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SARomics Biostructures 2018 Company Presentation


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The slides describe the details of the company services: X-ray structure determination of protein and protein complexes, protein NMR spectroscopy, antibody-antigen crystallization and structure determination, etc. A special focus this year is dedicated to our fragment-based drug discovery using the proprietary weak affinity chromatography (WAC) method.

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SARomics Biostructures 2018 Company Presentation

  1. 1. SARomics Biostructures at a glance • Strategic focus on early drug discovery • Proprietary discovery platform - Unique expertise in protein structure determination and in silico drug discovery - Fragment-based hit generation • Hybrid business model - CRO generating revenues - Proprietary discovery projects • Experienced and skilled team - Multifaceted team of 17 persons (12 PhDs) - Entrepreneurial and scientific expertise • Significant pharma, biotech and academic clients & partners world-wide • Sales representatives in Boston & Japan • Six major EU R&D grants • Mission to become significant hit generation player
  3. 3. SARomics Biostructures Integrated drug discovery solutions Gene-to-structure platform Structure-based drug design solutions Fragment-based hit generation solutions* Construct design & cloning Protein expression & purification (E. coli & BEVS) 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 *In collaboration with Red Glead Discovery
  4. 4. Synchrotron access We currently ship crystals to DLS, Oxford, and ESRF, Grenoble, on average every second week Our lab is located close by MAX IV and when it opens we will have extremely rapid access to synchrotron beamtime
  5. 5. Our future light source • The MAX IV Laboratory is the new Swedish national synchrotron facility • SARomics will be in a very advantageous position for access to MAX IV in 2018 • Access to the world’s most advanced synchrotron source will substantially increase our competitiveness • SARomics will be a key player in leveraging industrial usage of MAX IV May, 2015
  6. 6. State-of-the-art crystallization lab SARomics Biostructures performs high-throughput low volume crystallization using liquid handling, crystallization and imaging robotics Microlitre robot Crystallization robotics Plate hotel/imaging robotics
  7. 7. A wide range of technologies are used to characterize ligand binding and protein behavior using biophysical principles Biophysics-based screening and protein characterization Ligand binding studies • 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
  8. 8. Ultra high field NMR instruments Access to NMR instruments, 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)
  9. 9. Don’t work in the dark! Access to structural information increases your understanding and enables you to execute projects faster Why Fab–antigen structures? Use structural information for: 1. Epitope definition to file stronger IP 2. Understanding MoA 3. Structure-based design 4. Antibody engineering: affinity maturation 5. Antibody engineering: humanization 6. Antibody engineering: ADC 7. Structural characterization of protein drugs
  11. 11. FastLane™ structures Standard *Please see for a complete list. Proteins* ready to be expressed, purified and crystallized according to existing verified protocols (complexes delivered within 4 to 10 weeks) • >50 kinases • >30 phosphatases • >20 bromodomains • >20 other epigenetic targets • >25 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, CLK3, DAPK3, EphA7, MAP2K4, PFKFB3, PIP4K2A, PLK4, STK10, STK17B, Vps34 Epigenetics targets: KDM4C, ATAD2A & 2B, SMARCA2B, SMARCA4 Proteases: USP8, Cathepsin C, DPP4, Thrombin Other: AR, BCAT2, Bfl-1, BlaC, DHODH, Gal-3C, GMPR2, HER2, Hsp90, IL-17A, IL-23, LDHA, PDE4d, PTP1B, RORg, S100A4, S100A9, S100A12, TIM-3, TNFα, Tubulin Premium Soon also: Hif2α, KDM4a-d, KDM6a, USP7
  12. 12. FastLane™ structures Case study I From FastLane™ Standard to 3D structure in 2.5 weeks • Expression and crystallization of ATAD2A bromodomain • Followed verified protocols from The SGC • Final structure resolution 1.65 Å (compared to published 1.95 Å) ATAD2AATAD2A with bound thymine
