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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


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).


Epigenetics in biology

• Cell differentiation

Lunyak V V , Rosenfeld M G Hum. Mol. Genet. 2008;17:R28-R36




• X chromosome silencing

Wolf Reik & Annabelle LewisNature Reviews Genetics 6, 403-410 (May 2005)





• 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)





• 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





• 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).

*


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


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.


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.


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).

*


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


AlphaScreen Assay Platform


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


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


• ~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


James et.al., (2013) Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain. Nat Chem Biol 9:184-191.


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.


Acknowledgments

Stephen Frye

University Cancer Research Fund


Sponsored by:

June 25, 2014


Webinar Series




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%



HDAC4 Substrate B 0.5 38%

HDAC5 Substrate B 1 17%




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


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


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


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


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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