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

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?

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

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

Example of Simulated Annealing in Biological System

• Adaptive Immunity

Molecular components of adaptive immunity

• Somatic hypermutation

Molecular components of adaptive immunity

• DNA break and repair

AID at the center of adaptive immunity

•AID converts C to U, causing U:G mispairs.

•The mispairs are repaired through the base excision repair (BER) or the mismatch repair (MMR) pathways

•Mutations are introduced through the intervention of translesion DNA polymerases.

How does AID works?C U

UNG regulates transition/transversion ratio

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).

A Problem:

How to restrict AID function within the targeted sequence? The genomic damage must be avoided!

Possible solution:

Mimic the Immunoglobin structure?

in vivo evolution application based on adaptive immunity

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?

AID can work in yeast

An Example

Class of drug target SpeciesNumber of

molecular targets

Targets of approved drugs Pathogen and human 324

Human genome targets of approved drugs

Human 266

Targets of approved small-molecule drugs

Pathogen and human 248

Targets of approved small-molecule drugs

Human 207

Targets of approved oral small-molecule drugs

Pathogen and human 227

Targets of approved oral small-molecule drugs

Human 186

Targets of approved therapeutic antibodies

Human 15

GPCR, deorphanization and drug discovery

• GPCR: G protein coupled receptors

• A huge gene family

• Important pharmacological target

Sexual Reproduction in yeast-- a GPCR signaling pathway

How to get it done in yeast?

GPCR signaling mating pathway expression of heterologous GPCRs

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 pr

operties of mammalian subunits into a Gpa1 subunit that retains efficient interaction with the yeast G beta gamma

Four modification for heterogolous GPCRs

Knockout some native genes and incorporating reporter genes

knock out Ste2, Sst2, Far1

combine reporter genes behind PRE

Autocrine system establish an autocrine system

combine the ligand to a factor or alpha factor facilitating its secreting

but restrict on the membrane

What can we do with it ??

Our Plan …

hAID

lacZ lacI

lacO

IRES

PRE

Peptide-alpha factor

One example using GPCR protocol for artificial evolution

Ade2

Ade2

His3

IRES

Protocol…

The whole system

Signalling

No binding between peptide and GPCR

x

Initially………………..

hAIDlacO

Peptide-alpha factorAde2

Ade2

xlacZ lacI

IRES

PRE His3

IRES

No GPCR signalling: hAID is expressed to mutate peptide ligand

Signalling

Binding between peptide and GPCR

Until the peptide become an agonist of GPCR….

hAIDlacO

PRE

Peptide-alpha factor

GPCR is activated, AID is silenced…

Ade2

Ade2

x

Fus1

lacZ lacI

IRES

PRE His3

IRES

His3 lacZ

lacZ readout with fluorescence……… or visual detection directly

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

Applications for drug discovery

• Peptidergic ligand for specific GPCR

• Optimizing peptidergic ligand hits

• Finding conserved motif for agonist/antagonist

Assay procedure: it is easy!

• Transform GPCR to ready-knockout lines• Assay for constitutive activity• Transform the peptide-encoding vector libr

ary into a nice coupled GPCR line• Grow the transformant in large vial with ev

olution medium (His-, 3AT+)• After sometime, collect the solution and pl

ate for colonies• Sequence individual colony for hits

Further development on compound structure

GPCR other than ligand

Taking the complexity of the GPCR pathway into account

We can first use the simple yeast two- or three- hybrid systems for a test.

Yeast two-hybrid system

hAID

lacZ lacI

lacO

IRES

UAS

Peptide-Gal4-AD

For example,the core circuit could be adopted into Y2H

Ade2

Ade2

His3

IRES

The methods in two-hybrid systemsGenerally, the cDNA encoding the DBD-X fusion prot

ein and the cDNA encoding the AD-Y fusion protein are inserted into two plasmids, respectively, and then both transformed into the yeast cells.

Sexual Reproduction in yeast

Interaction mating methods can also be used in two-hybrid systems.

The AD and DBD fusion proteins begin in two different haploid yeast strains with opposite mating types, a and α, respectively.

To test for interaction,the hybrid proteins are brought together by mating, a process in which two haploid cells fuse to form a single diploid cell.

Further, Yeast

three-hybridsystem

• RNA aptamer screen

• … or: RNA-interacting protein?

Application of Y3H

Applications other than GPCR

• Nuclear receptor ligand screen

• Protein interaction screen

• Novel bacterial transcriptional biosensors?

• Whatever you can think about! :)

Summary

• We present a simple core genetic circuit which can evolve any desired target in vivo

• We present a unified, inexpensive solution for both academical and industrial needs

• In vivo evolution brings greater capacity and flexibility to screening

• Further assay development based on mammalian systems such as immune cell lines

Acknowledgements

• Wang Yiping

• Youri Pavlov

• Rao lab members

• iGEM 2007 members :)

• PKU iGEM 2008 society :)