structural genomics - asbmb · structural genomics – aids in understanding biochemical mechanisms...
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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
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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