STRUCTURAL GENOMICSAled Edwards

SGC, Midwest Center for Structural Genomics, Center for Structural Genomics of Infectious Diseases

SGC OxfordSGC Toronto SGC Stockholm

Structural genomics projects

Protein Structure InitiativeTo increase our understanding of the relationship between sequence and structure so that a protein’s structure and function can be predicted from sequence

SPINETo develop and disseminate the technologies to determine the structures of protein complex

CSGIDTo increase the number of 3D structures of proteins of biological importance from Class A-C pathogens

SGCTo promote drug discovery by increasing the number of 3D structures of human proteins of therapeutic relevance

Targeting the protein universe

What US and Swedish scientists think

Canada: Leading the way

STRUCTURAL GENOMICS

Contributions (present and future) to human structural and chemical

biology

SGC OxfordSGC Toronto SGC Stockholm

Progress in Structural Annotation of the Human Proteome

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Year 2000 Year 2008

UnstructuredSignal peptideTransmembraneCoiled-coil

To be determined

Structural genomicsStructural biology

SG Contributions to Structural Universe

Structural genomics of human proteins• SG is cost-effective

– SG contributes >30% of global output of human structures each year and >15% of total output

– SG produces each human structure for ~$125,000

• SG is not a synonym for “no impact”

Cell 134:793 (2008); Cell 136:352 (2009) PNAS 103:7829 (2006); PLoS Biology5:1063 (2007); PLoS Biology 7:e43; Nature 448:87 (2007); Nature Methods 4:1019 (2008); Nature Methods. 5:135 (2008); Nature Methods. 6:477 (2009); EMBO J.28:969 (2009); J. Med. Chem. 52:3108 (2009); Nature 440:833 (2006); Nature448:613 (2007); Nature Struct. and Mol. Biol.14:1229 (2007); EMBO J. 25:4245 (2006); PNAS 103:7637 (2006); PNAS 103:15835 (2006); Nature Chemical Biology 5:436 (2009); Nature 455:822 (2008); J. Med. Chem. 51:7053 (2009)

• SG promotes faster science

– All structures and clones made available immediately (no “hold til publication”)– SG promoting open access science – to enable drug discovery

I was trying to be a good boy (but evidently did not succeed)

Structural genomics contributions• Methodology

– Methods to increase success rates of protein structure determination– Quantifying the probability of success for experimental choices

• Genomics

– Largest (and most cost-effective) contributor to our understanding of relationship between sequence and structure

• SG promotes open science

– Structural biologists are highly secretive with their structures; Science accelerated ~12-18 months by the immediate release of structural information

SGC OxfordSGC Toronto SGC Stockholm

Design of Selective Inhibitors using Large Scale Structural Comparison

Stefan KnappStructural Genomics Consortium

Phosphorylation Dependent Signalling GroupOxford University, Nuffield Department of Medicine

Oxford, United Kingdom

September 29 2009

A world of lemmings

Kinases: > 500 000 papers in PubMedCovering ~10% KinomePatents follow public data

RNAi hits

Kinase myopia

The need for tool compounds

RNAi hits

Synthetic Lethality Screens identify novel targets that are relevant in a certain disease genetic background

Make undruggable targets druggable

RNAi Targets

Kinome WideScreening

Kinome wideSBDD/screening

ChemicalProbe

FunctionalAnnotation

DiseaseTissue/Cells

Impact of structural genomics

46 Human Kinase Structures by SG since 2004 (42 by SGC)

54 Structure in PDB from Academic Labs (within 95% seqID)34 Structures in PDB from Industrial Labs

Human Kinase Structures

SG and chemical biology

Many “probes” are non selectiveLarge differences in inhibitor sensitivity of different kinasesNo hits for certain kinasesBinding profile for most commercial inhibitors available online

PNAS Dec. 2007

Chemical Probes as Tools: PIM-1

High levels of surface CXCR4 expression on blasts from patients with AML are associated with elevated levels of PIM1 expression.

