Protein Production for StructueBased Drug Design基于結構的藥物的設計的蛋白質(zhì)的生產(chǎn)

1、protein production for structure-based drug design stephen chambershead of gene expressionvertex pharmaceuticals incorporatednigms 2004 psi protein production & crystallization workshopmarch 29-31, 2004vertex: building a major drug companyfocus:small molecule drugs for major diseases established

2、: 1989; public: 1991 common stock: nasdaq: vrtx 700 employees 3 sites cambridge, us (headquarters) oxford, uk san diego, catraditional vs. vertex approach to drug discoverytherapeutic area perspectivesingle target approachtarget family perspectivechemogenomics multi-target approachexpanding parallel

3、 drug discovery into gene familiescontent 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

4、families integration into a broader process with other disciplinesstructure 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-ou

5、tput (cf high-throughput) failure not an optionprotein expression bottleneckcl oni ngexpr es s i onpur i f i cat i oncr ys t al l ogr aphydnaenzymol ogyconsumers: protein biochemistry soluble, purifiable protein enzymology soluble, active protein 0.1-10 mg of protein crystallography soluble, crystal

6、lizable protein 5-100 mg of proteinexpression process triage expression prior to production: expression systems growth conditions cell lines constructs mutantspbev: dual-system expression vector(chambers et al 2004)expression systemse.coliyea stin sectcell sm amm aliancell spr oteolyticcleavage+/-+/

7、-+glycosylation-+secre tion+/-+foldi ng+/-+/-+pho spho ry lation-+yield (% )based on dry wt1-5130agcstecmgccamkmodel behavior in e.coli(/)(http:/www.hgmp.mrc.ac.uk/software/emboss/apps/cai.html)effect of protein size on expression & solubility in e.coliquantitative analys

8、is of expression strategies:decision tree analysis of kinase expression86% structures14% structures actual payoff in kinase structuresnovel kinase structures in pdb(yon & jhoti 2003)ht-expression allows the exploration of diversity: rapid identification of well expressing proteinspim-1 2.4 gsk3

9、2.7 (ter haar et al 2001)crystal structure of map kappa 2 kinase(meng et al 2002)mapk2k46mapk2kmapk2k 46 2.8 crystal structure of aurora-2 kinaselimited proteolysis(cheetham et al 2003)aurora2k 107 2.9 crystal structure of flt-3 kinase(griffith et al 2004)flt-3(h564-s993) 2.1 domaindeletiondupli cat

10、ionmutanth564-s993i tdh564-s993h564-s993h711-v782h564-s993f723-h761h564-v958i tdh564-v958h564-v958d835yh564-v958h711-v782h564-v958h711-h761h564-v958f723-v782h564-v958f723-h761h564-f936h564-f936h711-v782h564-f936f723-h761n587- s993i tdn587- s993n587- s993h711-v782n587- s993f723-h761n587- v958i tdn587

11、- v958n587- v958h711-v782n587- v958h711-h761n587- v958f723-v782n587- v958f723-h761n587- f936i tdn587- f936n587- f936h711-v782n587- f936f723-h761w603-s993w603-s993h711-v782w603-s993f723-h761w603-v958w603-v958h711-v782w603-v958f723-h761w603-f936ht-expression allows the exploration of diversity:mutatio

12、nsht-expression allows the exploration of diversity:utilizing different cell linesinsect cell expressed structures in pdbe.coli expressed structures in pdbfrom the vertex portfoliosource of proteinssource of structuresexpressing difficult proteins in insect cells: proteasescathepsinsserine proteases

13、metallo proteasesht-expression allows the exploration of diversity: protein families (phosphatases)ht-cloning & expression processht-purification & crystallography processno protein left behind: rescuing insoluble proteinscdc25a 1.7 ice 2.6 no protein left behind: rescuing soluble proteinsin

14、tegrated platform serving structural biologyminiaturizedautomatedcrystallizationmultiple inhibitor structures for drug designnovel kinase structures: not as interesting as the active sitean active site with various inhibitorsconclusionsdemonstrated efficient protein production integrated into a plat

15、form for structure-based drug-designinsect cells expression negates many of the deficiencies observed in e.coli expressionhigh-throughput expression used to identify soluble expressed protein proteins that are difficult to express and insoluble are usually difficult to purify and crystallizeparallel expression in e.coli and insect cell