NanoBRET® TE PRMT5 Assay
Detect Multiple Modes of PRMT5 Engagement in Live Cells
- Measure intracellular compound affinity for PRMT5
- Evaluate target engagement at both the peptide-substrate binding pocket and the cofactor binding pocket
- Characterize the binding of MTA-cooperative inhibitors to PRMT5-MTA complex in live cells
- Optimized assay using a simple, addition-only workflow
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Catalog Number: CS3489A02
Catalog Number: CS3489A03
Catalog Number: CS3489A22
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Measure Target Engagement to PRMT5 in Live Cells
Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation for both histone and non-histone substrates and uses S-adenosylmethionine (SAM) as the cofactor and methyl group donor. PRMT5 regulates a wide spectrum of cellular processes, such as cell signaling and differentiation, RNA splicing and DNA damage response, and has been shown to be a promising synthetic lethality target for MTAP (methylthioadenosine phosphorylase)-deleted cancer cells.
The NanoBRET® TE PRMT5 Assay can be used to quantify affinity of PRMT5 inhibitors with different modes of action in live cells. The assay is based on the NanoBRET® technology, an energy transfer technique designed to measure molecular proximity in living cells.
Want to accelerate your drug discovery workflow? Our Tailored R&D Solutions team offers services using the NanoBRET® TE Assay for PRMT5.
How the NanoBRET® TE PRMT5 Assay Works
NanoBRET® TE Assays quantify target engagement using bioluminescence resonance energy transfer (BRET). These BRET-based assays rely on the transfer of luminescent energy from a NanoLuc® luciferase-target fusion protein expressed in cells to a cell-permeable fluorescent NanoBRET® tracer that binds to the target protein.
In the NanoBRET® TE PRMT5 Assay, PRMT5 is fused to NanoLuc® Luciferase and co-expressed with WDR77, the partner protein of PRMT5, in mammalian cells. To generate a BRET signal, a fixed concentration of a cell-permeable fluorescent NanoBRET® TE tracer is added to these cells and binds to the NanoLuc-PRMT5 fusion protein. Addition of compounds that bind to PRMT5 results in a dose-dependent decrease in BRET signal. This data allows quantitation of the intracellular affinity of compounds for PRMT5 in live cells.
Evaluate Target Engagement at Substrate and Cofactor Binding Pockets
PRMT5 contains two distinct substrate and cofactor binding pockets (Panel A), and both binding pockets have been targeted for developing PRMT5 inhibitors. The NanoBRET® TE PRMT5 Assay uses a single tracer that binds to the substrate pocket to detect compound engagement at either the substrate binding pocket (Panel B) or the cofactor binding pocket (Panel C).
A
Measure Compound Binding to PRMT5-MTA Complex
In MTAP-deficient cancer cells, there is an accumulation of 5′-Methylthioadenosine (MTA), an intermediate in the methionine salvage pathway. An elevated level of MTA leads to inhibition of PRMT5, sensitizing the cancer cells to further loss of PRMT5 activity and exposing a vulnerability in MTAP-deficient cancers. Developing inhibitors that preferentially engage the PRMT5-MTA complex can exploit this synthetic lethal interaction to selectively target MTAP-deficient cancer cells. The NanoBRET® TE PRMT5 Assay can be used to assess target binding by MTA-cooperative PRMT5 inhibitors in live cells.
See the PRMT5 TE Assay Scientific Poster on how the assay can be used to evaluate the impact of MTA on inhibitor-PRMT5 interactions and characterize MTA-cooperative inhibitors.
What You Need to Perform the NanoBRET® TE Assay
We supply:
- NanoBRET® Tracer PRMT5*
- NanoLuc®-PRMT5 Fusion Vector
- WDR77 Expression Vector
Additional reagents needed for performing the NanoBRET® TE Assay are listed below. A full list of required materials can be found in the Application Note.
*Each vial is sufficient for at least 1,000 assays when used as recommended in the Application Note.
Patents and Disclaimers
Materials may be covered by pending or issued patents or may have certain limitations.
Information is available upon request and will be included on applicable quotes.
Protocols
Specifications
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Resources
Featured Resource
A live cell PRMT5 NanoBRET® Target Engagement Assay querying competitive and uncompetitive modes of inhibition
A live-cell PRMT5 NanoBRET® Target Engagement (TE) Assay is now developed that enables the analysis of PRMT5 inhibitors with a variety of modes of action, including inhibitors that are MTA-cooperative. Engagement potency of MTA-cooperative inhibitor MRTX1719 measured by the TE assay correlates with disease-state MoA.
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