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Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase
Anna K Duell1, Daniel J Sanderson1, Michael S Cohen2
1Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA.
Abstract:
PARP7 is an enzyme that catalyzes mono-ADP-ribosylation (MARylation), is a critical regulator of type I interferon signaling, and has emerged as an immune-oncology drug candidate. PARP7 is a labile protein that is regulated in a proteasome-dependent manner. Indeed, endogenous PARP7 levels are undetectable by western blot in most cells. Intriguingly, treatment of cells with orthosteric small molecule inhibitors of PARP7 can increase endogenous PARP7 protein to detectable levels. This characteristic of PARP7 inhibitors could potentially be exploited to assess target engagement-and thus cellular efficacy-of PARP7 inhibitors; however, no method exists to quantitatively monitor endogenous PARP7 levels in a high-throughput manner. In this protocol, we describe an assay using a split Nanoluciferase (NanoLuc) system for quantifying endogenous PARP7 protein levels and PARP7 inhibitor target engagement in cells in a 96-well plate format. We show that this assay can be used to quantify PARP7 protein levels under various cellular treatments and can assess cellular PARP7 inhibitor target engagement. We envision this split NanoLuc PARP7 assay can be used not only for evaluating the cellular efficacy of PARP7 inhibitors in a high-throughput manner but also for uncovering the mechanisms regulating PARP7 protein levels in cells.
Insights
A new assay quantifies poly(ADP-ribose) polymerase 7 (PARP7) protein levels and inhibitor engagement in cells. This method enables high-throughput evaluation of PARP7 inhibitors for immune-oncology applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Poly(ADP-ribose) polymerase 7 (PARP7) catalyzes mono-ADP-ribosylation (MARylation), regulating type I interferon signaling and emerging as an immune-oncology target.
- PARP7 is a labile protein, typically undetectable in cells due to proteasomal degradation, complicating target engagement assessment.
- PARP7 inhibitors can stabilize the protein, offering a potential biomarker for cellular efficacy.
Purpose of the Study:
- To develop a high-throughput assay for quantifying endogenous PARP7 protein levels.
- To establish a method for assessing PARP7 inhibitor target engagement in cellular models.
- To facilitate drug discovery and mechanistic studies of PARP7 in immune-oncology.
Main Methods:
- Utilized a split Nanoluciferase (NanoLuc) system for protein quantification.
- Developed a 96-well plate assay format for cellular analysis.
- Validated the assay for measuring PARP7 levels under different cellular conditions.
Main Results:
- Successfully quantified endogenous PARP7 protein levels in cells using the split NanoLuc assay.
- Demonstrated the assay's capability to assess PARP7 inhibitor target engagement.
- Showcased the assay's utility in evaluating PARP7 protein modulation by various treatments.
Conclusions:
- The split NanoLuc PARP7 assay provides a robust method for high-throughput quantification of PARP7 and its inhibitor engagement.
- This assay can accelerate the development of PARP7-targeted immune-oncology therapeutics.
- The assay may also aid in elucidating the regulatory mechanisms governing PARP7 protein stability.
