Here in Technical Services we often talk with researchers at the beginning of their project about how to carefully design and get started with their experiments. It is exciting when you have selected the Luciferase Reporter Vector(s) that will best suit your needs; you are going to make luminescent cells! But, how do you pick the best way to get the vector into your cells to express the reporter? What transfection reagent/method will work best for your cell type and experiment? Do you want to do transient (short-term) transfections, or are you going to establish a stable cell line?
For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.
Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.
The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.
The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:
Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.
You can read the Top 10 article in The Scientisthere.
A paper published in on August 8 in ChemBioChem has identified a number of small molecule kinase inhibitors that may have potential as antimalarial drugs. The authors, Derbyshire et al from Duke University, used a panel of human kinase inhibitors to screen for activity against malaria parasites. Using a high-throughput screening approach, they were able to identify several potential drug targets among the kinases of Plasmodium sp.,—most of which were effective against the parasite during both it’s blood-borne and liver-based life cycle stages.
Liver and blood-stage malaria parasites have different gene expression profiles and infect different host cells. The authors exploited these differences to try to specifically identify compounds that were active against the parasite while it was still in the liver, the idea being that any drug-based prevention strategy needs to be effective against the parasites in the liver in order to eradicate infection.
The authors screened a library of over 1300 kinase inhibitors that included several compounds already being used in clinical trials for anti-cancer activity. Initial screening was performed in human liver-derived HepG2 cells infected with Plasmodium berghei expressing a luciferase reporter. Compounds that decreased parasite load by more than 95% were further characterized in dose-response experiments, and promising hits were tested in using luminescent and fluorescent cell based assays to identify compounds that were not toxic to liver cells. Continue reading “Protein Kinase Inhibitors Show Promise in Malaria Study”
Numerous pharmaceutical companies have adopted assays for detecting activation of pregnane X receptor (PXR), a nuclear receptor known to regulate expression of cytochrome P450 (CYP450) drug-metabolizing enzymes (1). PXR is a transcription factor that has been designated the “master xenosensor” due to its ability to upregulate cellular levels of a variety of drug-metabolizing enzymes in response to drugs and foreign chemicals. Elevated levels of CYP450 enzymes can elicit alterations in the pharmacokinetics of co-administered drugs, which can result in adverse drug-drug interactions (DDI) or diminished bioavailability. By assessing PXR activation and CYP450 enzyme induction early in the drug development process, many companies hope to reduce late-stage clinical failures and minimize the high costs associated with bringing a new drug to market.
A recent paper by Shukla et al. (2) examined over 2,800 clinically used drugs for their ability to activate human PXR (hPXR) and rat PXR (rPXR), induce human cytochrome P450 3A4 enzyme (CYP3A4) at the cellular level, and bind hPXR at the protein level. Several studies have identified PXR as playing a key role in regulating the expression of CYP3A4, an enzyme involved in the metabolism of more than 50% of all drugs prescribed in humans. Continue reading “Screening for Drug-Drug Interactions with PXR and CYP3A4 Activation”