Development of High-Development of High-throughput Screening Assay throughput Screening Assay

for Small Molecule Inhibitor(s) for Small Molecule Inhibitor(s) of PIAS1of PIAS1

Bourns College of Engineering

Department of Bioengineering

Vicente Nunez

Dr. Jiayu Liao

OutlineOutline

BackgroundBackground Our PlanOur Plan ResultsResults Future workFuture work

JAK-STAT PathwayJAK-STAT Pathway

D. Levy and J. Darnell, Nat Rev Mol Cell Biol (2002) 3: 651-662

Focus on

STAT1-PIAS1

Interactions

What is STAT1?What is STAT1?

SSignal ignal TTransducers and ransducers and AActivators of ctivators of TTranscriptionranscription

A member of a family of related A member of a family of related proteinsproteins

Several roles in immune cell Several roles in immune cell regulations and transcription of anti-regulations and transcription of anti-viral genesviral genes

What is PIAS1?What is PIAS1?

Protein Inhibitor of Activated STATProtein Inhibitor of Activated STAT Negative regulator of STAT1 Negative regulator of STAT1

Why Inhibit PIAS1-STAT1 Why Inhibit PIAS1-STAT1 Interactions?Interactions?

UCLA Mice StudyUCLA Mice Study Control of Immune ResponseControl of Immune Response Potential therapy development for immune system Potential therapy development for immune system

deficienciesdeficiencies

Our PlanOur Plan

Have STAT1 & PIAS1 proteins Have STAT1 & PIAS1 proteins expressed in mammalian cellsexpressed in mammalian cells

Tag these proteins with other Tag these proteins with other proteins that show fluorescence proteins that show fluorescence

Determine what molecules inhibit Determine what molecules inhibit the binding of PIAS1 to STAT1the binding of PIAS1 to STAT1

Our Plan Our Plan

Transfect fusion plasmid into mammalian cellsTransfect fusion plasmid into mammalian cells Transfect the smaller plasmids:Transfect the smaller plasmids:

(YFP-STAT1 & PIAS1-CFP)(YFP-STAT1 & PIAS1-CFP)

OR

Our PlanOur Plan

Why Two Alternatives?Why Two Alternatives? ONE COMPLEX:ONE COMPLEX:

HypothesisHypothesis JAK1KD will phosphorylate STAT1 and JAK1KD will phosphorylate STAT1 and

initiate PIAS1-STAT1 interactioninitiate PIAS1-STAT1 interaction No need to introduce IFNNo need to introduce IFNγγ to activate STAT1 to activate STAT1

TWO SEPARATE PLASMIDS:TWO SEPARATE PLASMIDS: More straight forward approach to More straight forward approach to

experimentexperiment Need IFNNeed IFNγγ to activate STAT1 to activate STAT1

Our PlanOur Plan

Detect changes of FRET (Förster Detect changes of FRET (Förster Resonance Energy Transfer) signalsResonance Energy Transfer) signals

http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg

PIAS1

STAT1 PIAS1

STAT1

440 nm

480 nm 440 nm

527 nm

Hypothetical Inhibitor(s)Hypothetical Inhibitor(s)

Inhibitor “X” separates the two proteins Inhibitor “X” separates the two proteins Resulting in loss of energy transferResulting in loss of energy transfer We want to find what “X” isWe want to find what “X” is

XPIAS1 STAT1 PIAS1 STAT1

440 nm 480 nm

440 nm

527 nm

http://www.rowland.harvard.edu/labs/bacteria/images/fret2.jpg

What We AccomplishedWhat We Accomplished

YFP STAT1

pcDNA 3.1

pcDNA 3.1

PIAS1 CFP

pcDNA 3.1

What We AccomplishedWhat We Accomplished

Transfected the YFP-STAT1Transfected the YFP-STAT1 Transfected PIAS1-CFPTransfected PIAS1-CFP

What We AccomplishedWhat We Accomplished

Observed fluorescence from cells Observed fluorescence from cells expressing the YFP-STAT1 proteinexpressing the YFP-STAT1 protein

Em Wavelength in nm (Ex: 476nm)

490 500 510 520 530 540 550 560 570 580 590 6000

50000

100000

150000

Well M3

Lambda at Maximum

Future WorkFuture Work

Finish characterizing the PIAS1-CFP Finish characterizing the PIAS1-CFP plasmidplasmid

Finish building the 5 fragment Finish building the 5 fragment constructconstruct

Transfect it into mammalian cellsTransfect it into mammalian cells Undertake FRET Assay on both Undertake FRET Assay on both

strategiesstrategies Develop into high throughput Develop into high throughput

screening assayscreening assay

In ConclusionIn Conclusion

Finding PIAS1 inhibitor(s) will Finding PIAS1 inhibitor(s) will potentially benefit human healthpotentially benefit human health Potential development of treatments for Potential development of treatments for

immune system deficienciesimmune system deficiencies Further our understanding of PIAS1’s Further our understanding of PIAS1’s

role in the JAK-STAT pathway of role in the JAK-STAT pathway of immune regulationimmune regulation

AcknowledgemenAcknowledgementsts

Dr. Jiayu LiaoDr. Jiayu Liao Yang SongYang Song Vipul MadaharVipul Madahar Xiulin ShenXiulin Shen Adam ChengAdam Cheng Dr. V. G. J. RodgersDr. V. G. J. Rodgers BRITE ProgramBRITE Program