years of testing
• Represents 51,000 potency curves
Brandi BaughmanVictoria Korboukh Tim Wigle
Chemical Biology Approach
Center For Integrative Chemical Biology & Drug Discovery 20
Center For Integrative Chemical Biology & Drug Discovery
Center For Integrative Chemical Biology & Drug Discovery
James et.al., (2013) Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain. Nat Chem Biol 9:184-191.
Center For Integrative Chemical Biology & Drug Discovery
Wigle et.al, (2010) Screening for Inhibitors of Low Affinity Epigenetic Peptide-Protein Interactions: An AlphaScreen™-Based Assay for Antagonists of Methyl-Lysine Binding Proteins, Journal of Biomolecular Screening 15: (1) 62-71
Wigle et.al. Accessing Protein Methyltransferase and DemethylaseEnzymology Using Microfluidic Capillary Electrophoresis, Chem Biol.17(7):695-704.
Center For Integrative Chemical Biology & Drug Discovery
Center For Integrative Chemical Biology & Drug Discovery
Center For Integrative Chemical Biology & Drug Discovery
Acknowledgments
Stephen Frye
University Cancer Research Fund
Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
June 25, 2014
Participating Experts
Webinar Series
New Tools, New TargetsNovel Approaches for Identifying and Characterizing Epigenetic Modifications
William P. JanzenUniversity of North CarolinaChapel Hill, NC
Yan-Ling Zhang, Ph.D.Broad Institute of MIT and HarvardCambridge, MA
Kinetic Characterization of Inhibition of HistoneDeacetylase by Isoform Specific Inhibitors with Microfluidic Mobility Shift Assay
Yan‐Ling ZhangBroad Institute of MIT and Harvard06‐25‐2014
Histone Acetylation and Chromatin Structure
Adapted from Grayson DR et al (2010) Molecular Pharmacology 77, 126
3 Classes of Zinc-Dependent HDACs
Current Known HDAC InhibitorsCompound Class
Example Structure target potency
Hydroxamic Acid SAHA Classes I, II uM-nM
Cyclic peptides Depsipeptide (FK-228)
Class I nM
Short-chain fatty acids
Butyrate ClassI,II mM
Benzamides MS-275 HDAC1,2,3 uM-nM
• none of these inhibitors are HDAC isoform specific
Traditional HDAC assay format:trypsin‐coupled HDAC enzymatic assay
excitation =355 nm emission = 460 nm
[Histone H4K12-Ac]
Caliper microfluidics LabChip:A non‐coupled in vitro HDAC enzymatic assay
Time
Fluo
resc
enc
e
P
S
Substrate Peak Height
Product Peak Height
1) Incubate HDAC withacetylated peptide substrate
3) Read fluorescence
2) Separate substrate and product by capillary electrophoresisand microfluidics
HDAC2(200nM)
Low HDACs 1,2 activity with commercial available caliper substrates
HDAC3(20nM)
Marker
P S
S
P
Substrate Conversion<10% /1hrfor HDAC1,2 @200nM with H212,H218 or H219
H-212
H-219
H-218
In house Caliper substrate is >40 fold more active than commercial Caliper substrate for HDAC1,2
HDAC1(5nM)
HDAC1(200nM)
HDAC2(5nM)
HDAC2(200nM)
HDAC3(0.5nM)
HDAC3(20nM)
S
P
S
PP
P PPS
S
SSin house
substrate
commercial Substrate
H218
Caliper SAR assay: Genedata Analysis:
HDAC Enzyme
Substrate HDAC Conc. (nM)
Substrate Conversion%
@1hr
HDAC1 Substrate A 5 27%
HDAC2 Substrate A 3 20%
HDAC3 Substrate A 5 30%
HDAC4 Substrate B 0.5 38%
HDAC5 Substrate B 1 17%
HDAC6 Substrate A 2 29%
HDAC7 Substrate B 0.5 45%
HDAC8 Substrate B 0.5 22%
HDAC9 Substrate B 1 25%
In-house Caliper Substrate for HDAC 1-9
HDAC Enzyme
LBH-589 SAHA CI-994 Merck60
HDAC1 <0.005 0.008 0.5 0.02HDAC2 <0.003 0.030 0.7 0.05HDAC3 <0.005 0.007 0.6 3.7HDAC4 0.065 >30 >30 >30HDAC5 0.022 18 >30 >30HDAC6 0.002 0.002 >30 >30HDAC7 0.76 >30 >30 >30HDAC8 0.025 0.72 >30 >30HDAC9 0.39 >30 >30 >30