PIM1 co-localizes with CXCR, phosphorylates CXCR4 at S339 which regulates surface expression

PIM1-/- mice cannot home BM cells

PIM1 is a direct regulator of CXCR4/SDF1 signaling and is essential for migration of leukemic stem cells and solid cancer cells

Bone marrow cells from PIM1-/- mice express significantly less surface CXCR4

Inhibition of PIM (K00486/CARB13 or RNAi demonstrate regulation of CXCR4 surface expression by PIM1

SGI-1776 K00135

Clinical Trials started for AML & Prostate Cancer

Structural genomics as a path to generate chemical probes in “pioneer”

areas of drug discovery

SGC OxfordSGC Toronto SGC Stockholm

Structural genomics and chemical probes

Outline

1. What is a chemical probe?

1. Chemical probes to promote research in epigenetics

2. Chemical probes and the kinome

Why chemical probes?

• Temporal resolution– rapid exposure and elimination of effects are possible

• Mechanistic flexibility– can potentially target separate functions of a protein, as

opposed to ablating them all• Ease of delivery

– freely cell permeable, potential for oral activity• Applicability to drug discovery

– transition from target validation to therapeutic intervention is more direct

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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.

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What is a quality chemical probe?• Molecular Profiling: Sufficient in vitro potency and selectivity

data to confidently associate its in vitro profile to its cellularor in vivo profile.

• Mechanism of Action: Activity in a cell-based or cell-freeassay influences a physiologic function of the target in adose-dependent manner.

• Identity of the Active Species: Has sufficient chemical andphysical property data to permit interpretations of results tobe attributed to its intact structure or a well characterizedderivative.

• Proven Utility as a Probe: Cellular activity data available toconfidently address at least one hypothesis about the role ofthe molecular target in a cell’s response to its environment.

• Availability: Is readily available to the academic communitywith no restrictions on use.

Biologically attractive, “pioneer target area”• Play a key role in development, differentiation and stem cell biology• Underlie many chronic diseases: cancer, inflammation, psychiatric

disorders• Directly impact transcriptional programs, DNA repair & metabolism• Intense area of research for which there is a receptive community to

test chemical probes and protein capture reagents

Epigenetic targets appear to be Druggable• SAHA (HDAC inhibitor) approved for cutaneous T-cell lymphoma• Inhibitors of DNA MTases shown to reactivate silenced genes• nM inhibitors of Bromo domains have been developed and can affect

transcriptional programs.

Opportunity for discovery of new biology and new drug targets using chemical biology approaches

Targeting Epigenetic & Chromatin‐Related Proteins

Structural genomics– Aids in understanding biochemical mechanisms– Structures used to Assess potential binding pockets

(druggability)– Guide medicinal chemistry and selectivity

Access to recombinant proteins facilitates med chem– Ligand-based libraries– Secondary, tertiary assays– Rapid feedback loop < activity/structure/chemistry>

Engage with the biomedical community to characterize selective, potent and cell permeable compounds to link biology with inhibition of an individual target or group of targets

The Strategy

IndustryPublicDomain

Public/PrivatePartnership

ChemicalProbes

ScreeningChemistryStructureBioavailability

TargetValidation

No IPNo restrictionsPublication

DrugDiscovery

(re)ScreeningChemistryLead optimizationPharmacologyDMPKToxicologyChemical developmentClinical development

Model for Pre-Competitive Chemistry

Creative commons Proprietary

Objective: make 37 probes and data publicly available (no restriction on use) over 4 years

Participants: Funder• SGC – Toronto (HMTs, Royal Family, HATs) Ontario $4.6M• SGC – Oxford (KDMs, Bromo domains) Wellcome T. $8M• GSK Exploratory Chemistry (8 med chemists)• NIH Chemical Genomics Center (20 HTS)• OICR medicinal chemistry (3 FTE)• Frye Lab, UNC (2 FTE)• Pending SGC, Stockholm; 2 pharmaceutical companies

