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Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

Tim J Wigle1, W David Church1, Christina R Majer1

  • 1Ribon Therapeutics Inc., Cambridge, MA, USA.

SLAS Discovery : Advancing Life Sciences R & D
|December 20, 2019
PubMed
Summary
This summary is machine-generated.

Developing robust biochemical assays for mono(ADP-ribosyl)transferases (monoPARPs) is crucial for discovering new drugs targeting cancer and inflammation. This study presents a novel family-wide assay approach for 12 monoPARPs and 3 polyPARPs, enabling inhibitor screening.

Keywords:
dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA)mono(ADP-ribosylation) (MARylation)nicotinamide adenine dinucleotide (NAD+)poly(ADP-ribose) polymerase (PARP)self-modification

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are critical posttranslational modifications.
  • While PARylation is therapeutically targeted, MARylation's roles in immunity, inflammation, and cancer are emerging, yet lack specific chemical probes.
  • Understanding mono(ADP-ribose) polymerase (monoPARP) substrate engagement is essential for assay development.

Purpose of the Study:

  • To develop robust, high-throughput biochemical assays for all 12 enzymatically active monoPARPs.
  • To establish assays for 3 poly(ADP-ribose) polymerase (polyPARP) enzymes.
  • To enable hit identification and characterization of inhibitor potency and selectivity across the monoPARP family.

Main Methods:

  • Developed a family-wide assay strategy immobilizing monoPARP enzymes.
  • Utilized biotinylated-nicotinamide adenine dinucleotide (NAD+) to induce enzyme self-modification.
  • Employed dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for sensitive detection.

Main Results:

  • Successfully developed and validated assays for 12 monoPARPs and 3 polyPARPs.
  • Demonstrated the utility of these assays for evaluating inhibitor potency and selectivity.
  • Provided a foundation for chemical probe development for monoPARPs.

Conclusions:

  • The developed assays represent a significant advancement for studying monoPARP enzymology.
  • This work facilitates the discovery of novel therapeutics targeting MARylation pathways.
  • The family-wide approach offers a scalable platform for future drug discovery efforts.