The Myc family of proteins regulates many aspects of cell fate. Myc genes are deregulated in several human neoplasias as a result of genetic and epigenetic alterations (1,2). Most biological functions of Myc require heterodimerization with its activation partner Max (3,4). Inhibition of the protein-protein interactions between Myc and Max by small molecules is a plausible approach towards the inhibition of Myc functions (2–4). Here we evaluated two cell-based assay systems from Promega using NanoLuc® luciferase-based technologies for Myc-Max protein-protein interaction (PPI) studies. Both the BRET- (bioluminescence resonance energy transfer) based assay (NanoBRET™) and NanoBiT® (NanoLuc® Binary Technology) assay, a structural complementation reporter assay, can be used in high-throughput screening of compounds to identify PPI inhibitors. Here we present the results from the optimization of these two assays and subsequent screening to identify novel chemicals that inhibit Myc–Max heterodimerization.
Materials and Methods
For NanoBRET™ assays, we used the MCF10A cell line (ATCC Cat.# CRL-10317) due to its low endogenous c-Myc levels and also its amenability for high-throughput screening (HTS). We followed the standard operating procedure developed by Promega with HEK293 cells for designing fusion constructs and transfecting them into cells for expression in the assay. Figure 1 is a schematic representation of a NanoBRET™ PPI assay. We tested different fusion constructs for the assay with varied orientations of fusion tags on the amino- and carboxy-terminus of Myc and Max proteins. We also used a mock donor construct (provided by Promega) without our test protein to check for nonspecific inhibition of BRET by the compounds tested. Furthermore, we tested different ratios of fusion plasmids during assay optimization, finding that a 100:1 ratio of acceptor:donor was ideal for the assay. The substrates for NanoLuc® and HaloTag® ligand were obtained from Promega (Cat.# N1663). NanoBRET™ signal was measured using a PHERAstar FSX plate reader (BMG Labtech) using filters to separate donor and acceptor signals. NanoBiT® Complementation Assay reagents were obtained by custom synthesis of large and small NanoBiT® subunits (LgBiT and SmBiT) fused with Myc and Max, respectively, from Promega. Interaction of the LgBiT and SmBiT subunits was measured using Nano-Glo® Live Cell Reagent (Cat.# N2013). Data was analyzed using Genedata Screener® (Version 13; Genedata AG). NanoBRET™ results are reported as the ratio of NanoBRET™ acceptor/donor signals. NanoBiT® results are reported as relative luminescence.
Figure 1. Schematic illustration of the NanoBRET™ PPI Assay using Myc tagged with NanoLuc® Luciferase as a donor and HaloTag®-fused Max as an acceptor for energy transfer. Both Myc and Max fusion proteins with either of the tags were used for the assay validation. For screening efforts, Max-HaloTag® and Myc-NanoLuc® constructs were used.
As there were no suitable potent compounds available as tool compounds, we validated the assay with non-HaloTag® ligand treated cells as negative control for the NanoBRET™ assay. Initially we did a checkerboard test for assay variability. Z’ was 0.5 or greater, suggesting good reproducibility of the assay (Figure 2).
Figure 2. Checkerboard with or without HaloTag® ligand. MCF10A cells were transiently transfected with protein fusion constructs in 150ml tissue culture-treated dishes overnight. The transfected cells were plated in 384-well assay plates (Corning Cat.# 3570). The NanoBRET™ ratio was calculated from raw values for NanoLuc® luciferase and HaloTag® signals for all wells, 5 hours after plating the cells. This ratio, with or without HaloTag® ligand, was normalized to 100% and 0%, respectively, using Genedata Screener® (Genedata AG).
Initially we tested a library of biologically active compounds (a commercial and in-house collection). A hit cutoff rate of 80% activity for PPI inhibitors was set giving a hit rate of about 1.5%, as shown in Figure 3. There is also the possibility of picking up fluorescent compounds as indicated in Figure 3, which will give increased signal in the acceptor channel and thus artificially increase the NanoBRET™ ratio. Encouraged by the initial results, we screened additional libraries. We selected some of the “actives” or “hits” from this screen and ran the dose response experiment in the same assay as shown in Figure 4 to demonstrate that inhibition of the Myc-Max assay was dose dependent.
Figure 3. Screening of compounds in the NanoBRET™ Assay using a biologically active set of compounds. The compounds in DMSO stock were used for screening in singlicates at a 20µM final concentration in assay medium (Lonza Cat.# CC-3153, without phenol red and with growth factors). The NanoBRET™ ratio was calculated from raw values for NanoLuc® and HaloTag® signals. This ratio with or without HaloTag® ligand was normalized to 100% and 0%, respectively, using Genedata Screener® (Genedata AG). The hit cutoff for the compounds was 80% or less of the activity in NanoBRET™ PPI with DMSO alone as 100%. The wells showing activity of greater than 100% NanoBRET™ ratio suggest the possibility of fluorescence artifact values from some compounds.
