uHTS of Mcl-1/Noxa 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: 3334
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.
Noxa is a specific Mcl-1 binding BH3-only protein that exhibits selectivity to Bcl-2 family proteins. To screen for small molecular inhibitors that selectively disrupt the interaction of Mcl-1 protein and Noxa, 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 26-residue Noxa-BH3 (residues 18-43) peptide. Mcl-1 protein with a 6xHis tag was indirectly labeled with a terbium through a terbium conjugated anti-6XHis antibody. Noxa peptide was synthesized with an N-terminal rhoamine tag. Terbium and rhodamine comprise a fluorescence energy transfer pair. Interaction of Mcl-1 protein with Noxa peptide brings two conjugated fluorophores into proximity, leading to an energy transfer from terbium to rhodamine and the generation of FRET signals. FRET signal is detected in an Envision Multilabel plate reader (Ex 340 nm laser, Em545 nm and Em572 nm) and expressed as FRET signal ratio (F572nm / F545nm * 10000). 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.
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. Terbium-anti-His-Tag antibody from Invitrogen corporation.
1. Make assay reaction buffer for uHTS that contains Mcl-1 protein (62.5 nM, final concentration), Noxa-rhodamine peptide (125 nM, final concentration), and terbium-anti-His-Tag antibody (2 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 545 nm and 572 nm are used with a dural dichroic mirror of D400.
1. FRET signals are expressed as FRET ratios:
FRET = F572 nm / F545 nm * 10000
2. Assay data are analyzed using CambridgeSoft BioAssay. 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 Noxa-rhodamine peptide only; FRET signal control is an average FRET ratio from wells containing Mcl-1 protein and Noxa-rhodamine peptide, which defines maximal FRET.
3. Compounds that cause > 50% inhibition are defined as positives.
1. Artifacts of this assay could be resulted from, but are not limited to, intrinsic fluorescence of some compounds, compounds that can quench terbium or rhodamine fluorescence, dust or lint.
2. All data reported were normalized on a per-plate basis.
Data Table (Concise)