Inhibitors of TCP-1 ring complex (TRiC) of Methanococcus maripaludis (MmCpn): qHTS
The eukaryotic chaperonin TRiC (for TCP-1 ring complex) is an essential and conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. TRiC is indispensable for cell survival, as folding of an essential subset of cytosolic proteins, including cytoskeletal components, cell cycle regulators and tumor suppressor proteins, exhibits a strict more ..
BioActive Compounds: 101
The eukaryotic chaperonin TRiC (for TCP-1 ring complex) is an essential and conserved chaperone required for the folding and activation of 10-15% of the proteins encoded by the human genome. TRiC is indispensable for cell survival, as folding of an essential subset of cytosolic proteins, including cytoskeletal components, cell cycle regulators and tumor suppressor proteins, exhibits a strict requirement for TRiC which cannot be substituted by other cytosolic chaperones. The TRiC complex consist of two stacked 7-9 membered rings, each containing a central cavity. Substrates are folded within the cavity in an ATP-dependent manner. TRiC is a complex of eight different, essential proteins. As each of the genes encoding the eight subunits is essential for cell viability and no protocol has been developed to over-express this enzyme to high levels. That is why several groups studying the TRiC chaperonin have turned to the archaeal homolog of TRiC, Methanococcus maripaludis (Mm-CPN), and have used this enzyme as a model system for the study of the type II chaperonin family. Small molecule inhibitors of the Mm-CPN enzyme would be extremely useful to study the mechanism of chaperonin activity. Importantly, TRiC and Mm-CPN both require ATP hydrolysis for function. Furthermore, the ATP binding sites are highly conserved among these type II chaperonins. This provides us with an easy, well tested and straightforward assay to identify inhibitors of chaperonin activity.
The Mm-CPN assay was developed in 1536-well plate format using the HTRF Transcreener ADP assay kit (Cisbio, Bedford, MA). This assay applied the time resolved fluorescence resonance energy transfer (TR-FRET) principle. ATP in the presence of Mm-CPN is converted to ADP + Pi. An antibody labeled with Eu3+-Cryptate specific and bound to ADP labeled with deuterium (d2) competes with the unlabeled ADP generated by Mm-CPN. The resulting TR-FRET signal is inversely proportional to the concentration of ADP in the sample. In this assay a reduction in TR-FRET signal is observed with ATP turnover.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030554-01A1
Assay Submitter (PI): Judith Frydman, Stanford University
Purified Mm-CPN was supplied by the laboratory of Dr. Judith Frydman. Protein samples were stored at -80C as single use aliquots. Protein was thawed in ambient temperature water bath and placed on ice once thawed. Buffer was prepared fresh day of screen. The buffer composition below was used to prepare the working solution of the Mm-CPN protein and ATP substrate. The buffer is comprised of 30 mM TRIS pH 7.4, 100mM KCl, 5mM MgCl2, and 1mM DTT. A final concentration of 25nM Mm-CPN and 10uM ATP per well were used in the screen. Instructions according to the manufacturer were followed for the ADP Transcreener kit. Used 1536 well Greiner white, medium binding (MB), solid bottom plates. Plates were read on the PerkinElmer EnVision plate reader using Excitation = 320 nm and Emission = 590, 665 nm.
1.Dispense 1.5ul per well of working [Mm-CPN] = 50nM (or 75 ng / well)
2.Spin plate at 1000 rpm for 30 seconds
3.23nl of compound transferred per well
4.Incubate at 37degC for 10 minutes
5.Dispense 1.5ul per well of working [ATP] = 20uM
6.Incubate at 37degC for 30 minutes
7.Dispense 3ul per well of Anti-ADP Cryptate / ADP-d2 mix (prepared as instructed by Cisbio)
8.Incubate at ambient for 30 minutes
9.Read using EnVision
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 Description". 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 all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)