NanoBRET® TE Assays for PARPs and PARG

Measure PARP and PARG Target Engagement in Live Cells

Quantitate intracellular affinity of compounds for PARG and multiple PARP family members
Ready-to-use, scalable, addition-only assays
Profile compound selectivity across multiple PARPs
See a list with ordering options below

Measure Target Engagement to PARPs and PARG in Live Cells

NanoBRET® Target Engagement (TE) is a biophysical method of assessing the binding between a compound and a target protein in live cells. Based on bioluminescent resonance energy transfer (BRET), NanoBRET® TE can be applied to multiple classes of target proteins, including poly (ADP-ribose) glycohydrolase (PARG) and members of the poly (ADP-ribose) polymerase (PARP) family, which play critical roles in cellular processes such as transcription, cell division and DNA repair.

Want to accelerate your drug discovery workflow? Our Tailored R&D Solutions team offers services using the NanoBRET® TE Assays for PARPs and PARG.

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How It Works

In the NanoBRET® TE Assays for PARPs and PARG, the target PARP or PARG protein is expressed as a NanoLuc® Luciferase fusion. A cell-permeable, fluorescent NanoBRET® PARP or PARG tracer is added. The tracer reversibly binds to the PARP or PARG target-NanoLuc® fusion protein.

**Overview of NanoBRET® Target Engagement Assay.** Binding of a test compound results in loss of NanoBRET® signal between a target-NanoLuc® fusion protein and a cell-permeable fluorescent NanoBRET® Tracer inside intact cells.

When in proximity to one another, the tracer and NanoLuc® Luciferase produce a BRET signal. When a test compound binds to the PARP or PARG target proteins, it competes with the tracer, and the BRET signal is lost.

Quantitate Cellular Affinities of PARP and PARG Small-Molecule Inhibitors

PARPs

The poly-ADP-ribose polymerase (PARP) family of proteins regulate numerous cellular processes, including DNA repair and transcription. Development of small-molecule inhibitors for PARPs are of therapeutic interest. The NanoBRET® TE PARP Assay can be used to study >10 PARP family members, specifically those circled in Panel A. The NanoBRET® TE PARP Assay allows you to get started quickly studying target engagement with your compounds in live cells because a protocol with recommended assay conditions and data is provided for each PARP. A panel of compounds was profiled against PARP1 and PARP5A using this assay, and representative data is shown in Panels B and C.

Profiling Chemical Matter Against PARP1 in Live Cells

Profiling Chemical Matter Against PARP5A in Live Cells

NanoBRET® TE PARP Assay coverage and representative data in HEK293 cells. PARP TE assay can be used with PARPs circled (Panel A). The PARP assay was used to determine intracellular affinity for various compounds against PARP1 (Panel B) and PARP5a (Panel C).

PARG

Poly (ADP-ribose) Glycohydrolase (PARG) catabolizes poly (ADP-ribose) units, facilitating the recycling of PARP and other modified effector proteins in PARP-mediated processes. PARG can be a complementary target in modulating the PARP-mediated DNA damage response, potentially providing a strategy for overcoming resistance to PARP inhibitors. The NanoBRET® TE PARG Assay can be used to directly quantify compound binding to PARG in live cells using an addition-only, microplate-compatible workflow. A protocol with recommended assay conditions and a list of required materials is provided with the assay reagents, allowing quick adoption of the assay.

Line graph showing measurement of test compound affinity using NanoBRET® TE Tracer. — For analysis of target engagement by a test compound, cells are treated with a fixed concentration of NanoBRET® tracer that is near the EC₅₀ value of the NanoBRET® tracer dose response curve. The assay protocol provides a representative tracer dose response curve and a final recommended tracer concentration.

Line graph showing cellular engagement (binding affinity measurements) of various target compounds. — To determine test compound affinity, cells are titrated with varying concentrations of the test compound in the presence of tracer at the recommended concentration. Binding to the target by the test compounds results in a loss of BRET signal. Ethacridine showed no displacement of the tracer, in agreement with the proposed mechanism of action of ethacridine binding directly to PAR instead of PARG [Tavassoli et al. (1985) *Biochim. Biophys. Acta.* **827**, 228–34].

View the NanoBRET® TE PARG Assay Protocol.

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.

Early Access Materials for NanoBRET® TE PARP and PARG Assays

These products are available under our Early Access program - Learn More

*Each vial of NanoBRET® Tracer PARG is sufficient for at least 1,000 assays when used as recommended in the Application Note.

Assays	Size	Part Number
NanoBRET® TE PARP Kit #1	1,000 assays	CS1810C551
NanoBRET® TE PARP Kit #1	10,000 assays	CS1810C621
Tracers	Size	Part Number
NanoBRET® Tracer PARG*	50µl	CS3489A25
Vectors	Size	Part Number
PARG-NanoLuc® Fusion Vector	20µg	CS3489A06
PARP1-NanoLuc® Fusion Vector, Iso1	20µg	CS1810C736
PARP2-NanoLuc® Fusion Vector	20µg	CS1810C688
PARP3-NanoLuc® Fusion Vector	20µg	Coming Soon
NanoLuc®-PARP4 Fusion Vector	20µg	Coming Soon
PARP5a-NanoLuc® Fusion Vector	20µg	CS1810C689
PARP5b-NanoLuc® Fusion Vector	20µg	Coming Soon
PARP7-NanoLuc® Fusion Vector	20µg	CS1810C687
PARP8-NanoLuc® Fusion Vector	20µg	Coming Soon
PARP10-NanoLuc® Fusion Vector	20µg	Coming Soon
PARP11-NanoLuc® Fusion Vector	20µg	CS1810C691
PARP12-NanoLuc® Fusion Vector	20µg	CS1810C692
Thaw-and-Use Cells	Size	Part Number
PARP1, Iso1, TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E107 CS1810E108
PARP2 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E77 CS1810E78
PARP3 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E80 CS1810E81
PARP4 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E74 CS1810E75
PARP5a TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E83 CS1810E84
PARP5b TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E86 CS1810E87
PARP7 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E47 CS1810E48
PARP8 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E89 CS1810E90
PARP10 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E92 CS1810E93
PARP11 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E50 CS1810E51
PARP12 TE HEK293 Thaw-and-Use Cells	1X 5X	CS1810E53 CS1810E54

Ordering Options

Contact us to get started with the NanoBRET® TE PARP Assay, PARP-NanoLuc® Fusion Vectors, and/or PARP Thaw-and-Use Cells.

MS 2022 Early Access Materials for NanoBRET TE PARP Assay

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