The Chemical Biology Consortium

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DNA methylation

Histonemodification

Histone

DNA

Lysine

Chemical biology of chromatin regulation

GOAL = Chemical Probes

Modification Read Write Erase

Acetyl (Ac) KMTBromo HDAC

Methyl (Mt) KMTRoyal KDM

* K = Lysine, R = Arginine, E = Glutamate

Domain Family Typical substrate class* Total domains

Solved by SGC

Purified by SGC

Histone Lysine demethylase

Histone/Protein K/R(me)n/ (meCpG)

53 2 24

Bromodomain Histone/Protein K(ac) 55 15 36ROYAL

Tudor domain Histone Rme2s25 4 13

Tandem Tudor Histone K(me)2/3

Chromodomain Histone/Protein K(me)3 35 6 19MBT repeat Histone K(me)3 11 5 9

PHD finger Histone K(me)n 78 0 22Acetyltransferase Histone/Protein K 25 10 15Methyltransferase Histone/Protein K&R 50 10 34PARP Histone/Protein E 17 7 13

TOTAL 349 59 185

Other (WD40, PWWP, E3, DUb, PPI, kinase)

DNA, protein, etc. >100 10 >50

SGC Progress for Epigenetics Targets

Histone Methyl Transferases

Jian Jin, CICBDDMasoud Vedadi, SGC

Overview of HMT’s• > 50 human HMTs identified since the first HMT discovered in 2000

• Histone lysine methylation recognized as one of most important PTMs. Essential function in many biological processes.

• Growing evidence suggests HMTs involved in various human diseases including cancer

• Lack of chemical probes: only 2 selective KMT inhibitors reported (BIX-01294 and chaetoxin)

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H3K4

H3K4

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Data from Kouzarides, Cell. (2007)128:693-705. Solved by others

Solved by SGC

Figure: Matthieu Schapira &Yong Zhao, SGC

Assays available in SGC

Design of UNC0224 as a Potent G9a Inhibitor

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BIX-01294G9a (Thioglo): IC50 = 0.11 μMGLP (Thioglo): IC50 = 0.062 μMKubicek, et al. 2007, Mol Cell, 473

UNC0123G9a (Thioglo): IC50 = 0.33 μMReduced MW and lipophilicity while maintaining potency

Array-basedoptimization

GLP-BIX-01294 complexAdopted from Chang, et al. 2009, Nat. Stru. Mol. Bio., (16), 316

UNC0224G9a (Thioglo): IC50 = 0.015 μMGLP: IC50 = 0.020 μM, inactive vs SETD7 & SETD8Selective over a panel of 30 non-Epi targets(except hitting M2 at 82% inh at 1 μM)

Feng Liu & Xin Chen, CICBDDAbdellah Allali-Hassani, SGC

Characterization of UNC0224

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ITC confirmation: UNC0224 binds better to G9a than BIX-01294 BIX-01294 UNC00000224A

SGC data

Co-crystal Structure of G9a + small molecule: G9a-UNC0224 complex

PDB code: 3K5K

• 7-Dimethylaminopropoxy side chain binds to the lysine binding channel, validating the binding hypothesis

Matthieu Schapira, SGCGregory Wasney, SGCAiping Dong, SGCDmitri Kireev, CICBDD

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What are Bromodomains?

• Small domain (~110 residues) that selectively binds to acetylated lysine residues

• Bundle of four α-helices, Z, A, B and C plus two loops forming a pocket with a conserved Asn residue

• A recognition domain forming part of numerous chromatin modifying proteins, including histone acetyltransferases (eg CREB & PCAF) and transcriptional coactivators/repressors

• 55 unique bromodomains identified to date

InflammationCancerMetabolic diseaseNeurological diseasesCardiovascular diseases

< 10 < 50 < 100< 500

Publications on Bromo targets

Clinical Relevance of Bromodomains

• Structures for 24 bromodomains now available

• 19 solved by SGC and deposited in PDB

• Further 18 in pipeline

Bromodomain Structures

• At least 1 probe from each major subfamily

• <100 nM Kd by ITC or displacement assay

• >30-fold selectivity vsrepresentative proteins from other subfamilies (highlighted in red)

