Quantitation and detection of proteins is fundamental in understanding their methods of regulation, synthesis, and modification within a cellular system. Therapeutics that dictate the degradation (PROTACs and molecular glues) or stabilization (DUBTACs) of their protein targets are becoming more common within the drug discovery industry, thus the need to develop accurate, reliable methodologies to examine protein expression are essential.
A variety of assay formats currently used at Charnwood Discovery to examine protein expression and changes in response to stimuli and therapeutic treatment. Immunological based detection including dot blotting, immunoblotting and ELISA are sensitive methods for investigating protein expression, however higher throughput techniques such as flow cytometry and AlphaLISA are becoming increasingly popular. Such methods utilize antibodies specifically targeted against the protein of interest with detection based upon chemi-luminescence and fluorescence.
Automated and Quantitative Approach
Immunoblotting has been used routinely used since the 1970’s. The specificity of the interaction allows the protein expression of the target to be determined within complex mixtures, however this technique is not truly quantitative. The next step in the evolution of western blot, the Simple-Western system uses capillary-based separation, requires only a small sample size and can implement multiplexed/multichannel detection. The Simple-Western system allows for a faster, automated, and importantly truly quantitative approach to protein expression analysis.
Figure 1) Cells treated with increasing concentrations of SNS-THAL-032 prior to lysis and subsequent analysis by Simple Western. Quantification of CDK9 (normalised to β-Actin) showed that SNS-THAL-032 is a potent and reproducible degrader of CDK9 with an average DC50 of 16.1 nM ± 7.6 nM and Dmax of 99% across three biological replicates.
The desire to increase throughput while maintaining physiological relevance can be met with the use of flow cytometry, allowing protein expression within individual cells to be determined. At Charnwood Discovery, our high-throughput flow cytometry capability enables the measurement of protein expression of multiple targets within mixed cell populations and to also analyze other key cell health markers simultaneously, such as apoptosis. Flow cytometry is therefore a valuable tool for PROTAC screening.
Figure 2) PROTAC treatment results in a dose dependent shift in the median fluorescence intensity (MFI) of the target protein as measured by flow cytometry. This shift can be quantified to determine DC50 constants for degrader molecules.
The enzyme-linked immunosorbent assay (ELISA) is a commonly used technique to determine the concentration of an analyte, whether that be a protein or antigen. Using specialized plates, the analyte is immobilized to the well surface, enabling quantification.
The bead based AlphaLISA® immunoassay works in a similar manner as the ELISA but has been specifically designed with drug discovery in mind. The AlphaLISA® is a chemiluminescent, no wash assay, with greater sensitivity, wider dynamic range, and smaller sample size than an ELISA. At Charnwood Discovery, this technology has been used to screen against targets of interest, producing highly reproducible data and allowing effective small molecule development.
Figure 4) AlphaLISA schematic example illustrating transfer of energy from donor to acceptor when in close proximity. Quantification of the protein of interest in response to therapeutic treatment demonstrates a dose dependent response.