CharacterizationofaDirectNF‐{kappa}BLigand

AmalchiCastillo‐Rodriguez08/07/09

CrystalstructureofNF‐kBp50/p65heterodimerboundtoDNANature391,410‐413(22January1998)|doi:10.1038/34956

FiveproteinsoftheNF‐{kappa}BFamily

Cell.2008Feb8;132(3):344‐62.

ThetwofacesofNF‐{kappa}Bingeneregulation

Cancercells

Cellsurvival

Metastasis

Proliferation

Inflammation

Normalcells

Celldeath

TumorSuppressors

Antiproliferativeeffects

Cell.2008Feb8;132(3):344‐62

TheNF‐{kappa}Bcanonicalpathway

Small-molecule microarrays (SMMs)

fluorescent features reveal putative binding interactions

1 mm

compute composite Z-scores

SMM containing 10,800 features and probed with protein

…proteins…

…co

mpo

unds

…

specificity heat map

fluor-labeled antibody

tag

protein

AGAisastrongerNF‐{kappa}Bbinderasshownbysurfaceplasmonresonance

80%BRD‐K92758126

KD=226nMp50‐SPRP65‐SPR

purified80%pure

nottestedKD=2,300nMKD=13,000nM

AGA

KD = 76 nM KD = 6,340 nM

AGAinhibitsNF‐{kappa}B translocation

Translocationassay,p50Ab(green)andHoechst(blue)

EdgarIbarraRakheeBusanelli

p50 Ab (green) and Hoechst (blue)

p50 Ab (green) and Hoechst (blue)

Maingoals

•  TostudytheinhibitoryeffectofAGAonallfiveNF‐{kappa}Bproteins.

•  TodeterminetheeffectofAGAoncellviability.

MeasuringtheeffectofAGAinNF‐{kappa}Bproteintranslocation

1.  Non‐stimulatedcells

3.TNF‐astimulated+AGA(10nM)

2.TNF‐astimulated

a

AGATransAMhasapreferenceforp65

1 hour stimulation

10nM AGA concentration

a

c

a

c

a

c

!"!#

HEK293cellviabilityisnotaffecteduponadditionofAGA

a

Conclusions

•  AGAtemporarilyinhibitsNF‐{kappa}Btranslocationtothenucleus.

•  TheinhibitoryeffectofAGAappearstovaryindifferentcancercelllines,butmighthaveapreferenceforp65.

•  AGAdoesnotaffectcellviability.

Futuredirections

•  Use of a gene expression database to explain ELISA data.

•  Use of higher concentrations of AGA to test for inhibition.

•  SynergystudiesinvolvingcompoundsthattargetotherpartsoftheNF‐kBpathway.

•  Dockingexperimentswillhelptheorizesitesofinteraction.

Acknowledgements

Dr. Angela Koehler

Rakhee N. Busanelli

Katie Doud

Jack Kelly

David Pearlman

Nathan West

Stuart Schreiber

SRPG program

30minuteincubation 1hourincubation

Thereisnosignificantincreaseinabsorbanceupon30minuteincubationover1hourincubation.

Pulsatile Stimulation Determines Timing and Specificity of NF-kB-Dependent Transcription. L. Ashall, C. A. Horton. Science 324, 242-246

VaryingdegreeofAGAselectivityamongdifferentcancercells

3T3L1 HeLa C2C12