  13. 13. FastLane™ structures Case study II From FastLane™ Standard to 3D structure in 10 days • Purification and crystallization of KDM4C histone demethylase • Followed verified protocols from The SGC • Final structure resolution 2.50 Å (compared to published 2.55 Å) KDM4C (or JMJD2C) KDM4C (or JMJD2C) in complex with (2,4-PDCA)
  14. 14. FastLane™ structures Case 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
  15. 15. FastLane™ structures Case study 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
  17. 17. Schrödinger Computational Technology Glide - Complete solution for ligand-receptor docking Phase - High-performance program for ligand-based drug design Strike - Powerful software for statistical modeling and QSAR Prime - Powerful and innovative package for accurate protein structure predictions SiteMap - Fast, accurate, and practical binding site identification LigPrep - Versatile generation of accurate 3D molecular models MacroModel - Versatile, full-featured program for molecular modeling Liaison - Efficient and accurate ligand-receptor binding free energy prediction QikProp - Rapid ADME predictions Canvas - Cheminformatics Jaguar - Rapid ab initio electronic structure calculation Epik - Rapid and robust pKa predictions SARomics currently holds a license to the following modules:
  19. 19. FRAGMENT SCREENING IN LEAD DISCOVERY BY WEAK AFFINITY CHROMATOGRAPHY (WAC™) Red Glead Discovery AB & SARomics Biostructures AB Medicon Village, Lund, Sweden
  20. 20. Fragment-based lead discovery Fragment screening Screening technologies In silico screening Thermoshift assay (DSF) NMR WAC™ Biochemical screening (HCS) X-ray crystallography MST Compounds Proprietary library Specifically ordered sets Client libraries • Medicinal chemistry • Synthesis: small molecules & peptides • Analytical chemistry (NMR & MS) • In vitro biology • In vitro ADME & physchem • HTS/FBLG expertise • Structure-based drug design • X-ray crystallography • Computational chemistry • NMR screening • Biophysics • Protein chemistry
  21. 21. Conventional HTS vs. fragment screening in drug discovery Duong-Thi, 2013, Thesis.
  22. 22. Fragment screening & qualification of hits NMR screening Thermal shift assay / DSF Weak Affinity Chromatography ΔTm at 62.5 μM RG200001 7.3 RG200001 6.4 RG200002 10.4 RG200002 10.7 RG200003 -0.48 RG200003 -0.76 Primary screening techniques Follow-up techniquesX-ray crystallography MST, microscale thermophoresis Crystallization robotics MAX IV EC50=44 µM HCS – high concentration biochemical screening Follow-up techniques Suitable combinations of orthogonal techniques designed for each target
  23. 23. Fragment library – possibilities • Client libraries • Internal library – Collection of 1300 fragments (available as neat sample, DMSO and DMSO-d6 solutions) – General purpose, (not target-directed) covering diverse chemical space – Focus on low-molecular weight fragments (<220) – Solubility data (DMSO, water) on >80% of fragments – Analytical data (LCMS, 1H-NMR) on all fragments – MedChem friendly (HBDs, HBAs and ring count, ClogP, functional groups, etc) – >90% commercially available -> rapid SAR- generation • Exclusive small libraries – an option available through our academic MedChem collaborations 0 50 100 150 200 cLogP distribution (1016 fragments) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 50 100 150 200 MW distribution (1016 fragments) Number Accum%
  24. 24. Weak affinity chromatography (WAC™) • WAC™ attractive as primary fragment screening assay • Invented by Prof. Sten Ohlson (Linnaeus University, Sweden) • Commercial rights by Transientic Interactions AB (TI) • SARomics and RGD in unique collaboration with TI offering exclusive service platform
  25. 25. Fragment mix Affinity LC-MS column Fragment binding Column with immobilized protein Column without protein (blank column) Extracted ion chromatograms Schematic representation of screening using WAC™ Column cross section • Target protein immobilized on HPLC column • Retention time of fragments related to selective interactions with target • Physiological buffer used as mobile phase • Kd can directly be calculated from retention time Principles of WAC™
  26. 26. WAC™ – data analysis Affinity constant (KD) is calculated from the retention time difference (∆tret) for the compounds on target and blank columns. KD = Btot/(∆tret × flow rate)
