1. Protein Production for Structure-Based Drug Design Stephen Chambers ~ Head of Gene Expression Vertex Pharmaceuticals Incorporated NIGMS 2004 PSI Protein Production & Crystallization Workshop March 29-31, 2004

2. Vertex: Building a Major Drug Company Focus: Small molecule drugs for major diseases Established: 1989; Public: 1991 Common stock: NASDAQ: VRTX >700 employees 3 sites – Cambridge, US (headquarters) – Oxford, UK – San Diego, CA Focus: Small molecule drugs for major diseases Established: 1989; Public: 1991 Common stock: NASDAQ: VRTX >700 employees 3 sites – Cambridge, US (headquarters) – Oxford, UK – San Diego, CA

3. Traditional vs. Vertex Approach to Drug Discovery Therapeutic Area Perspective Single target approach Target Family Perspective Chemogenomics multi-target approach

4. Expanding Parallel Drug Discovery into Gene Families

5. Content Structural Genomics vs Structure Based Drug Design –Much in common Expression strategy for higher output –Parallel expression of E.coli & insect cells –Quantitative analysis –Illustrated using examples from Vertex Kinase program –Application to other protein families Integration into a broader process with other disciplines

6. Structure Based Drug Design Drug Discovery (& Development) Human proteins –Complex post-translationally modified proteins –Heterogeneous proteins –‘Difficult’ proteins Highest value structures contain inhibitor Premium given to high-output (cf high-throughput) Failure not an option

7. Protein Expression Bottleneck Consumers: Protein Biochemistry –soluble, purifiable protein Enzymology –soluble, active protein –0.1-10 mg of protein Crystallography –soluble, crystallizable protein –5-100 mg of protein

8. Expression Process Triage Expression Prior to Production : Expression systems Growth conditions Cell lines Constructs Mutants

9. pBEV: Dual-System Expression Vector (Chambers et al 2004)

10. Expression Systems (Valk & Keus 1990)

11. Automated baculo-viral production qPCR used to determine viral titer

12. Wave Reactor (Wave Biotech) HiGro Shaker (Genomic Solutions Inc) Highly Engineered Process Using Standard Equipment Ni-NTA Magnetic Agarose Beads Genesis (Tecan) Cup-horn sonicator (Misonix Inc)

13. HT-Expression & Automated Purification

14. Full-Length Kinases in E.coli & Insect cells Increasing solubility in E.coli: TK>AGC>STE>>CMGC>CAMK

15. Model Behavior in E.coli (http://www.biotech.ou.edu/)(http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/cai.html)

16. Effect of Protein Size on Expression & Solubility in E.coli

17. Quantitative Analysis of Expression Strategies: Decision Tree Analysis of Kinase Expression 86% Structures 14% Structures Actual Payoff in Kinase Structures

18. Novel Kinase Structures in PDB (Yon & Jhoti 2003)

19. HT-Expression Allows the Exploration of Diversity: Rapid Identification of Well Expressing Proteins Pim-1 2.4 Å GSK3  2.7 Å (ter Haar et al 2001)

20. Crystal Structure of MAP Kappa 2 Kinase (Meng et al 2002) MAPK2K∆46 MAPK2K MAPK2K ∆46 2.8 Å

21. Crystal Structure of Aurora-2 Kinase Limited Proteolysis (Cheetham et al 2003) Aurora2K ∆107 2.9 Å

22. Crystal Structure of FLT-3 Kinase (Griffith et al 2004) FLT-3(H564-S993) 2.1 Å

23. HT-Expression Allows the Exploration of Diversity: Mutations

24. HT-Expression Allows the Exploration of Diversity: Utilizing Different Cell Lines

25. Insect Cell Expressed Structures in PDB

26. E.coli Expressed Structures in PDB

27. From the Vertex Portfolio Source of Proteins Source of Structures

28. Expressing Difficult Proteins in Insect Cells: Proteases CathepsinsSerine proteasesMetallo proteases

29. HT-Expression Allows the Exploration of Diversity: Protein Families (Phosphatases)

30. HT-Cloning & Expression Process

31. HT-Purification & Crystallography Process

32. No Protein Left Behind: Rescuing Insoluble Proteins CDC25a 1.7 Å ICE 2.6 Å

33. No Protein Left Behind: Rescuing Soluble Proteins

34. Integrated Platform Serving Structural Biology Miniaturized Automated Crystallization Multiple Inhibitor Structures for Drug Design

35. Novel Kinase Structures: Not as Interesting as the Active Site

36. An Active Site with Various Inhibitors

37. Conclusions Demonstrated efficient protein production integrated into a platform for structure-based drug-design Insect cells expression negates many of the deficiencies observed in E.coli expression High-throughput expression used to identify soluble expressed protein –proteins that are difficult to express and insoluble are usually difficult to purify and crystallize Parallel expression in E.coli and insect cells, providing greater number of soluble expressed protein, increases operational efficiency Process applicable to a range of gene families

38. Acknowledgements Molecular Cloning Protein Expression Protein Biochemistry X-Ray Crystallography Enzymology