SARomics Biostructures 2017 presentation
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Transcript of SARomics Biostructures 2017 presentation
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SARomics Biostructures at a glance • Hybrid business model
- CRO generating revenues- Proprietary discovery projects
• Strategic focus on early drug discovery with a proprietary discovery platform- Unique expertise in protein structure
determination, fragment screening and in silico drug discovery
- ProPHECY™ protein optimization technology
• Experienced and skilled team- Multifaceted team of 12 persons (10 PhDs)- Entrepreneurial and scientific expertise
• Significant pharma, biotech and academic clients & partners world-wide
• Sales representatives in Boston & Japan• Five major EU R&D grants• Mission to become significant hit
generation player
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GENE-TO-STRUCTURE
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SARomics BiostructuresIntegrated drug discovery solutions
Gene-to-structure platform
Structure-based design solutions
Fragment-based hit generation solutions
Construct design
Cloning
Protein expression & purification
Protein characterization
Crystallization
Synchrotron-based data collection
• In silico screening• Protein production & characterization• Co–crystal lead structure determination
• Biophysics-based screening• Hit identification & SAR exploration• Co–crystal fragment structure
determination
Off-the-shelf protein structures• FastLane™ library• Focus on kinases and epigenetic
targets
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State-of-the-art crystallization labSARomics Biostructures performs high-throughput low volume
crystallization using liquid handling, crystallization and imaging robotics
Microlitre robot
Crystallization roboticsPlate hotel/imaging robotics
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Synchrotron accessWe currently ship crystals to Diamond Light Source (DLS), Oxford,
and BESSY, Berlin, on average twice/monthWe are located in close vicinity to the Swedish synchrotron MAX IV
and when it opens we will have rapid access to beamlines
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A wide range of technologies are used to characterize ligand binding and protein behavior using biophysical principles
Biophysics-based screeningand protein characterization
Ligand binding• NMR screening (96 tube sample changer)• NMR analysis/titration• DSF (thermofluor-based method, 96 wells)• ITC• MST
DLSDSF NMRCDITC
Protein characterization• DSF (thermofluor-based method, 96 wells)• DLS (96 well format)• Buffer screen (DLS+DSF)• CD• NMR
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Access to structural information increases your understanding and enables you to execute projects faster
Extensive experience in Fab–antigen crystallization & structure determination
Use structural information for:1. Epitope definition to file stronger IP2. Understanding MoA3. Structure-based design4. Antibody engineering: affinity maturation5. Antibody engineering: humanization6. Antibody engineering: ADC7. Structural characterization of protein drugs
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FASTLANE™ LIBRARY
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FastLane™ structures
Standard
*Please see www.saromics.com for a complete list.
Proteins* ready to be expressed, purified and crystallized according to existing verified protocols (complexes delivered within 4 to 10 weeks)
• >55 kinases
• >30 phosphatases
• >20 bromodomains
• >20 demethylases
• >30 other targets
Crystallization system up and running and ready to be co-crystallized with customers compounds (complexes delivered within 2 to 6 weeks)
Kinases:BTK, CK2, DAPK3, PFKFB3, PIP4K2A, PLK4, STK17BEpigenetics targets:KDM4C, ATAD2A & 2B (bromodomains)Proteases:USP8, Cathepsin C, ThrombinOther:AR, BCAT2, BlaC, DHODH, Gal3C, Hsp90, IL-17A, LDHA, PDE4d, PTP1B, S100A4, S100A9, S100A12, TIM3, TNFα
Premium
Soon also: Hif2α, KDM4a-d, KDM6a, LSD1, RORg
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FastLane™ structuresCase study I
From FastLane™ Standard to 3D structure in 2.5 weeks• Expression and crystallization of ATAD2A bromodomain
• Followed verified protocols
• Final structure resolution 1.65 Å (compared to published 1.95 Å)
ATAD2AATAD2A with bound thymine
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FastLane™ structuresCase study II
From FastLane™ Standard to 3D structure in 10 days• Purification and crystallization of KDM4C histone demethylase
• Followed verified protocols
• Final structure resolution 2.50 Å (compared to published 2.55 Å)
KDM4C (or JMJD2C) KDM4C (or JMJD2C) in complex with (2,4-PDCA)
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FastLane™ structuresCase study III
Delivery of refined structure 3 days after receiving compound• PFKFB3 established as FastLane™ structure
• Tuesday: Received compound by FedEx
• Wednesday: Soaking of compound into available crystals
• Thursday: Data collection to 2.8 Å (911-3 beamline)
• Friday: Structure refinement and delivery of results to customer
PFKFB3 – 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
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FastLane™ structuresCase study IV
Proprietary crystallization system for IL-17A• Generated several small molecule ligand complexes
• Enables both co-crystallization and soaking into apo crystals
• Resolutions ranging from 2.2 – 3 Å
