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In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement
Tim J Wigle1, Danielle J Blackwell1, Laurie B Schenkel1
1Ribon Therapeutics, 35 Cambridgepark Drive, Suite 300, Cambridge, MA 02140, USA.
Cell Chemical Biology
|July 18, 2020
Summary
Researchers developed novel active site probes for Poly(ADP-ribose) polymerase (PARP) enzymes. These probes enable precise characterization of inhibitors targeting NAD+ binding in vitro and in live cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Poly(ADP-ribose) polymerase (PARP) enzymes catalyze the modification of proteins using nicotinamide adenine dinucleotide (NAD+).
- PARP enzymes are classified as monoenzymes (MARylation) or polyenzymes (PARylation), with diverse but often uncharacterized substrates.
- Developing specific inhibitors for PARP enzymes is crucial for therapeutic applications, yet hindered by substrate ambiguity and assay limitations.
Purpose of the Study:
- To develop active site probes for characterizing inhibitors of PARP monoenzymes and polyenzymes.
- To establish robust in vitro and cellular biophysical assays for evaluating inhibitors that compete with NAD+ binding.
- To overcome substrate specificity limitations in current PARP enzyme activity assays.
Main Methods:
- Development of active site probes for PARP enzymes.
- Implementation of in vitro biophysical assays using minimal enzyme quantities.
- Establishment of cell-based assays for potency and residence time measurements in live cells.
- Assays designed to be agnostic of specific PARP protein substrates.
Main Results:
- The developed assays enable the characterization of active site-directed inhibitors that compete for NAD+ binding.
- In vitro assays achieve single-digit nanomolar potency discrimination with reduced enzyme usage.
- Cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
- The substrate-agnostic nature of the assays broadens their applicability across different PARP enzymes.
Conclusions:
- Novel active site probes and associated biophysical assays provide a powerful tool for characterizing PARP enzyme inhibitors.
- These assays facilitate the development of selective tool compounds and potential therapeutics targeting PARP enzymes.
- The substrate-agnostic approach overcomes a significant hurdle in PARP research, enabling broader inhibitor discovery and characterization.
