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Mladen Vinkovi ć

Robotic crystallisations in high throughput crystallography. Mladen Vinkovi ć. Presentation overview. Astex – company, facilities, technologies Crystallisation experiments in HTX ® Automation of crystallisation process Micro-batch ( m b) with PixSys. Astex Technology.

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Mladen Vinkovi ć

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  1. Robotic crystallisations in high throughput crystallography Mladen Vinković

  2. Presentation overview Astex – company, facilities, technologies Crystallisation experiments in HTX® Automation of crystallisation process Micro-batch (mb) with PixSys

  3. Astex Technology • Established in December 1999 (Founders - Sir Tom Blundell, Harren Jhoti, Chris Abell) • First-round financing raised over US$45M in 2001 (Abingworth, Oxford Bioscience, Advent Intl, Alta Partners, GIMV, Cambridge University) • Based in Cambridge (UK) with 92 employees • Protein Technology (18) • Structural Biology & Biophysics (18) • Computational Chemistry & Informatics (16) • Medicinal Chemistry (20) • Biology (8)

  4. PRESS RELEASE Astex announces Alzheimer’s drug discovery collaboration with AstraZeneca Cambridge UK, 3rd March 2003 Astex Technology, the structure-based drug discovery company, today announced a collaboration with AstraZeneca. The agreement relates to the discovery of novel drugs against a key protein target implicated in Alzheimer’s disease. PRESS RELEASE Astex Technology announces first deal with a major pharmaceutical company. Amsterdam, April 16th2000 At the European Life Sciences Conference, Astex announced that it had signed its first research collaboration with a major pharmaceutical company. Harren Jhoti, Astex’s VP of Research, while describing the company’s high throughput X-ray crystallography technology………….. PRESS RELEASE Astex announces structure-based drug discovery collaboration with Mitsubishi Pharma Corporation Cambridge, UK, 10 June 2002 - Astex Technology, the UK structure-based drug discovery company, today announced a structural biology research agreement with Mitsubishi Pharma Corporate partnersStructure-based Lead Discovery

  5. PRESS RELEASE Astex announces structure-based drug discovery collaboration with Aventis Pharmaceuticals Cambridge, UK, and Bridgewater, NJ, USA, March 2002 - Astex Technology, the private, structure based drug discovery company, and Aventis Pharmaceuticals today announced a structural biology research agreement to facilitate the rapid design and development of drug candidates. PRESS RELEASE Astex announces drug discovery collaboration with Mitsubishi Pharma Corporation Cambridge, UK, 21st October 2002 Astex Technology, the structure-based drug discovery company, today announced a drug discovery collaboration in the area of metabolic disorders with Mitsubishi Pharma Corporation. The collaboration has been established to identify novel lead compounds against a key protein target and will then be extended to other targets. PRESS RELEASE Astex and AstraZeneca announce structural biology research agreement on cytochrome P450 Cambridge, UK, and Molndal, Sweden, 3 May 2001 - Astex Technology Ltd, a private structure-based drug discovery company, and AstraZeneca AB…. Corporate partners Structure-based metabolic optimisation

  6. Astex facilities • Protein production • BV & E. coli expression • - Novel re-folding methods • Protein NMR • 500 MHz • X-ray crystallography • 4 detectors • ACTOR robot • crystallisation robot • crystal visualisation robot Medicinal chemistry - MS/NMR analysis • Computational chemistry • 80 node PC farm

  7. Astex facilities – X-ray analysis • Crystallisation robot • Crystal visualisation robot • ACTOR robot • 4 X-ray detectors

  8. AutoSolve Protein/ligand complexes Technology HTX Pyramid Initial structure Fragment-based lead discovery

  9. N N N N N N N N N N N N R R1 R R2 R3 R Structural screeningof drug fragments 100R1x100R2x100R3 = 1,000,000 cmpds 100R1 + 100R2 + 100R3 = 300 cmpds • Higher hit rate • Detect unique structures (mM) • Precise structural data (validate/prioritise the hits) • Rapid structure based optimisation • Room to optimise • High re-use -protein family specific scaffolds

  10. Pyramid process

  11. Crystallisation Database

  12. Crystallisation experiments New proteins (clones, preps) commercial screens – VD, mb protein specific in house screens (96) – VD, mb additive screens Production of xtals for soaking experiments Co-crystallisations commercial screens - VD, mb limited screens – 4-24 optimised conditions

  13. Automation of crystallisation process Problems with manual crystallisations repetitive stress injury low throughput at peak times Automation of dispensing (PixSys 4200) plate reservoirs filed manually with electric multi-channel pipettor increased throughput (2000 experiments/hour) reduced costs & plate storage space option to extend automation to reservoir filling and mother liquor preparation

  14. PixSys 4200

  15. PixSysInitial problems & solutions Problem – air gap (protein-hydraulic water) protein dilution, affects accuracy, significant mixing of water and protein in valve tubing Solution – oil gap (1cst silicone oil) no measurable protein dilution for V10 ml avoid volumes between 10 and 20ml (use 2 channels instead) reduced mixing in valve tubing sample loss typically 5%

  16. Crystallisation protocols (PixSys) VD sitting drop Greiner low profile plate(reservoirs pre-filled with ML) up to 900 exper./hour

  17. Crystallisation protocols (2) VD sitting drop Greiner standard profile plate (3 wells/reservoir) (reservoirs pre-filled with ML) up to 2000 exper./hour

  18. Crystallisation protocols (3) mb method mother liquor (ML) stocks in 96 well plate Greiner 384 or 1536 clear bottom plate

  19. ML 7-12 ML 1-6 8*Si-oil 8 compound stocks C1 C4 C2 C3 C5 C6 C7 C8 Crystallisation protocols (4) VD co-crystallisation screen of 12 ML 8 compounds Si-oil gap 5 min/plate (8 comp.) Ch6 Ch5 Ch4 Ch3 Ch2 Ch1 1 protein Ch6 Ch5 Ch4 Ch3 Ch2 Ch1 Ch1 6+6 mother liquors 8 compounds

  20. mb with PixSys - problems Paraffin (mineral) oil mixture of saturated hydrocarbons (25-80cst) PixSys can’t shot drops through Al’s oil mixture of paraffin and 1cst silicone oil 1cst Si-oil evaporates within 6 hours Nunc HLA and DI Vapor Batch plates not in SBS standard Solutions interface frame & complex programs to use non-SBS plates new oils – non volatile, water permeable

  21. Oils for (modified) mb Silicone oils = Polydimethylsiloxane =

  22. Silicone oils:evaporation and water permeability 2mm oil 20°C, 35% rel. humidity H2O oil

  23. Lysozyme mb test Lysozyme 10 mg/ml, Wizard 1 & 2 screens 0.5 ml + 0.5 ml

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