uHTS of Mcl-1/Bid interaction inhibitors
The Bcl-2 protein family includes anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1 and pro-apoptotic proteins such as Bak, Bax, Bim, Bid and Bad. All members of the Bcl-2 protein family contain at least one conserved Bcl-2 homology (BH) domain. These domains have been demonstrated to be involved in the homo- and hetero-dimerizations among the Bcl-2 family proteins. Experimental more ..
BioActive Compounds: 2129
NIH Molecular Libraries Screening Centers Network [MLSCN]
Emory Chemical Biology Discovery Center in MLSCN
Assay provider: Nikolovska-Coleska, University of Michigan
MLSCN Grant: R21NS057014
The Bcl-2 protein family includes anti-apoptotic proteins such as Bcl-2, Bcl-xL and Mcl-1 and pro-apoptotic proteins such as Bak, Bax, Bim, Bid and Bad. All members of the Bcl-2 protein family contain at least one conserved Bcl-2 homology (BH) domain. These domains have been demonstrated to be involved in the homo- and hetero-dimerizations among the Bcl-2 family proteins. Experimental three-dimensional structures of Bcl-2, Bcl-xL and Mcl-1 show that these proteins all have a well-defined, hydrophobic surface binding groove, known as the Bcl-2 homology domain 3 (BH3) binding groove, into which the pro-apoptotic proteins bind. Several BH3 peptides from proapoptotic proteins have been demonstrated to bind Bcl-2 family of proteins (Bcl-2, Bcl-xL, Mcl-1). Although there are several compound classes reported as inhibitors of Bcl-2/Bcl-xL, there is still a lack of small-molecule inhibitors of Mcl-1. Reports indicate that the most potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-xL and Bcl-w described to date, fail to bind to Mcl-1. This is evidence that Mcl-1 has critical structural differences compared with other anti-apoptotic proteins and strongly suggests that specific targeting of Mcl-1 should be possible.
Bid is a broad acting BH-3 only protein that binds Mcl-1 as well as other pro-survival proteins, including Bcl-2 and Bcl-xL To screen for small molecular inhibitors that disrupt the interaction of Mcl-1 protein and its binding partners, a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed and miniaturized to a 1536 well format to measure the interaction between Mcl-1 protein and the 21-residue Bid-BH3 (residues 79-99) peptide. Mcl-1 protein with a 6xHis tag was indirectly labeled with a europium chelate through a europium conjugated anti-6XHis antibody. Bid peptide was synthesized with an N-terminal Dy647 tag. Europium and Dy647 comprise a fluorescence energy transfer pair. Interaction of Mcl-1 protein with Bid peptide brings two conjugated fluorophores into proximity, leading to an energy transfer from europium to Dy647 and the generation of FRET signals. FRET signal is detected in an Envision Multilabel plate reader (Ex 340 nM, Em615 nM and Em665 nM) and expressed as FRET signal ratio (F665nm / F620nm * 104). The assay is robust with a consistent Z' factor of 0.5-0.8 in a 1536-well plate format and is used for the screening of the NIH/DPI library for Mcl-1/Bid interaction inhibitors.
1. Assay buffer: 20 mM Tris buffer, pH 7.5, 50 mM NaCl, and 0.01% NP40
2. Mcl-1 protein: 6xHis-tagged Mcl-1 protein
3. Europium Chelate Anti-6xHis-Antibody (His-Eu): LANCE Eu-W1024 from Perkin Elmer Life Sciences
1. Make assay reaction buffer for uHTS that contains Mcl-1 protein (100 nM, final concentration), Bid-Dy647 peptide (30 nM, final concentration), and His-Eu (1 nM, final concentration).
2. Dispense 4.5 uL of assay reaction buffer to 1536-well black assay plate.
3. Add 100 nL of library compound (1 mM in DMSO) to each well and incubate plates at room temperature for 2 hr. Final concentration of each compound is 21.74 uM.
4. Record FRET signals with an Envision Multilabel plate reader (Perkin Elmer Life Sciences) with laser. An laser excitation at 340 nm and emission filters at 615 nm and 665 nm are used with a dural dichroic mirror of D400/630.
1. FRET signals are expressed as FRET ratios:
FRET = F665 nm / F620 nm * 10000
2. Assay data are analyzed using CambridgeSoft's BioAssay software. Percentage of inhibition is calculated with the following equation based on data from each plate.
% of Inhibition = 100 - ((FRETcompound - FRETpeptide only) / (FRETcontrol - FRET peptide only)) * 100
Where FRETcompound is the FRET ratio from a well with a test compound, FRET peptide only is an average FRET signal from wells with Bid-Dy647 peptide only; FRET signal control is an average FRET ratio from wells containing Mcl-1 protein and Bid-Dy647 peptide, which defines maximal FRET.
PUBCHEM_ACTIVITY_OUTCOME: Compounds that cause > 50% inhibition are defined as positives.
PUBCHEM_ACTIVITY_SCORE: The percent inhibitions are rounded to 0 decimal places. Pct Inhibitions > 100 are rounded to 100, and those < 0 are rounded to 0.
1. Artifacts of this assay could be resulted from, but are not limited to, intrinsic fluorescence of some compounds, compounds that can quench europium or Dy647 fluorescence, dust or lint.
2. All data reported were normalized on a per-plate basis.
** Test Concentration.
Data Table (Concise)