Discovery Research via in vivo Evolution

1. Discovery Research via in vivo Evolution Huang Lei, Tian He, Wen Ya, and Zhang Yi Peking University, and National Institute of Biological Sciences, Beijing 2008 03 02

2. Discovery Research in Biology • To answer the question: ‘what’/‘whether’ • Example 1: what activates receptor X? • Whether drug alpha activates X? • Example 2: what suppresses gene Y? • Whether gene beta suppresses Y? • Example 3: what maintains stem cell state? • Whether kinase gamma maintains stem cell state?

3. Strategies for Discovery Research • Two strategies towards the goal: • Guess: answering whether, intelligent but very few novel insight • Screen: answering what, laboring but can give anti-intuition insight • Hereinafter we concentrate on screening

4. Complexity Theory for Screen • You always have it in your first 100 lines or you never have it -- Seymour Benzer on flies • Complexity theory: when dimension grows, for serial screening, complexity grows in geometrical metrics • Monte Carlo method complexity • Simulated annealing: decelerating Monte Carlo method

5. Example of Simulated Annealing in Biological System • Adaptive Immunity

7. Molecular components of adaptive immunity • Somatic hypermutation

8. Molecular components of adaptive immunity • DNA break and repair

9. AID at the center of adaptive immunity

10. U C How does AID works? • AID converts C to U, causing U:G mispairs. • The mispairs are repaired through the base excision repair (BER) or the mismatchrepair (MMR) pathways • Mutations are introduced throughthe intervention of translesion DNA polymerases.

11. UNG regulates transition/transversion ratio

12. Limiting AID function • Transcription rate of the target gene: AID only targets ssDNA • AID promoters and enhancers • Epigenetic insulators • Specific sequence bias -Hotspots: DGYW/WRCH (R = A/G, Y = T/C, W = A/T, D = A/G/T).

13. A Problem: How to restrict AID function within the targeted sequence? The genomic damage must be avoided! Possible solution: Mimic the Immunoglobin structure?

14. in vivo evolution application based on adaptive immunity

15. Problems (and solutions?) • Mammalian cells grow slow • Bacteria/yeast grow fast • Mammalian cells are expensive • Bacteria/yeast are cheap • Eukaryote protein has to be correctly folded and glycosylated • Yeast better than bacteria?

16. AID can work in yeast

17. An Example

20. GPCR, deorphanization and drug discovery • GPCR: G protein coupled receptors • A huge gene family • Important pharmacological target

30. Sexual Reproduction in yeast -- a GPCR signaling pathway

31. How to get it done in yeast? GPCR signaling mating pathway expression of heterologous GPCRs

32. Four modification for heterogolous GPCRs • Introducing heterologous GPCRs add a cleavable leader sequence to aid transport to the plasma membrane remove regions not required for interacting with the ligand or G protein. • Modifying the G protein develop chimeric G alpha subunits to incorporate receptor binding properties of mammalian subunits into a Gpa1 subunit that retains efficient interaction with the yeast G beta gamma

33. Four modification for heterogolous GPCRs • Knockout some native genes and incorporating reporter genes knock outSte2, Sst2, Far1 combine reporter genes behind PRE • Autocrine system establish an autocrine system combine the ligand to a factoror alpha factor facilitating its secreting but restrict on the membrane

35. What can we do with it ?? Our Plan …

36. Protocol… Peptide-alpha factor Ade2 IRES IRES His3 lacZ PRE lacI hAID lacO Ade2 The whole system One example using GPCR protocol for artificial evolution

37. No binding between peptide and GPCR x Signalling Initially………………..

38. Peptide-alpha factor Ade2 x IRES IRES His3 lacZ PRE lacI hAID lacO Ade2 No GPCR signalling: hAID is expressed to mutate peptide ligand

39. Binding between peptide and GPCR Signalling Until the peptide become an agonist of GPCR….

40. Peptide-alpha factor Ade2 Fus1 IRES IRES His3 lacZ PRE PRE lacI x hAID lacO Ade2 His3 lacZ GPCR is activated, AID is silenced…

41. lacZ readout with fluorescence ……… or visual detection directly

42. Positive and negative selections • Positive selection: • his3 mediated histidine- survival • High lacZ activity • Negative selection: • Raise in complete medium (let it grow!) • Low or no lacZ activity

43. Applications for drug discovery • Peptidergic ligand for specific GPCR • Optimizing peptidergic ligand hits • Finding conserved motif for agonist/antagonist

44. Assay procedure: it is easy! • Transform GPCR to ready-knockout lines • Assay for constitutive activity • Transform the peptide-encoding vector library into a nice coupled GPCR line • Grow the transformant in large vial with evolution medium (His-, 3AT+) • After sometime, collect the solution and plate for colonies • Sequence individual colony for hits

45. Further development on compound structure

47. GPCR other than ligand

48. Taking the complexity of the GPCR pathway into account We can first use the simple yeast two- or three- hybrid systems for a test.

49. Yeast two-hybrid system

50. Peptide-Gal4-AD Ade2 IRES IRES His3 lacZ UAS lacI hAID lacO Ade2 For example, the core circuit could be adopted into Y2H