How it works Enzyme
Enzyme Fragment Complementation
Assay Overview
DiscoveRx’s proprietary Enzyme Fragment Complementation (EFC) technology is a proven and established screening platform that can be used to quantitatively measure the interactions between a test compound and it’s cognate receptor. This robust and reliable assay technology has been applied to every major class of drug target, including GPCRs, kinases, proteases, nuclear hormone receptors, transcription factors, and secreted proteins and can be used in both biochemical and cell based formats to provide unique solutions to enhance therapeutic drug discovery programs.
Assay Principle
Enzyme Fragment Complementation (EFC) is a homogenous detection system based on two genetically engineered fragments of β-galactosidase: a small peptide fragment called Enzyme Donor or ED (Panel A) and a large protein fragment, called Enzyme Acceptor or EA (Panel B). Separately, the β-galactosidase fragments are inactive. But in solution, the fragments rapidly recombine to form active β-galactosidase enzyme (Panel C) that is capable of hydrolyzing substrate and producing a chemiluminescent or fluorescent signal (Panel D).
Enzyme Donor (ED)
ED is a small (4-11 kD), non-isotopic tag, which can be chemically conjugated to a variety of small molecules or proteins without changing their properties and function. In addition, ED can be expressed recombinantly as a fusion protein and used for novel protein:protein interactions or protein trafficking in cells.
Enzyme Acceptor (EA)
EA is a truncated, inactive β-galactosidase enzyme fragment lacking the amino acids expressed in the ED peptide. In solution, EA can rapidly recombine with either conjugated ED or recombinantly expressed fusion protein to form active β-galactosidase enzyme capable of hydrolyzing a chemiluminescent substrate.
Active β-galactosidase Enzyme and Substrate
β-galactosidase (E.C. 3.2.1.23) specifically and reproducibly hydrolyzes numerous substrates. DiscoveRx assays are optimized with a chemiluminescent substrate which produces a high intensity signal with low background that is not affected by naturally fluorescent compounds.
EFC provides all of the following advantages
- Proven technology – over 600 targets
- High-throughput screening friendly format
- Rapid and reliable results
- Industry validated
Adaptable to Multiple Targets and Target Classes
DiscoveRx offers validated biochemical and cell based assay solutions for every major drug target class which includes over 600 assays for GPCRs, kinases, proteases, nuclear hormone receptors, transcription factors, and secreted proteins.
DiscoveRx EFC-based assays for various drug target classes
| Technology |
GPCRs |
Kinases
|
NHRs |
Proteases |
Pathways |
| Protein:Protein Interaction |
X |
X |
X |
|
X |
| Protein Degradation |
|
|
|
X |
X |
| Protein Translocation |
|
|
X |
|
X |
| Protein Secretion |
|
|
|
X |
X |
| Protein Trafficking |
|
|
|
|
X |
Target Panels, Services & Solutions
HitHunter® Biochemical Assays
Biochemical assays are formatted as competitive immunoassays, where analytes are detected in solution without separation or wash steps. This provides improved sensitivity and precision compared to conventional immunoassays.
PathHunter® Cell–Based Assays
The PathHunter whole cell assays use positional complementation of the β-galactosidase enzyme in live cells to monitor translocation events for targets such as transcription factors, arrestin proteins, kinases and nuclear hormone receptors or to monitor protein secretion, degradation or expression without the use of a specific antibody.
PathHunter Assays using ProLabel tagged fusion proteins
One recombinant form of ED, called ProLabel (or PL) complements very efficiently with EA and can be referred to as "high affinity complementation". In one example of this format, EA is specifically localized to the nucleus, while the ProLabel tagged target protein is localized in the cytoplasm. Positional Complementation occurs only when the ProLabel fusion proteins translocates to the nucleus in response to extracellular stimuli. Assays that measure protein degradation, secretion and membrane trafficking can also be easily configured.
PathHunter Assays using ProLink tagged fusion proteins
ProLink™ (or PK), a mutant variant of ProLabel sequence, has a lower affinity to EA. For complementation to occur, the two tagged proteins (EA and ProLink tagged proteins) have to interact following a biological response to stimulus. This leads to complementation and formation of active β-galactosidase. Assays that measure receptor activation and cytoplasmic interactions can easily be configured using this principle. Over 600 GPCRs, Receptor Tyrosine Kinases and Nuclear Hormone Receptor assays are available.
Robust and Reliable - Ideal for Profiling
EFC is an enzymatically amplified signal, resulting in large signal to background ratios and high precision with excellent Z' factors. EFC-based assays do not require washing, centrifugation or filtration steps and the assays have been optimized for robust and reliable results. The chemiluminescent assay signal minimizes interference from library compounds, introducing no artificial signal due to non-specific binding of beads or secondary labels. Together, features deliver reproducible, low hit rates and low batch to batch variation.