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T R -F R E T (6 6 5 /6 2 0 ) x 1 0 0 0 0 T R -F R E T ra tio (5 2 0 /4 9 0 ) x 1 0 ,0 0 0 FRET RATIO (520/490)X10000 S /B Development and validation of HTS assay 6 0 0 0 0 5 0 0 0 4 0 0 0 3 0 0 0 G S T -ta g C H E C K k it (C is B io ) G S T c o n tro l G S T -S U M O 1 G S T -S U M O 2 4 0 0 0 0 S 1 F H T R F F S 1 H T R F E C 5 0 = 8 .7 u M E C 5 0 = 1 .4 u M 6 4 S 1 -F H T R F F -S 1 H T R F 2 0 0 0 1 0 0 0 2 0 0 0 0 2 0 0 .1 1 1 0 1 0 0 1 0 0 0 [G S T d is p la c e r] (n M ) 0 0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 1 0 0 0 0 [F lu o re s c e in -P e p tid e ] (n M ) • Binding assay for GST-SUMO1/FITC-S1 pair developed with HTRF Lumi4®-Tb anti-GST antibodies for primary HTS • Developed counter screen assays • Pilot HTS was performed on three assays in 1536-well plates using PHERAstars • The project submitted into MLPCN 20000 15000 10000 0 1 0 1 0 0 1 0 0 0 1 0 0 0 0 5000 F L U O R E S C E IN (n M ) GST-SUMO1/FITC-S1 FITC-S1 (no SUMO1) IC 50 = 11.9 uM 0 0 20 40 [S1 peptide] (uM)
SUMO-PPI MLPCN screening project • Primary HTS (PubChem AID 602429) • TR-FRET assay using GST-SUMO1 and FITC-S1 peptide and Lumi4®- Tb anti-GST (1536-well plates) • 364K-compound MLSMR collection • Hits profiled against PubChem data to eliminate artifacts • Secondary assays: • FP assay SUMO1 and FITC-S1 (PubChem AID 624382) • TR-FRET SUMO2 and FITC-S1 (PubChem AID 624384) • FP assay SUMO2 and FITC-S1 (PubChem AID 624385) • PubChem Summary AID 602467 • PI lab is currently characterizing the compounds identified in the screening Danielle Key, Ada Kane, Siobhan Malany, PhD Eliot Sugarman, Kevin Nguyen, Eigo Suyama, PhD, Steve Vasile, PhD
Page 1 and 2: HTRF Symposium Avignon, France Apri
Page 3 and 4: • SBMRI and CPCCG • NIH Roadmap
Page 5 and 6: Development of Sanford-Burnham Medi
Page 7 and 8: Sanford-Burnham’s Discovery Resea
Page 9 and 10: Conrad Prebys Center for Chemical G
Page 11 and 12: Sanford-Burnham uHTS Facility (Orla
Page 13 and 14: Specifics of MLPCN projects • MLP
Page 15 and 16: MLPCN screening at CPCCG Detection
Page 17 and 18: MLPCN UBL projects performed at CPC
Page 19 and 20: SUMO PPI project • PI: Yuan Chen,
Page 21: Assay design for SUMO PPI project B
Page 25 and 26: SUMOylation project: HTRF Assay GST
Page 27 and 28: SUMOylation project: Probe compound
Page 29 and 30: • Assay Reagents: Assay captures
Page 31 and 32: R a tio (5 2 0 /4 9 0 )x 1 0 0 0 0
Page 33 and 34: Z' factor CUL1 neddylation project
Page 35 and 36: MLN4924 has a complex mechanism of
Page 37 and 38: Concluding remarks • HTRF approac

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