Known HDAC inhibitor selectivity profiling against Caliper HDAC selectivity panel
Caliper – IC50 Values (μM) for in-house cpds
Isoform IC50 (uM) Compd. 1
IC50 (uM)Compd. 2
IC50 (uM) Compd. 3
HDAC1 0.00273 1.08 9.57
HDAC2 0.033 1.15 1.68
HDAC3 2.35 0.064 11.7
HDAC4 > 35 > 35 > 35
HDAC5 > 35 > 35 7.86
HDAC6 > 35 > 35 0.021
HDAC7 > 35 > 35 14.2
HDAC8 > 35 > 35 0.056
HDAC9 > 35 > 35 > 35
Kinetic mechanism of slow, tight‐binding inhibitors and data analysis
Upon dilution, I 0, 1obsk k
Upon dilution, I 0, 2obsk k
competitive tight-binding mechanism
competitive tight-bindingwith conformational change
Dilution Experiment for Koff Measurement
~ ~
0 50 100 150 200 250
0
5
10
15
20
25
30
35 SAHA Time Course
SAHA 1uM (H-2) SAHA 0.5uM (H-2) SAHA 0.25uM (H-2) SAHA 0.125uM (H-2) SAHA 0.0625uM (H-2) SAHA 0.03125uM (H-2) SAHA 0.0156uM (H-2) SAHA 0.0078uM (H-2) SAHA 0.0039uM (H-2) SAHA 0.00195uM (H-2) SAHA 0.00097uM (H-2) SAHA 0uM (H-2)
% S
ubst
rate
Con
vers
ion
Time (mins)
0 100 200 300 400 500 600 70005
10152025303540 Reversibility Assay for SAHA
(100-fold dilution; 2.5nM final) at 1nM final HDAC2 09/06/2011
DMSO Buffer (H-2) SAHA dilution (H-2)
% S
ubst
rate
Con
vers
ion
Time (mins)
SAHA HDAC2Koff(min-1) >0.2T1/2 (min) <4
SAHA is a fast on/fast off inhibitor for HDAC2
summary of kinetic parameters
100x
0 50 100 150 200 250
0
5
10
15
20
25
30CI-994 Time Course with HDAC2
CI-994 10uM (H-2) CI-994 5uM (H-2) CI-994 2.5uM (H-2) CI-994 1.25uM (H-2) CI-994 0.625uM (H-2) CI-994 0.3125uM (H-2) CI-994 0.15625uM (H-2) CI-994 0.078uM (H-2) CI-994 0.039uM (H-2) CI-994 0.0195uM (H-2) CI-994 0.0097uM (H-2) CI-994 0uM (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2) koff (User) Fit of Sheet1 CI-994 (H-2)
% S
ubst
rate
Con
vers
ion
Time (mins)
0 2 4 60.00
0.02
0.04
0.06
0.08
0.10
Kob
s
CI994 uM
Equation y = a + b*x
Weight No Weighting
Residual Sum of Squares
6.84343E-5
Adj. R-Square 0.98383Value Standard Error
C14 Intercept 0.00651 0.00184
C14 Slope 0.01613 8.42819E-4
0 100 200 300 400
0
5
10
15
20
25 Reversibility Assay for CI-994 (100-fold dilution; 25nM final) at 1nM final HDAC2
% S
ubst
rate
Con
vers
ion
Time (mins)
CI994 HDAC2Kon(min-1, uM-1) 0.016Koff(min-1) 0.0036T1/2(min) 190Ki (nM) 223
summary of kinetic parameters
A linear trend indicates a competitive tight-binding mechanism
CI-994 is a slow, tight-binding inhibitor for HDAC2
Red : cpd dilutionBlack:DMSO dilution
100x
0 50 100 150 200 250
0
5
10
15
20
25
30 Merck 60 Time Course with HDAC2
Merck 60 1uM (H-2) Merck 60 0.5uM (H-2) Merck 60 0.25uM (H-2) Merck 60 0.125uM (H-2) Merck 60 0.0625uM (H-2) Merck 60 0.03125uM (H-2) Merck 60 0.0156uM (H-2) Merck 60 0.0078uM (H-2) Merck 60 0.0039uM (H-2) Merck 60 0.00195uM (H-2) Merck 60 0.00097uM (H-2) Merck 60 0uM (H-2) koff (User) Fit of Sheet1 Merck 60(H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60 (H-2) koff (User) Fit of Sheet1 Merck 60(H-2)
% S
ubst
rate
Con
vers
ion
Time (mins)
0.0 0.2 0.4 0.6 0.8 1.0
0.00
0.02
0.04
0.06
0.08 hdac2 Linear Fit of Sheet1 C19
Kob
s
Merck 60 uM
Equation y = a + b*x
Weight No Weighting
Residual Sum of Squares
3.27909E-5
Adj. R-Square 0.99363Value Standard Error
C19 Intercept 6.72795E-4 6.74731E-4
C19 Slope 0.07655 0.00194
0 100 200 300 400
0
5
10
15
20
25 Reversibility Assay for Merck 60 (100-fold dilution; 5nM final) at 1nM final HDAC2