• Demonstration of interaction with target protein in cells at <1uM

• Demonstration of functional effects in cells (desirable but not essential => will be done by scientific community)

Bromodomain Probes - Target Profile

Primary SAR / Hit ID

Family Selectivity Secondary assay

Cellular assays

Probe data package

Tm shift assay

Displacement(AlphaScreen), ITC, NMR

Broader selectivityPhyschem propertiesPermeability

CREBBPBRD2_1PCAFBAZ2BPB1_5LOC93349 FRET/FRAP

Expression profilingPathway specific

Bromodomain Screening Cascade

Tm shift data for Bromodomains

Bromo Subfamily 1 2 3 5 6 7 8 9 13 14

Bromodomain SMARCA2 LOC93349 BAZ2B ATAD2 BRD9 BRPF1 CREBBP BRD2 BRD4 CECR2 PCAF FALZ

Most potent hit

ΔTm / ˚C5.6 12.9 3.0 3.8 5.6 12.1 6.7 4.8 8.4 3.6 5.3 3.9

• Rapid, low protein consumption• Partially validated by comparison with ITC• Screened ~10K compounds: fragments, VLS, pharmacophores• Hits for >10 bromodomains

Thermal stability (DSF) 

Model: OneSitesChi^2/DoF = 2640N 1.01 ± 0.00152K 6.17E7 ± 6.02E6KD 16.20 ± 0.4 nMΔH -7215 ± 20.15ΔS 10.6

Temperature

A Potent BRD4 Ligand

ΔTm=7˚C

• Identified through focused screening in Tm shift assay

• High potency revealed by ITC

Selectivity for the BET subfamily

• BDGBJ000086 shows affinity only for closely related bromodomains in the BET subfamily, and shows no interaction with bromodomains across the remaining subfamilies

Summary

• Team in place to generate well-characterized tool compounds

• Structure-guided chemistry in progress

• Open access philosophy enables wide collaboration net

• Cellular assays accessed via collaboration

• Three probes will be released in next 4 months and (hopefully!) they will keep rolling out

• Probes will allow for perturbation experiments, and subsequent modelling of epigenetic regulation

ACKNOWLEDGEMENTS

FUNDING PARTNERSCanadian Institutes for Health Research, Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Knut and Alice Wallenberg Foundation, Merck & Co., Inc., Novartis Research Foundation, Ontario Innovation Trust, Ontario Ministry for Research and Innovation, Swedish Agency for Innovation Systems, Swedish Foundation for Strategic Research, and Wellcome Trust. www.thesgc.org

SGC (Oxford)Tom HeightmanChas BountraCheryl ArrowsmithJohan WeigeltUdo OppermannPaul BeswickStan NgAlice GrabbeMichelle DanielStefan KnappPanagis FillipakopoulosSarah PicaudTracy KeatesIldiko Felletar

SGC cont.Brian MarsdenMinghua WangSree VadlamudiFrank von DelftOliver KingMartin PhilpottFrank NiesenTony TumberJing Yang

GSKTim WillsonRyan TrumpIan BaldwinMike RenoCunyu ZhangChun‐wa ChungIan FillmoreGemma WhiteRoy KatsoRachel GrimleyChampa Patel

Oxford ChemistryChris SchofieldNathan RoseAkane KawamuraOliver King

Oxford BiochemistryRob KloseShirley Li

NCGCAnton SimeonovDave MaloneyAjit JadhavAmy Quinn

….your name here

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Acknowledgements (cont’d)

• UNC– Stephen Frye– Tim Wigle – Martin Herold– Bill Janzen– Dmitri Kireev– Jian Jin – Feng Liu– Xin Chen

• SGC (Toronto)– Cheryl Arrowsmith– Masoud Vedadi– Natalie Nady– Peter Brown– Matthieu Schapira– Abdellah Allali-Hassani– Taraneh Hajian– Gregory Wasney– Aiping Dong

• NCGC - Anton Simeonov