• No Fab fragment present
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FRAGMENT-BASEDSCREEINING
STRUCTURE-BASEDDRUG DESIGN
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FRAGMENT LIBRARY SCREENING, VALIDATION & FRAGMENT EXPANSION/ELABORATIONCollaboration between SARomics Biostructures & Red Glead Discovery
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FragmentscreeningScreeningtechnologies
InsilicoscreeningThermoshiftassay(DSF)
NMRWAC
Biochemicalscreening(HCS)X-raycrystallography
MST
CompoundsProprietarylibrary
SpecificallyorderedsetsClientlibraries
• Medicinalchemistry
• Synthesis:smallmolecules&peptides
• Analyticalchemistry(NMR&MS)
• Invitrobiology• InvitroADME&physchem
• HTS/FBLGexpertise
• Structure-baseddrugdesign• X-raycrystallography• Computationalchemistry
• NMRscreening
• Biophysics• Proteinchemistry
A collaborative innovative screening platform
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Crystallographic fragment screening
• High-throughput low volume crystallization capability
• Soaking pools of fragments
• Co-crystallization of selected fragments
• High-throughput data collection (at automated synchrotron beamlines)
• Structure determination and refinement through automated data pipelines
Crystallization robotics
MAX IVDLS
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In silico screening using Schrödinger computational technology
Glide - Complete solution for ligand-receptor docking
Phase - High-performance program for ligand-based drug designStrike - Powerful software for statistical modeling and QSARPrime - Powerful and innovative package for accurate protein structure predictionsSiteMap - Fast, accurate, and practical binding site identificationLigPrep - Versatile generation of accurate 3D molecular modelsMacroModel - Versatile, full-featured program for molecular modeling
Liaison - Efficient and accurate ligand-receptor binding free energy prediction
QikProp - Rapid ADME predictions
Canvas - CheminformaticsJaguar - Rapid ab initio electronic structure calculationEpik - Rapid and robust pKa predictions
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Weak binding fragments require binding to be detected by biophysical principles
Typical fragment screening technologies:• CFS - crystallographic fragment screening• DSF - differential scanning fluorimetry (or thermal shift, TSA)• WAC - weak affinity chromatography• NMR - ligand detected• NMR - protein detected• [SPR - surface plasmon resonance (BIAcore)]• MST - microscale thermophoresis• HCS - high concentration biochemical screening
DSF NMR CFS
Biophysics-based screening
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Ultra high field NMR instrumentsAccess to NMR instruments at the Swedish NMR Centre
Automatic assignment, NUS sampling, fragment screening etc.
• 900 MHz Bruker Avance III HD– 4 RF channels– Triple-axis pulsed field gradients– TCI cryoprobe (1H/13C/15N, 5 mm)– Triple resonance probe (1H/13C/15N)
• 800 MHz Bruker Avance III HD– 4 RF channels– Triple-axis pulsed field gradients– TCI cryoprobe (1H/13C/15N, 3 mm)– SampleJet
• 800 MHz Bruker Avance III HD– 4 RF channels– Triple-axis pulsed field gradients– TXO cryoprobe (1H/13C/15N, 5 mm)– Triple resonance probe, 8 mm
• 3x600 MHz Varian Inova/Bruker– 3-4 RF channels– Triple-axis pulsed field gradients– Tripple resonance cryo-probe (1H/13C/15N)– Triple resonance probe (1H/13C/X)– 4 mm NANO probe– Diffusion probe (1H/2H/19F)
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Ligand detected NMR screening
• Sample changer screens 96 samples overnight
• Cryo probe equipped 800 MHz NMR instrument
• Ligand observed NMR spectra
• NMR analysis/titration of selected hits
• Typically pools of 4-6 cmpds
• Capacity: ~500 cmpds per day
• Performed at the Swedish NMR Centre
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NMR screening – verification
(S1):compound in buffer
(S2):compound in buffer +target
(S3):compoundinbuffer+target+refcmpd
• Automatic compound QC• 500 MHz in-house and 600 MHz at nearby Lund University• Solid NMR expertise
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Differential Scanning Fluorimetry (DSF)Initial screening (1ary or 2ndary assay)
Dose response curves
Biochemical assay and/or NMR
X-ray – soaking/co-crystallization(very predictive for X-ray success)
0.1 mg /ml 0.2 mg/mlΔTm at 62.5 µM ΔTm at 62.5 µM
RG200001 5.95 7.3RG200001 6.3 6.4RG200002 9.6 10.4RG200002 10.2 10.7RG200003 -1.3 -0.48RG200003 -0.9 -0.76
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MicroScale Thermophoresis (MST)
• Quantification of molecular interactions• Measures the motion of molecules along microscopic
temperature gradients• Access affinities (Kd, dissociation constant): nM to mM range• Rapid assay optimization• Fast measurement: Kd in 10 min• Low sample consumption: minimal concentration (nM) and
small volume (< 4 µl)
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• WAC™attractiveasprimaryfragmentscreeningassay
• InventedbyProf.Sten Ohlson (LinnaeusUniversity,Kalmar,Sweden)
• CommercialrightsbyTransienticInteractionsAB(TI)
• SARomicsandRGDinuniquecollaborationwithTItosetupexclusiveserviceplatform
Screening using Weak Affinity Ahromatography (WAC™)
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• TargetproteinimmobilizedonHPLCcolumn• Retentiontimeoffragmentsrelatedtoselectiveinteractionswithtarget• Physiologicalbufferusedasmobilephase• Kd candirectlybecalculatedfromretentiontime• Highthroughputpotential(upto3000-4000fragmentsperday?)