Figure 4. Dose-response data for the actives in the Myc-Max NanoBRET™ screen. A few of the compounds that showed reduction in the NanoBRET™ ratio in the primary screen were retested in a 14-point dose-response experiment in MCF10A cells transiently transfected with Myc- and Max-fusion constructs. The NanoBRET™ ratio was calculated from raw values for NanoLuc® and HaloTag® signals. This ratio with or without HaloTag® ligand was normalized to 100% and 0%, respectively using Genedata Screener®. IC50 values are indicated for the compounds 1 and 2.
NanoLuc® Counterscreen Assay
We noticed that some of our compounds were reducing the donor luciferase signal to an extent more than that was observed with the NanoBRET™ positive samples. Hence, we were interested to know whether our compounds had an inhibitory effect on donor signal thereby causing a corresponding decrease in acceptor signal resulting in reduced NanoBRET™ ratio. This may help to avoid false positive hits. As shown in Figure 5 there was a reduction in luminescence in response to compounds when there was no HaloTag® ligand, suggesting an effect on NanoLuc® luciferase instead of protein-protein interactions between Myc and Max. The ideal profile for specific PPI inhibitors in the NanoBRET™ Assay is that of compound 3.
Figure 5. NanoBRET™ screen and counterscreen for compounds with Myc or a mock donor, respectively. Myc and Max fusion proteins were expressed in MCF10A cells. Also, we used a mock NanoLuc® donor obtained from Promega along with Max acceptor fusion protein to express in the cells to look for any nonspecific effects of compounds on the assay readout. Compound 3 showed desirable outcome as it is specific without an inhibitory effect on the NanoLuc® signal in a dose-dependent manner. Both compounds 4 and 5 showed a reduction in NanoLuc® signal in which compound 4 was not active in the NanoBRET™ screen but compound 5 showed reduction in the NanoBRET™ ratio. X axis values show concentration of compounds in log10 [M] and y axis values show normalized NanoBRET™ ratio.
NanoBiT® Complementation Assay
There have been literature reports related to the use of enzyme complementation assays to evaluate PPI of Myc-Max heterodimers (5). Hence, we investigated the use of the NanoBiT® complementation assay for the validation of this interaction. As shown in Figure 6, we see complementation when the two NanoBiT® subuits were fused with Myc and Max proteins (0 level of titrated partner). Next we tested whether this complementation could be disrupted by co-expressing a range of either Myc or Max proteins with HaloTag® ligands that were used with NanoBRET™ assay. As shown in Figure 6, we observed a dose-dependent competition of Myc or Max proteins with respective partners, suggesting the feasibility of detecting disruption of heterodimerization.
Figure 6. Overview of the NanoBiT® Complementation Assay and competition with Max- and Myc- HaloTag® fusion expression constructs co-expressing Myc- and Max- NanoBiT® vectors. Panel A. Proteins A and B are fused to LgBiT and SmBiT and expressed in cells. Interaction of the two proteins leads to structural complementation of LgBiT and SmBiT, and generation of luminescence. For expression of the Max- (Panel B) and Myc- (Panel C) HaloTag® fusion expression constructs, indicated ratios of plasmid DNA amounts were transfected in HEK293 cells in 100ml tissue culture dishes. The NanoBiT® complementation assay was performed about 20 hours after transfection with the transfected cells replated in 384-well plates (Corning Cat.# 3570). Background refers to luminescence values obtained from wells containing cells without NanoLuc® substrate. All other wells received NanoLuc® substrate before luminescence was measured.
NanoBiT® Complementation Counterscreen Assay
To look for specificity of the NanoBiT® Myc/Max assay, we used an unrelated p53-Mdm2 NanoBiT® assay as a counterscreen. Nutlin-3, a specific p53-Mdm2 interaction inhibitor, disrupted the PPI between p53 and Mdm2 in this assay, as shown in Figure 7. We also tested nutlin-3 in cells expressing Myc- and Max- NanoBiT® contructs, which did not disrupt complementation of these two protein partners. We ran the two assay systems in parallel to test the specificity of the possible Myc-Max PPI inhibitors.
Figure 7. Counterscreen for Myc-Max PPI screen in the NanoBiT® assay. We used the respective fusion protein pairs for expression in HEK293 cells and looked for activity of nutlin-3 (Sigma Cat. # N6287), a PPI inhibitor of p53-Mdm2 interaction, in those cells in a dose-response format.
Both the NanoBRET™ Assay and the NanoBiT® complementation assay are homogeneous and allow for high-throughput screening platforms for PPI in live cells. These are powerful tools to investigate PPI in a cellular environment that can complement the existing biochemical approaches. Both NanoLuc® luciferase-based systems are simple to use and provide very robust and sensitive detection methods. The two systems can also be used together, allowing for counter screening for nonspecific and false positive signals.
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