% S
ubst
rate
Con
vers
ion
Time (mins)
Model koff (User)
Equationy=vs*x+(vr-vs)*(1-exp(-kob*x))/kob+y0
Reduced Chi-Sqr
0.00739
Adj. R-Square 0.98217Value Standard Error
Merck 60 (H-2) vs 0.043 0Merck 60 (H-2) vr 0.0014 0Merck 60 (H-2) kob 5.02633E-4 8.68846E-6Merck 60 (H-2) y0 0.26754 0.01176 MERCK60 HDAC2
Kon(min-1, uM-1) 0.077Koff(min-1) 1.2e-4T1/2(hour) ~80Ki (nM) 1.5
summary of kinetic parameters
A linear trend indicates a competitive tight-binding mechanism
Red : cpd dilutionBlack:DMSO dilution
100x
Merck60 is a pseudo-irreversible inhibitor for HDAC2
Kinetic Parameters
CI994 Compd. 4 Compd.5
HDAC1 Kon(min-1,uM-1) 0.25 0.15 0.44
Koff(min-1) 0.0094 0.0083 0.039T(1/2) min 74 83 18Ki(nM) 37 55 89IC50(nM) @3hr 46 23 29
HDAC2 Kon(min-1,uM-1) 0.016 0.0083 0.015
Koff(min-1) 0.0036 0.0028 0.0084T(1/2) min 190 250 82Ki(nM) 223 340 560IC50(nM) @3hr 154 108 81
HDAC3 Kon(min-1,uM-1) 0.18 0.00028 0.0013
Koff(min-1) 0.0044 0.0025 0.0027T(1/2) min 160 280 260Ki(nM) 25 9,200 2,100IC50(nM) @3hr 49 6,700 970
SAR Summary HDAC1,2 Selective Compound(IC50,Ki,Koff)
• Compd.4 and 5 are HDAC1,2 selective inhibitors with >10 fold selectivity against HDAC3
Kinetic Parameters
CI994 Compd._6
HDAC1 Kon(min-1,uM-1) 0.25 ~0.020Koff(min-1) 0.0094 ~0.27T(1/2) min 74 ~2.5Ki(nM) 37 5,100**IC50(nM) @3hr 46 1,380
HDAC2 Kon(min-1,uM-1) 0.016 ~0.0082Koff(min-1) 0.0036 0.052T(1/2) min 190 13Ki(nM) 223 ~6,300IC50(nM) @3hr 154 1,340
HDAC3 Kon(min-1,uM-1) 0.18 0.3Koff(min-1) 0.0044 0.0088T(1/2) min 160 79Ki(nM) 25 29IC50(nM) @3hr 49 55
• CI994 is a HDAC1,2,3 pan inhibitorwhile compd.6 is a HDAC3 selective inhibitor with >20 fold selectivity against HDAC1,2.
• CI994 has slow off rate for HDAC1,2,3 while Compd. 6 has fast off rate for HDAC1,2 but slow off rate for HDAC3
SAR Summary for HDAC3 Selective Compound(IC50,Ki,Koff)
Summary
High efficiency HDAC Caliper substrates have been characterized for HDAC1‐9
HDAC1‐9 Caliper assays have been optimized for HDAC SAR support and HDAC inhibitor selectivity profiling.
HDAC caliper assay has also been used for study inhibitor reversibility and inhibition kinetics
Quantitative SAR (including affinity, selectivity, inhibition kinetics and stability) has been successfully used to guide lead optimization and prioritization in developing isoform specific HDAC inhibitors
Acknowledgment
Broad InstituteChemical Biology/Stanly Center Jennifer GaleFlorence WagnerEdward HolsonDan FassStephen HaggartyMichelle PalmerEdward Scolnick
Caliper/Perkin ElmerLaurel ProvencherSeth Cohen
MITLi-Hui Tsai’ Lab
MGHStephen Haggarty’s Lab
Thank you!
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Participating Experts
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series
June 25, 2014
New Tools, New TargetsNovel Approaches for Identifying and Characterizing Epigenetic Modifications
William P. JanzenUniversity of North CarolinaChapel Hill, NC
Yan-Ling Zhang, Ph.D.Broad Institute of MIT and HarvardCambridge, MA
For related information on this webinar topic, go to:www.perkinelmer.com/learnepigenetics
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To provide feedback on this webinar, please e‐mailyour comments to [email protected]
Brought to you by the Science/AAAS Custom Publishing Office
Sponsored by:
Webinar Series
June 25, 2014
New Tools, New TargetsNovel Approaches for Identifying and Characterizing Epigenetic Modifications