Fragment screening by WAC™
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ITID Benefits with WAC™
WAChassignificantadvantagescomparedtotheothermethodsintermsofspeedandinformationprovided.
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• GoodcorrelationwithNMR,SPRandX-ray
• MoresensitivethanSPR• IndicativeforX-ray
Screeningof111fragmentsbyWAC,NMR,SPR,FPandTmshift.Selected
fragmentsalsobyITCandX-raycrystallography.Fragmentslisted
withdecreasingaffinitiesasmeasuredbyWAC.
(Meiby etal.2013,Anal.Chem.,85,6756)
WAC™ versus other techniques
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Casestudy:Screening155fragmentsonaBRD4-column
• UsingaWAC-cutoffofΔtret >5min,allfragmentsverifiedbyothertechniquesexcept:• RG200054andRG200067(”Pfizer-hit”)• RG210010nottestedinotherassays.
A=active;NA=notactive;NT=nottested
CompoundID Δtret (min)WAC Biochem DSF NMR X-rayRG210073 138.1 A(dose-response) A NT YESRG210080 87.2 A(dose-response) A NT YESRG210074 71.4 A(dose-response) A NT YESRG210069 52.1 Tendencyofactivity A NT YESRG210070 42.1 Tendencyofactivity A NT YESRG210081 35.1 A A NT YESRG210079 28.3 A A NT YESRG210019 10.7 NA A NA NT
RG200067 9 NA NA NA NT
RG100104 7.6 NT NA A NT
RG200054 7.3 NA NA NT NT
RG100056 6.8 NT NT A NT
RG210010 5.9 Nottestedinanyotherassay
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Fragment library
WAC
DSF + QC NMR/MST (if needed)
Fragment in crystal structure
Expansion of fragments
Screening
Validation of Actives
Furthervalidation
Crystallization & structure determination
Analysis of fragment binding
Hit IDEffect in
bioassay?Hit
quality?
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Recent collaborative publicationsAstraZeneca/Royal Institute of Technology
“Crystal structures of the Chromobacteriumviolaceum ω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.” Humble et al., 2012, FEBS J, 279, 779-79.
Lund University/ESS
“Structural basis for carbohydrate-binding specificity -a comparative assessment of two engineered carbohydrate-binding modules.” von Schantz et al., 2012, Glycobiology, 22, 948-61.
“Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH-π interactions.” von Schantz et al., 2014, Proteins, 82, 3466-75.
“Neutron Crystallographic Studies Reveal Hydrogen Bond and Water-Mediated Interactions between a Carbohydrate-Binding Module and Its Bound Carbohydrate Ligand.” Fisher et al., 2015, Biochemistry, 54, 6435-8.
Arsanis Biosciences
“Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH.” Badarau et al., 2015, J Biol. Chem., 290, 142-56.
“Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for the generation of neutralizingantibodies.” Badarau et al., 2016, Mabs. 8, 1347-1360.
Galecto Biotech/Lund University
“The carbohydrate-binding site in galectin-3 is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics.”Saraboji et al., 2012, Biochemistry, 51, 296-306.
“Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.” Diehl et al., 2010, JACS, 132, 14577-89.
Cancer Research Technology, UK
“Fragment library design considerations.”Boyd et al., 2012, WIREs Comput. Mol. Sci., 2, 868-885.
“Fragment-based drug discovery and protein-protein interactions.”Turnbull et al., 2014, Research and Reports in Biochemistry, 4, 13-26.
ETH
“De Novo Fragment Design for Drug Discovery and Chemical Biology.”
Rodrigues et al., 2015, Angew Chem Int Ed Engl., 54, 15079.
Sahlgrenska Cancer Center
“Cancer differentiation agent hexamethylene bisacetamide inhibits BET Bromodomain proteins.”
Nilsson et al., 2016, Cancer Res., 76, 2376-83.
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MD
Fragment screening
Fragment expansion
Hit seriesexpansion
Lead series identification
Lead optimization Partner
Internal discovery pipelineC
ance
r
merozyne
SBX-1401 TAKTIC NIK kinase inhibitor
SBX-1301 BRD4 epigenetic inhibitor
SBX-1501 Pim-1 kinase inhibitor
KINOMED Kinase inhib.
Merozyne Therapeutics
SBX-1402 mAb
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HeadquartersMedicon Village • SE-223 81 Lund • Sweden
Tel: +46 46 26 10 470
US branch245 First Street • Cambridge • MA 02142
Tel: 508 269 9048
Björn WalseCEO
SARomics Biostructures [email protected]
Tel: +46 46 26 10 470
www.saromics.com
Japanese distributorCarna Biosciences. Inc.
1-5-5 Minatojima-MinamimachiChuo-ku • Kobe • Japan
Tel: +81 78 302 7091