qHTS Assay for Disrupters of an Hsp90 Co-Chaperone Interaction
Hsp90 (heat shock protein 90) is the essential molecular chaperone and it accounts for 1-2% of all cytosolic proteins and is critical for the activity of diverse cellular proteins that are involved in a variety of cellular processes, including development, cell cycle, and steroid hormone signaling. Its client proteins include signaling kinases such as IGF1R, Akt, v-Src, Raf-1; regulators of cell more ..
BioActive Compounds: 300
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
NCGC Assay Overview:
Hsp90 (heat shock protein 90) is the essential molecular chaperone and it accounts for 1-2% of all cytosolic proteins and is critical for the activity of diverse cellular proteins that are involved in a variety of cellular processes, including development, cell cycle, and steroid hormone signaling. Its client proteins include signaling kinases such as IGF1R, Akt, v-Src, Raf-1; regulators of cell survival such as Bcr-Abl; and transcription factors such as mutated p53, estrogen and progesterone receptors. Hsp90 has emerged as an important target for cancer treatment due to its essential role in maintaining transformation and regulating the function of oncogenic signaling proteins.
The in vivo function of Hsp90 is regulated by the binding and hydrolysis of ATP at its N-terminal. Geldanamycin binds to ATP site and inhibit Hsp90 function. Geldanamycin and its analogues are under clinical trials in cancer patients.
Recent data indicate the C terminal domain of Hsp90 as an important regulator as the N terminus and may represent a second site for pharmacological intervention in chaperone function. This domain contains the binding site for tetratricopeptide repeat (TPR) domains on HOP (Hsp Organizing Protein). TPR is a structural motif used by a significant number of cochaperones by which the in vivo function of Hsp90 is regulated. TPR2A on HOP binds specifically to MEEVD motif of the C-terminal of Hsp90.
A homogeneous AlphaScreen is developed to assess the interaction between TPR2A and MEEVD motif of the C-terminal of Hsp90 by using the isolated TPR2A domain and a synthesized 20mer Hsp90 C terminal peptide. AlphaScreen is two bead-based proximity-dependent chemical energy transfer luminescent assay platform. TPR2A-His-6 and biotinylated Hsp90 C terminal peptide will bring streptavidin donor beads and nickel chelate acceptor beads to a proximity range (less than 200 nm), and subsequently lead a chemical luminescent reaction upon exciting donor beads with 680 nm laser. Inhibition of TPR2A and MEEVD motif interaction by small molecules could attenuate or abolish this chemical luminescent reaction.
Counter screening: Biotinylated-His-6 (PerkinElmer) can be utilized as a counter screen, as it serves as a linker to bring streptavidin beads and nickel chelate beads together. The counter screen can exclude alpha screen platform false positives due to small molecules being metal chelators, biotin analogues and imidazole analogues, or due to interfering with oxygen release from donor beads. Such an assay should be utilized to qualify any activity observed in this screen.
NCGC Assay Protocol Summary:
The assay was performed on 1536-well white plate (untreated solid bottom). Liquids were dispensed by 1536-well plate washer (Kalypsys, San Diego, CA). Plates were read on EnVision (PerkinElmer, Boston, MA).TPR2A-His6 was produced using a bacteria expression system and purified using a well developed method (Ghosh et al., 2000). Hsp90 C terminal 20-mer peptide with or without an N-terminal biotin group are synthesized using automated solid phase synthesis by the Yale Keck facility. The composition of assay buffer was (in mM) 25 HEPES, 100 KCl, 2 MgCl2 and 2 DTT; 0.1% BSA and 0.01% Tween-20. TPR2A-His6 binding to biotin-Hsp90 will bring streptavidin donor beads and nickel chelate acceptor beads (PerkinElmer) together and can be measured by Alpha reading by EnVision (PerkinElmer). Free Hsp90 blocks biotin-Hsp90 binding to TPR2 and serves as a positive control in the current asssay.
The assay was performed at room temperature. All compounds were screened as titrations from 0.6 nM to 46 uM final concentration. The assay was performed in 1536-well format plates (untreated white solid bottom, Corning Inc.) as follows:
1,Reagent,3 uL TPR2A-His6,Final concentration was 5nM
2,Compound,23 nL,Final titration was from 0.7 nM to 46 uM
3,Time,15 minutes,Room temperature
4,Reagent,1 uL biotin-Hsp90,With and without free Hsp90. Final concentration was 10 nM for biotin-Hsp90 and 10uM for free Hsp90.
5,Time,1 hour,Room temperature
6,Reagent,1 uL bead mixture,10 ng/well for each bead
7,Time,2 hour,Room temperature
Keywords: Hsp90, TPR2A, AlphaScreen, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Classification". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For inconclusive compounds, PUBCHEM_ACTIVITY_SCORE is 10.
Data Table (Concise)