  27. 27. Beneficial WAC™ characteristics Throughput fast setup, mixes, detection of multiple binders Robustness low variation between replicates, long column life time Cost efficiency low consumption of protein and fragments, standard equipment Quality automatic quality control, not sensitive for impurities Ranking immediate ranking Low concentration fragments screened at low concentrations (1-5 µM)
  28. 28. Limitations • Immobilization – Co-factors / protein complexes – Potential distortion of protein structure and/or active site • Requirement on MS-response in used buffer – Method development in progress
  29. 29. Overview of tested WAC™ targets Target class Specific targets Reference / comment Proteases Thrombin, trypsin Collaboration with AstraZeneca Kinases Cyclin-G associated kinase Confidential, not published NHRs Non-disclosed Confidential, not published Epigenetic targets and PPIs BRD4 D1, Pin1, 2 non- disclosed targets BRD4: 12% hit rate (150 screened) Chaperones Hsp90 (several screens) Duong-Thi et al., 18(2) 160-171 (2013) plus non-disclosed screens Channels Aquaporin 1 Duong-Thi et al., Analyst, 141,981 (2016) Other enzymes Non-disclosed synthase 38 hits (1697 screened) Other targets Cholera toxin B, non- disclosed growth factor Ramos-Soriano et al., Chemistry - A European Journal, 19, 52, 17989-18003 (2013) Confidential, not published - 53 hits (2500 screened)
  30. 30. Hit rate 86% 8% 3% 2% 1% 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% < 0.25 min 0.25 to 0.5 min 0.5 to 1 min 1 to 2 min 2-5 min > 5min PPI 61% 12% 9% 6% 7% 5% 0% 10% 20% 30% 40% 50% 60% 70% < 0.25 min 0.25 to 0.5 min 0.5 to 1 min 1 to 2 min 2-5 min > 5min BRD Screens performed in duplicate Followed up screening hits
  31. 31. WAC™ data on bromodomain (BRD4)
  32. 32. BRIEF SUMMARY WAC™-screening of 155 fragments • Fragment lib. MW: 123-308, average MW = 239 • 10-fragment-mixtures • 20 mM sodium acetate buffer Robustness • Unchanged performance of BRD4-column over time (>1 month at least) • Re-test with every two 10-mixes pooled into one 20-mix provided very similar results • RG210073 (IC50 = 40 µM in biochemical assay) included in all mixes as control; Retention time = 138 ± 5 minutes (%CV = 3.9%) on BRD4- column (n=18)
  33. 33. HSP90 screening hits Comparison with other techniques BRD4 screening hits Screening of 111 fragments by WAC, NMR, SPR, FP and Tm shift. Selected fragments also by ITC and X-ray crystallography. Fragments listed with decreasing affinities as measured by WAC. (Meiby et al. 2013, Anal. Chem., 85, 6756) Using a WAC™-cutoff of ∆tret > 5 min, all fragments verified by other techniques except: • RG200054 and RG200067 (reference cmpd ”Pfizer-hit”) • RG210010 and RG200039 not tested in other assays Compound ID Δtret (min) WAC™ Biochem DSF NMR X-ray RG210073 138.1 A (IC50 ~50 µM) A NT YES RG210080 87.2 A (56%@100µM) A NT YES RG210070 84.1 Tendency of activity A NT YES RG210074 71.4 A (54%@100µM) A NT YES RG210069 52.1 Tendency of activity A NT YES RG210081 35.1 A (45% inhib@100µM) A NT YES RG210079 28.3 A (31% inhib@100µM) A NT YES RG200067 18.8 NA NA NA 4HBV RG210019 10.7 NA A NA YES RG100104 7.6 NT NA A YES RG200054 7.3 NA NA NT NT RG100056 6.8 NT NT A NT RG210010 5.9 Not tested in any other assay RG200039 5.0 Not tested in any other assay A = active; NA = not active; NT = not tested WAC™ hits verified by other techniques – maintained target properties upon immobilization
  34. 34. WAC™ for chiral separation min0 1 2 3 4 5 6 7 8 9 Detectorresponse void KD = 1.07 mM KD = 0.16 mM active thrombin column blocked thrombin column blank column ST058742 Target: Thrombin Column: 35 × 0.5 mm Sample: Fragment library Duong-Thi et al., Journal of Biomolecular Screening 18(2): 160-171, 2013
  35. 35. WAC™ offers All results are owned by the client Client evaluation • Protein and fragments from client • Screen of up to 50 fragments Ligandability • Evaluation of the target ligandability1 (incl. amenability for WAC™) • Limited screen of a fixed library of 190 fragments2 WAC™ screens • Two-step milestone process • Feasibility & setup • Screen • RGD/SBX collection and/or client fragments Ranking • Rank fragment hits obtained from other fragment techniques Follow up studies • NMR verification • Co-crystallization • Fragment evolution • And more … 1Fragment screening to predict druggability (ligandability) and lead discovery success. Edfeldt et al, Drug Discov Today. 2011 Apr;16(7-8):284-7 2 Essential Fragment Library, Enamine
