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

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

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

Expanding Parallel Drug Discovery into Gene Families

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

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

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

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

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

Expression Systems (Valk & Keus 1990)

Automated baculo-viral production qPCR used to determine viral titer

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)

HT-Expression & Automated Purification

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

Model Behavior in E.coli (

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

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

Novel Kinase Structures in PDB (Yon & Jhoti 2003)

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

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

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

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

HT-Expression Allows the Exploration of Diversity: Mutations

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

Insect Cell Expressed Structures in PDB

E.coli Expressed Structures in PDB

From the Vertex Portfolio Source of Proteins Source of Structures

Expressing Difficult Proteins in Insect Cells: Proteases CathepsinsSerine proteasesMetallo proteases

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

HT-Cloning & Expression Process

HT-Purification & Crystallography Process

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

No Protein Left Behind: Rescuing Soluble Proteins

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

Novel Kinase Structures: Not as Interesting as the Active Site

An Active Site with Various Inhibitors

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

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