The regulation and interaction between proteins and their binding partners can control and regulate complex biological processes. As such, dysfunction of these protein-protein interactions (PPIs) is frequently implicated in the development of diseases such as cancer and neurodegeneration.
Achieving Reproducible and Consistent Assays
Small molecule inhibitors, serving as biological probes or therapeutics and targeting these biomolecular interactions, have progressed significantly in helping us understand the impact of disrupting key PPIs. Conversely, stabilization of PPIs is a largely underrepresented area within research but is one with enormous potential in the process of drug discovery. Techniques to interrogate these interactions serve as important tools in novel therapeutic development.
Advances in high-resolution structures have revealed many PPI interfaces can be largely featureless, making molecule design difficult. Despite this, remarkable progression has been made to investigate the interaction between two proteins through numerous techniques, including but not limited to:
NanoBRET™
NanoBRET™ uses Bioluminescence Resonance Energy Transfer (BRET), a proximity-based assay that measures the transfer of energy from a donor to an acceptor within a live cell background (Figure 2).
The use of full-length proteins expressed within a cellular system allows for an appropriate reflection of physiological conditions to monitor the change in PPIs in response to therapeutic treatment.
Figure 2A) Overview of the NanoBRET™ system. Energy is transferred when the NanoLuc and HaloTag tags are in close proximity, driven by the interaction between proteins. 2B) Interaction between the two proteins is measured in milli BRET units (mBU). Therapeutics can modulate this in a concentration dependent manner.
Homogenous Time Resolved Fluorescence
Homogenous Time Resolved Fluorescence (HTRF) is a biochemical, cell-free, and high-throughput assay relying on fluorescence resonance energy transfer (FRET). FRET occurs when energy from an excited fluorophore (donor) is absorbed by another molecule (acceptor) within close proximity. The HTRF assay is not limited to PPIs and has been applied to cell signalling (phosphorylation and GPCRs), and cytokine and chemokine release from immune cells.
Figure 3A) HTRF schematic overview. Upon excitement (using light at the appropriate wavelength), the energy from the antibody-fluorophore conjugate binding protein A is transferred to a streptavidin-fluorophore conjugate bound to protein B. Energy transfer is only effective when fluorophores are in close proximity. 3B) The concentration of the inhibitor is directly proportional to the quantifiable HTRF signal, indicating PPI is disrupted by the inhibitor.
Surface Plasmon resonance (SPR)
Surface plasmon resonance (SPR) is a commonly used biophysical technique for detailed and quantitative studies of protein-protein interactions. SPR provides label-free, real-time investigation of PPIs to determine their binding equilibrium and kinetic parameters. SPR can be utilised to screen compounds that have potential to modulate PPIs, including fragment-based screening.
Co-immunoprecipitation (Co-IP)
Co-immunoprecipitation (Co-IP) is a popular technique to identify physiologically relevant protein-protein interactions by using target-specific antibodies to immobilise the protein of interest enabling retention of binding partners. Commonly used to identify novel PPIs, this technique can also be used to study the dynamics of the interaction in response to various stimuli and treatments with samples typically examined by immunoblot.
NanoBRET™ uses Bioluminescence Resonance Energy Transfer (BRET), a proximity-based assay that measures the transfer of energy from a donor to an acceptor within a live cell background (Figure 2).
The use of full-length proteins expressed within a cellular system allows for an appropriate reflection of physiological conditions to monitor the change in PPIs in response to therapeutic treatment.