  36. 36. Essential Fragment library • Approximately 190 compounds • The library was designed in cooperation with research group at University of Cambridge, UK, to be a universal tool for initial screen of novel targets. • All compounds in the library have been tested for water solubility and chemical stability in buffer solution • Essential Fragments are especially suitable for the purposes limited by different factors, e.g. validation of new targets, excessive cost of setting up a library for one or two targets, assay particularities etc. The following features make Enamine’s Essential Library an attractive tool for initial fragment screening: • Design is based on privileged fragments - frequently reported hits and scaffolds derived from experimentally determined structures of protein-ligand complexes reported in PDB. • Confirmed water solubility at least at 1mM concentration in PBS buffer, 1% DMSO. Nephelometry-based solubility assay. • Experimentally confirmed chemical stability in water buffer solution (pH 7.0±0.5) for 24 hours at stabilized 30°C. LC/GC-MS methods have been used. • Compounds with fluorescence interference at 488 or 520 nm and as well as compounds with affinity to CMD- coated surfaces were removed.
  37. 37. Applied Biophysics for Drug Discovery Chapter 7 Sten Ohlson and Minh-Dao Duong-Thi Weak Affinity Chromatography (WAC) Pages 107–130 Chapter 3 Björn Walse, Andrew P. Turnbull and Susan M. Boyd Tailoring Hit Identification and Qualification Methods for Targeting Protein–Protein Interactions Pages 29–59 Applied Biophysics for Drug Discovery, 2017 Eds. Donald Huddler, Edward R. Zartler
  38. 38. FAQ of WAC™ 1. What affinity range is detectable by WAC? Standard WAC screens are typically adjusted for 200 µM – 5 mM (30-60 min per run). But can adjusted down to sub- micromolar binders by adjusting protein load and/or flow rate 2. How robust is WAC? Provided stable protein (good stability normally found), normally high reproducibility: %CV of 0.5-5% in retention time (>95% of fragments) 3. How is the protein attached to the column? Amide couplings via solvent-exposed lysine residues (before column- packing) 4. Attachment points other than Lys possible? Yes, but not used so far 5. What if ΔRT is >60min or longer? Provided not too tightly bound, it will be observed in subsequent runs 6. What is considered a hit? What ΔRT? ΔRT ≥ 0.5 min (higher cutoff used if many hits) and ΔRT/RTprotein > 0.1 ΔRT = RTprotein – RTblank 7. What about the fragments non-detectable by MS? Use other techniques 8. What buffer is used? If its acidic, how does that affect binding? Typically acetate buffer pH ~5 9. How many columns with protein are required? Normally one 10. How much protein do we need to modify a column? 5 mg on average, some highly druggable targets we can go down to 1 mg 11. What is the protein kDa range allowed? No limitation with respect MW. Issues being around non-covalent complexes, co-factors, etc. 12. How much fragment do we use? What concentration? 1-10 µL of 1-5 µM solution 13. How much automation is there? Inject with robot possible, etc? Big mixes, not a problem by hand, more for data analysis 14. How much does it cost? Please inquire 15. Pilot project first or real project first? We have done several, academia have done several, at this point no need for pilot and its no problem to go directly into real project 16. What is the setup time, from the point when protein is provided/synthesized? Up to 2 weeks 17. Are predictable timelines possible for every stage of the project? Absolutely, right now we are doing a 1000 frag screen and the data will come within 1 week. Weekly updates, daily availability via e-mail and or/phone/skype
  39. 39. Headquarters Medicon Village • SE-223 81 Lund • Sweden Tel: +46 46 26 10 470 US branch 245 First Street • Cambridge • MA 02142 Tel: 508 269 9048 Björn Walse CEO SARomics Biostructures AB Tel: +46 46 26 10 470 Japanese distributor Carna Biosciences. Inc. 1-5-5 Minatojima-Minamimachi Chuo-ku • Kobe • Japan Tel: +81 78 302 7091